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Engineered Microtopographies to Induce in Vitro Endothelial Cell Morphologies Stable to Shear

Permanent Link: http://ufdc.ufl.edu/UFE0019660/00001

Material Information

Title: Engineered Microtopographies to Induce in Vitro Endothelial Cell Morphologies Stable to Shear
Physical Description: 1 online resource (174 p.)
Language: english
Creator: Carman, Michelle Lee
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: actin, adhesion, alignment, biomaterial, cells, contact, endothelial, flow, genipin, glutaraldehyde, graft, guidance, orientation, polydimethylsiloxane, shear, silicone, topography, vascular, wettability
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Large diameter ( > 6 mm) synthetic grafts have an established reoord of clinical success, but consistently occlude at smaller diamters. Attempts to improve patency by seeding grafts with endothelial cells have failed due to removal of cells under high shear. Endothelial cells are known to elongate with flow, requiring cells to break focal adhesion and form new ones. A means of inducing alignmnent of adhesions before exposure to flow could improve retention. This is the first known work to investigsate the influence of microscale topographies on inducing cellular alignment to improve retention. Initial studies examined the efficacy of existing wetting models. A series of engineered topographies were generated in polydimethylsiloxane elastomer (PDMSe) and contact angles of four solvents were measured. Results correlated strongly with classical models (y=0.99x) with a coefficiant of determination of 0.89. Data were compared with settlement of algae spores and porcine vascular endothelial cells. Packing density of algae spores and alignmnent of endothelial cells followed similar trends, suggesting wettability of topographies may be a strong factor in determing biological responses. Based on insights from the wetttability studies, topographies were designed to induce cytoskeletal alignment of endothelial cells. Gelatin was selected as a potential base material and glutaraldehyde and genipin were investigated as crosslinking agents. Mechanical properties of gelatin films with varying crosslinker concentrations were determined. Genipin stabilized gelatin more efficiently, exhibiting relatively high modulus and tensile strength while mainimising swelling during hydration. Porcine vascular endothelial cells were cultured on a series of microscale topographies in genipin-crosslinked gelatin and fibronectin-adsorbed PDMSe. Cells did not grow on gelatin, most likely due to cytotoxicity of unreacted genipin. Cells grew to confluence on topographies formed in fibronectin-treated PDMSe. Focal adhesions and overall cell shape aligned with the underlying topography. The topographies led to significantly smaller mean cell areas, more closely approaching that of cells in vivo. Microscale topographies enhanced cell spreading but not cell retention after 2 minutes of 2 Pa of flow-induced shear stress. After flow, cells on smooth controls decreased spreading by 60% and tended to form isolated aggregates. Cells on microtopographies maintained spreading, suggesting better viability.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Michelle Lee Carman.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Brennan, Anthony B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0019660:00001

Permanent Link: http://ufdc.ufl.edu/UFE0019660/00001

Material Information

Title: Engineered Microtopographies to Induce in Vitro Endothelial Cell Morphologies Stable to Shear
Physical Description: 1 online resource (174 p.)
Language: english
Creator: Carman, Michelle Lee
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: actin, adhesion, alignment, biomaterial, cells, contact, endothelial, flow, genipin, glutaraldehyde, graft, guidance, orientation, polydimethylsiloxane, shear, silicone, topography, vascular, wettability
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Large diameter ( > 6 mm) synthetic grafts have an established reoord of clinical success, but consistently occlude at smaller diamters. Attempts to improve patency by seeding grafts with endothelial cells have failed due to removal of cells under high shear. Endothelial cells are known to elongate with flow, requiring cells to break focal adhesion and form new ones. A means of inducing alignmnent of adhesions before exposure to flow could improve retention. This is the first known work to investigsate the influence of microscale topographies on inducing cellular alignment to improve retention. Initial studies examined the efficacy of existing wetting models. A series of engineered topographies were generated in polydimethylsiloxane elastomer (PDMSe) and contact angles of four solvents were measured. Results correlated strongly with classical models (y=0.99x) with a coefficiant of determination of 0.89. Data were compared with settlement of algae spores and porcine vascular endothelial cells. Packing density of algae spores and alignmnent of endothelial cells followed similar trends, suggesting wettability of topographies may be a strong factor in determing biological responses. Based on insights from the wetttability studies, topographies were designed to induce cytoskeletal alignment of endothelial cells. Gelatin was selected as a potential base material and glutaraldehyde and genipin were investigated as crosslinking agents. Mechanical properties of gelatin films with varying crosslinker concentrations were determined. Genipin stabilized gelatin more efficiently, exhibiting relatively high modulus and tensile strength while mainimising swelling during hydration. Porcine vascular endothelial cells were cultured on a series of microscale topographies in genipin-crosslinked gelatin and fibronectin-adsorbed PDMSe. Cells did not grow on gelatin, most likely due to cytotoxicity of unreacted genipin. Cells grew to confluence on topographies formed in fibronectin-treated PDMSe. Focal adhesions and overall cell shape aligned with the underlying topography. The topographies led to significantly smaller mean cell areas, more closely approaching that of cells in vivo. Microscale topographies enhanced cell spreading but not cell retention after 2 minutes of 2 Pa of flow-induced shear stress. After flow, cells on smooth controls decreased spreading by 60% and tended to form isolated aggregates. Cells on microtopographies maintained spreading, suggesting better viability.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Michelle Lee Carman.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Brennan, Anthony B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0019660:00001


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ENGINEERED MICROTOPOGRAPHIES TO INDUCE IN VITRO ENDOTHELIAL CELL
MORPHOLOGIES STABLE TO SHEAR























By

MICHELLE LEE CARMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007


































2007 Michelle Lee Carman

































To my family for their loving support.









ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Tony Brennan, for the patience and guidance he

showed me throughout my graduate studies. I would also like to thank Dr. Chris Batich, Dr.

Roger Tran-Son-Tay, and Dr. Mark Segal for serving on my supervisory committee.

I would like to acknowledge the support of my colleagues, both past and present. Their

friendships as well as their technical expertise were invaluable. I am especially grateful for the

efforts of Leslie Wilson, James Schumacher, Clay Bohn, Adam Feinberg and Thomas Estes. As

fellow members of Dr. Brennan's research group, Matthew Blackburn, Kenneth Chung, Amy

Gibson, Dave Jackson, Chris Long, Chelsea Magin, Sara Mendelson, Sean Royston, Jim Seliga

and Julian Sheets were also very helpful. I also thank the Goldberg and Batich group members

for all of their assistance along the way. In particular, Olajompo Maloye was instrumental in

helping me reassemble the cell culture lab so that my studies could be completed.

This work would not have been completed without the financial support of the Office of

Naval Research and the Alpha 1 Foundation.

Finally, I would like to thank my family for their endless love and support through this

experience. They managed to ground me even at the most chaotic moments.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ............................ ............... ..............................................................4

L IST O F T A B L E S ........................................................... ..................................... . 8

LIST OF FIGURES .................................. .. ..... ..... ................. .9

LIST OF A BBREV IA TION S............. ........... ...............................................................13

A B S T R A C T ......... ....................... ............................................................ 15

CHAPTER

1. INTRODUCTION ............... ................. ........... .............................. 17

2. BACKGROUND .................... .......... ...... .... ................22

In tro du ctio n ...................................... ...................................................... 2 2
Vessel Anatomy and the Endothelial Layer ........................................ ....................... 22
E ndothelial Seeding of G raft Surface ......................................................................... ..... 23
Shear-Induced Changes in Endothelial Cells ........................................ ...... ............... 24
M icropatterning of Cells ......... .... .. .... .... .... ...... ............... .. .. ..... 25
T opography and W ettability ............................................................................................. 28
A application to this W ork ............................................ .. .. ........... .... ..... .. 30

3. INFLUENCE OF TOPOGRAPHY ON WETTABILITY AND BIOADHESION ..............31

N otice of P reviou s Publication .................................................................... .....................3 1
Intro du action ................... ................... ...................................................... .. 3 1
M materials and M methods ...................................... .. .......... ....... ...... 34
M a te ria l ................... ...................3...................4..........
P pattern D designs ...................................................................................................... 34
Silicon W after P processing ........................................................................ .................. 35
Pattern Transfer and D ie Production .................................................................... ....35
Sam ple P reduction ......... ........ ......................................................................... .. ... 35
Contact Angle Measurements.......................................... 36
Com prison w ith M odel .......................................................... ..... ..........36
Predicted W getting on Novel Topographies .......................................... ............... 36
U lva Z oospore A ssay ............................................................................... ...... 37
Porcine Vascular Endothelial Cell (PVEC) Assay........................................................38
Statistical M methods .............................................. .. .. ........... ..... ...... 39
Results .........................................................................................................................................39
C contact A ngle M easurem ents............................................................... .....................39
Com prison w ith M odel .......... ................. .......... ... ................... .. ............... 40
Predicted W ettability on Novel Topographies ..................................... ............... 41









U lva Zoospore A ssay ............................................ ... .... ........ ......... 42
P V E C A ssay ................................................................4 2
D iscu ssion ............. .. ............. ........................................................ 43

4. CHARACERIZATION OF GLUTARALDEHYDE AND GENIPIN CROSSLINKED
GELATIN FILMS ................... ...................................... .. 55

Introduction ....................... ................................55
M materials and M methods ................................... ... .. .......... ....... ...... 57
P D M Se M old P reparation ....................................................................... ..................57
G elatin F ilm preparation ......................................................................... ...................59
Soxhlet Extraction of G elatin ................................................. ............................. 60
Postcuring of G elatin .......... .... ........ .... .... ......... ........ .... ..... 60
M mechanical Testing .................................. .. .......... .. ............60
Swelling Study................................................61
Evaluation of Microscale Gelatin Features ............................... .............. ...............61
Statistical A n aly sis ..................... .... .... ............................ ...... ......... .. ............ 62
R e su lts an d D iscu ssio n ..................................................................................................... 6 2
M echanical Testing of G elatin Film s ........................................ ......................... 62
Swelling Studies of Gelatin Films ............... ............... ................... 64
Evaluation of Microscale Gelatin Features ............................... .............. ...............66
C on clu sion ......... ..... ............. .......................................... .................................6 7

5. ENDOTHELIAL CELL GROWTH ON TOPOGRAPHICALLY PATTERNED
S U B S T R A T E S ................................................................................................................. 8 1

In tro d u ctio n ................... ................... ...................1..........
M materials and M methods ................................................................. .. ...... ... ... ..... .... .. 83
E engineered T opographies ................................................................. .......... ............... 83
P D M Se M old P reparation ....................................................................... ..................85
P reparation of G elatin F ilm s ........................................ .............................................85
Preparation of PDMSe Films....................................... ....... ...... ...................85
Characterization of Topographically Modified PDMSe and Gelatin Films....................86
Fibronectin A dsorption to Sam ples .................................................................... ....... 86
Cell Culture, Im aging and Processing .................................... .......................... ......... 86
Prelim inary assay ..................................... ......... .............. .. 87
Im m unofluorescent assay ............................................................. ............. .87
C ell culture assay 3 ............................................ ................. ........ 90
C ell culture assay 4 .......................................... ................... .... .. .. 9 1
Statistical M methods .............................................. .. .. ........... ..... ...... 92
R esu lts .................................... .. .... ... ....... ... ..................... ........................ 92
Characterization of Topographically Modified PDMSe and Gelatin.............................92
Prelim inary Cell Culture A ssay ........................................................................ 93
Im munofluorescent Cell Culture Assay ........................................ ....... ............... 93
Cell Culture Assay 3............... ....................... ...................95
Cell Culture Assay 4..................... .....................................96



6









D iscu ssio n ................... ...................9...................8..........
C o n clu sio n ................... ...................9...................9..........

6. INFLUENCE OF TOPOGRAPHY ON SHEAR STABILITY OF ENDOTHELIAL
C E L L S .............. ..... ..............................................................12 1

Introduction ................... ........... ......................121
M materials and M ethods ................................................................................. ..................... 122
Design of Parallel Plate Flow Chamber .............. ............ .............................122
Production of PDM Se Topographies........................................ .........................122
Prelim inary Shear Study........................ ............ .................... ............... 123
Sam ple gasket preparation ............................................. ............................ 123
Cell culture ..... ........... .. ......... ................... 124
Shear treatment .......................................................................... ......... .................. 124
Staining and im aging .... ..................................................................... ..... .. 124
F in al S h e ar S tu d y ..........................................................................................................12 5
Gasket preparation ......... .. ...... ..... .. ... ................. .............. 125
PD M Se culture w ell preparation ........... ................ ...........................................125
Sam ple preparation .................................................................................. 126
Cell culture ..... ........... .. ......... ................... 126
Shear treatment .......................................................................... ......... .................. 127
Staining and im aging ........ ................................................ ........ ... ......... 127
Statistical M methods ............................ ........................ .... .... ......... .. .... .. 127
Results ...................... ......................................128
Prelim inary Shear Study........................ ............ .................... ............... 128
F in al S h e ar S tu d y ..........................................................................................................12 8
D iscu ssion ......... ...... ... ............. ............................................130
C conclusion ......... ..... .... ........... ..........................................131

CONCLUSIONS AND FUTURE WORK ............................... ............... 140

C onclusions.....................................................................140
F utu re W ork ......................................................14 1

APPENDIX

A. SUMMARY OF LITERATURE ON CELLULAR RESPONSES TO TOPOGRAPHY....145

B. CALCULATION OF SHEAR IN PARALLEL PLATE FLOW ........................................155

L IST O F R E F E R E N C E S ....................................................................................................... 160

BIOGRAPHICAL SKETCH ................................................................... ........... 174









LIST OF TABLES


Table page

3-1 Dimensions of Topographies Used in Wettability and Bioadhesion Studies ....................52

3-2 Dimensions of Novel Theoretical Topographies................. ............. .................53

3-3 Measured Contact Angles on Microtopographies....................... ...............54

4-1 Diffusion Parameters Determined for GEN and GTA Crosslinked Gelatin....................79

4-2 Feature Dimensions of Topographically Modified Substrates Measured by WLIP..........80

5-1 N am es of Topographies ............................................................. ............... 118

5-2 Sam ples for Prelim inary A ssay .......................................................................... .... 119

5-3 Samples for Immunofluorescence Assay and Assay 3 ...................................................119

5-4 Sam ples for A ssay 4 ................................. ............ ............ ......... 119

5-5 Feature Dimensions of Topographies Determined by WLIP ........................................ 120

A Chronological Listing of Literature on Cellular Responses to Topography ....................145









LIST OF FIGURES


Figure page

3-1 SEM images of PDMSe microtopographies ........ .............. ................ 46

3-2 Glass mold used to make PDM Se samples................................. ......................... 46

3-3 AutoCad sketches of proposed topographies.................. .................. ..................47

3-4 Measurements taken to calculate nuclear form factor ................................................. 47

3-5 Layout of channel topographies............................................... .............................. 48

3-6 Change in wettability induced by the 20 atm spaced ridges (grey squares) and
Sharklet AFTM (black triangles) topographies compared to smooth PDMSe ....................48

3-7 Comparison of contact angles predicted by the model to contact angles measured on
th e su rfa c e s......................................................................... 4 9

3-8 SEM images of 2 [tm diameter pillars in PDM Se................................... ............... 49

3-9 Ulva settlement on smooth (SM) and textured PDMSe.................. .......................... 50

3-10 Ulva settled on smooth and textured PDM Se. ........................................ .....................50

3-11 PVEC alignment on smooth (SM) and textured PDMSe............................................51

3-12 Endothelial cells grown on smooth and textured PDMSe. ..............................................51

4-1 Chemical Reactions between gelatin and glutaraldehyde................... ..................68

4-2 C rosslinking m mechanism of genipin............................. ................................................69

4-3 Mold design for creating smooth PDMSe wells for casting gelatin ..............................70

4-4 Process for preparing topographically patterned PDMSe wells.......................................70

4-5 Process for crosslinking gelatin films with glutaraldehyde....................... ...............71

4-6 Representative stress-strain curves for GEN and GTA crosslinked gelatin ....................72

4-7 Initial Young's modulus versus crosslinker concentration for GEN and GTA
crosslinked gelatin.. .................................... .. ........... .. ............73

4-8 Elongation at break versus crosslinker concentration for GEN and GTA crosslinked
g elatin ................... ............................................................ ................ 7 3









4-9 Ultimate tensile strength versus crosslinker concentration for GEN and GTA
crosslinked gelatin. ..................................... .. ........... .. ............74

4-10 Stress-strain curves of GEN and GTA crosslinked gelatin.................................... 74

4-11 Effect of strain rate on the initial modulus of GEN crosslinked gelatin............................75

4-12 Effects of post-processing on the initial modulus of GEN crosslinked gelatin ...............75

4-13 Swelling of GEN and GTA crosslinked gelatin for 7 days in water...............................76

4-14 Swelling at 20 h of GEN and GTA crosslinked gelatin films.........................................77

4-15 Representative plot used to calculate diffusion coefficients............................................77

4-16 Representative plot used to calculate the time exponent for diffusion kinetics ...............78

4-17 Mass loss of GEN and GTA crosslinked gelatin samples after swelling for 7 days in
w ate r ................... ............................................................. ................ 7 8

4-18 Profilometry images of channel topographies replicated in different materials ..............79

5-1 Example of convention used for naming topographies...................................................100

5-2 Processing of DAPI images to measure cell density and nuclear orientation................100

5-3 Processing of images to measure cell area, elongation and orientation...........................101

5-4 Processing of Alexa fluor 488 images to measure alignment of focal adhesions...........102

5-5 SEM images of PDMSe replicates of silicon wafers patterned by different processing
methods....... .............. ................................. ........... 102

5-6 WLIP images of topographies formed by the DRIE process................... ............... 103

5-7 WLIP images of PDMSe topographies formed by the photoresist process.....................103

5-8 W LIP images of gelatin channels. ........................... ... ...... ........................ 104

5-9 PVECs grown on PDMSe topographies in the preliminary assay................................105

5-10 Density of PVECs on PDMSe topographies in the preliminary assay ............................106

5-11 Fluorescent images of PVECs grown on PDMSe and polystyrene syrfaces................. 107

5-12 Density of PVECs on topographies in the fluorescent assay ................ ....................108

5-13 Mean cell area for PVECs on topographies in the fluorescent assay.. ........................108









5-14 PVEC elongation on topographies in the fluorescent assay. .........................................109

5-15 PVEC orientation on topographies in the fluorescent assay ................. ................109

5-16 Orientation of PVEC nuclei on topographies in the fluorescent assay........................... 110

5-17 PVEC focal adhesion orientation on topographies in the fluorescent assay....................110

5-18 Histograms of alignment indices for focal adhesions on topographies in the
fluorescent assay .................................. ........................... ......... .......... 111

5-19 Light microscope images of PVECs grown on topographies for Assay 3.......................112

5-20 PVEC density on topographiess in Assay 3............... ................................113

5-21 PVEC coverage on topographies in Assay 3 .............. .... .... ................. 113

5-22 PVEC area on topographies in Assay 3 ................................... .....................114

5-23 Light microscope images of PVECs grown on topographies for assay 4.....................15

5-24 PVEC density on topographies in Assay 4 ......................... ........... .............. 116

5-25 PVEC confluence on topographies in Assay 4 .............................. ..................116

5-26 PVEC spreading on topographies in Assay 4 .......... .................................. 117

5-27 PVEC elongation on topographies in Assay 4.............................................. 117

5-28 PVEC orientation on topographies in Assay 4 .............................. ..................118

6-1 Original design of flow chamber. ......................................................... ...............132

6-2 M odified design of flow chamber ............................ ........... ................. ............... 132

6-3 Layout of samples for the final shear study. .......................................... .................. 133

6-4 PVECs on topographies before exposure to flow in the preliminary shear study ...........134

6-5 Density of PVECs on topographies before and after flow in the preliminary shear
stu d y ................... ......................... .. ............................. ................ 1 3 5

6-6 PVECs grown on topographies in the final shear study ...............................................136

6-7 Density of PVECs on PDMSe topographies before and after flow in the final shear
stu d y ................... ........................................................................... 1 3 7

6-8 Elongation of PVECs on topographies before flow in the final shear study .................137









6-9 Orientation of PVECs on topographies before flow in the final shear study.................138

6-10 Retention of PVECs based on topography in the final shear study................................138

6-11 Average area for PVECs on topographies before and after flow in the final shear
stu d y ................... ............................ .............................. ................. 3 9

B Diagram of flow between parallel plates. ............................................. ............... 159









LIST OF ABBREVIATIONS

ANOVA Analysis of Variance

AFM Atomic force microscope

ATS Allyltrimethoxysilane

BHK Baby hamster kidney

CH Channel

DAPI 4',6-Diamidino-2-phenylindole

DMF Dimethylformamide

DRIE Deep reactive ion etching

ECM Extra Cellular Matrix

FG Fibrinogen

FN Fibronectin

FOV Field of View

GEN Genipin

GTA Glutaraldehyde

HBSS Hank's balanced salt solution

HMDS Hexamethyldisilazane

Hyal Hyaluronic acid

HyalS Sulfonated hyaluronic acid

MDCK Madine Darby canine kidney

Mel Methyliodide

MEQ Molar equivalent

NFF Nuclear form factor

PBS Phosphate buffered saline









PDMS Polydimethylsiloxane

PDMSe Polydimethylsiloxane elastomer

PR Photoresist

PS Polystyrene

PVEC Porcine vascular endothelial cell

RFGD Radio frequency glow discharge

RGD Arginine-glycine-aspartate

SEM Scanning electron microscopy

SK Sharklet AFTM

SM Smooth

SMC Smooth muscle cell

TCP Tissue culture polystyrene

TRITC Tetramethylrhodamine isothiocyanate

UV Ultraviolet

WLIP White light interference profilometry

tCP Microcontact printing









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENGINEERED MICROTOPOGRAPHIES TO INDUCE IN VITRO ENDOTHELIAL CELL
MORPHOLOGIES STABLE TO SHEAR

By

Michelle Lee Carman

August 2007

Chair: Anthony Brennan
Major: Biomedical Engineering

Large diameter (>6 mm) synthetic grafts have an established record of clinical success, but

consistently occlude at smaller diameters. Attempts to improve patency by seeding grafts with

endothelial cells have failed due to removal of cells under high shear. Endothelial cells are

known to elongate with flow, requiring cells to break focal adhesions and form new ones. A

means of inducing alignment of adhesions before exposure to flow could improve retention.

This is the first known work to investigate the influence of microscale topographies on

inducing cellular alignment to improve retention. Initial studies examined the efficacy of

existing wetting models. A series of engineered topographies were generated in

polydimethylsiloxane elastomer (PDMSe) and contact angles of four solvents were measured.

Results correlated strongly with classical models (y=0.99x) with a coefficient of determination of

0.89. Data were compared with settlement of algae spores and porcine vascular endothelial cells.

Packing density of algae spores and alignment of endothelial cells followed similar trends,

suggesting wettability of topographies may be a strong factor in determining biological

responses.

Based on insights from the wettability studies, topographies were designed to induce

cytoskeletal alignment of endothelial cells. Gelatin was selected as a potential base material and









glutaraldehyde and genipin were investigated as crosslinking agents. Mechanical properties of

gelatin films with varying crosslinker concentrations were determined. Genipin stabilized

gelatin more efficiently, exhibiting relatively high modulus and tensile strength while

minimizing swelling during hydration.

Porcine vascular endothelial cells were cultured on a series of microscale topographies in

genipin-crosslinked gelatin and fibronectin-adsorbed PDMSe. Cells did not grow on gelatin,

most likely due to cytotoxicity of unreacted genipin. Cells grew to confluence on topographies

formed in fibronectin-treated PDMSe. Focal adhesions and overall cell shape aligned with the

underlying topography. The topographies led to significantly smaller mean cell areas, more

closely approaching that of cells in vivo.

Microscale topographies enhanced cell spreading but not retention after 2 minutes of 2 Pa

of flow-induced shear stress. After flow, cells on smooth controls decreased spreading by 60%

and tended to form isolated aggregates. Cells on microtopographies maintained spreading,

suggesting better viability.









CHAPTER 1
INTRODUCTION

Cardiovascular disease is the leading cause of death in the United States. In 2003 685,089

U.S. deaths were attributed to the disease which correlates to one death every 45 seconds [1].

Although synthetic large diameter grafts have an established record of clinical success, they

consistently fail if used at vessel diameters of 6 mm or less. Currently, the preferred treatment of

partially occluded vessels is minimally invasive angioplasty and stenting. However, for severe

cases angioplasty and stenting are not an option and bypass grafts are necessary. The current

standards for coronary bypass grafts are autologous mammary artery followed by saphenous vein

despite drawbacks including donor site morbidity and limited supply.

As early as the 1970s, researchers sought to improve the antithrombogenic nature and

hence the patency of artificial vascular grafts though the incorporation of endothelial cells onto

the inner lumen of the graft surface. They have shown that endothelial cells grow to confluence

on a wide variety of substrates, but are removed easily when exposed to shear stresses equivalent

to those present in natural human arteries.

Attempts to improve endothelial cell adhesion have included surface modifications of the

graft materials. Substrates have been treated though adsorption of adhesion proteins (e.g.,

albumin, extracellular matrix, gelatin and fibronectin), carbon deposition, photo discharge, and

plasma discharge. Protein adsorptions were aimed at providing selective sites for cell adhesions.

The remaining techniques were focused on increasing the density of non-specific reactive surface

groups or altering surface wettability to influence the adsorption of proteins. Although these

methods have been successful at increasing cell density and improving adhesion, there is still a

need to develop a surface capable of supporting endothelial cells under high shear stress.









Endothelial cells in vivo elongate in the direction of blood flow in regions of high shear

stress. It is logical to assume that endothelial cells would tend to alter their morphology in this

way when exposed to shear in vitro as well. In fact, researchers report that cells maintain

attachment and undergo elongation if subjected initially to low shear rates followed by

increasing rates up to physiological shear rates [2, 3]. It seems likely that the problem with

current attempts to seed vascular grafts with endothelial cells might lie in the fact that cells are

seeded in static conditions. It is hypothesized here that these cells are removed when exposed to

flow because they must break focal adhesions to adapt their morphology. This implies that a

method of increasing cellular alignment under static conditions could improve endothelial cell

retention after flow-induced shear stress is applied.

Cells alter their shape based upon the underlying surface morphology, a phenomenon

referred to as contact guidance [4]. This has been widely studied over the past several decades

with a diverse selection of cell types, surface materials, and topographical features. The design

and formation of microscale features has been made possible through advances in fabrication

techniques by the microelectronics industry. Simple geometries such as channels and pillars are

often used, although more complex geometries have been investigated as well. In particular,

channels topographies have been shown to result in cell elongation parallel to the long axis of the

channels. For endothelial cells and fibroblasts, cells adhere almost exclusively to the valleys of

channels with widths of 5 tm or greater. Consequently, gaps in cell coverage exist wherever the

channels are separated by ridges.

The work presented here builds upon these studies in order to increase cytoskeletal

alignment of endothelial cells without a disruption in cell confluence. Initially, results of

colleagues studying both endothelial cells and marine fouling by algae are examined. Surfaces









used in their studies are evaluated for the influence of topography on surface wettability. A

correlation between the topographical influences on wettability and biological settlement is then

made. Based upon this work as well as the diverse literature on contact guidance, topographies

are designed with the goal of supporting shear-stable endothelial layers.

Topographical dimensions are selected based on their ability to influence the formation of

focal adhesions rather than their ability to influence the shape of the cells as a whole. Focal

adhesions are typically between 2 and 3 .im in diameter, and so topographical features are

developed with lateral dimensions of 2 rim. The height of topographical features is minimized

in order to prevent disruptions in cell confluence.

Polydimethylsiloxane elastomer (PDMSe) surface modified with adsorbed fibronectin has

been widely used by colleagues in the study of contact guidance of mammalian cells. PDMSe is

examined here as well as a hydrogel material system. Gelatin is derived from collagen which is

a natural component of vascular walls. Crosslinked gelatin is investigated for its mechanical

stability, ability to replicate microscale topographical features and potential to support cell

growth. Glutaraldehyde and genipin were the crosslinking reagents investigated.

Glutaraldehyde is commonly used as a fixative for protein systems but is known to be cytotoxic

in its unbound state [5]. Genipin is a natural plant extract and suppliers claim a lower level of

toxicity.

The primary objective of this work is to determine if microscale topographies induce

alignment of endothelial cells and whether this morphological change results in improved

resistance to removal by flow induced shear stress. This is the first known attempt to evaluate

the influence of engineered topographies to endothelial cell retention during exposure to flow.

To achieve this goal, the influence of microscale topographies on biologic settlement was









correlated with changes in surface wettability. Then gelatin was investigated as a potential

substrate material and mechanical properties and diffusion kinetics were improved through the

optimization of crosslinking. Microscale topographies were next created in the crosslinked

gelatin as well as fibronectin-treated polydimethylsiloxane elastomer. The surfaces were

evaluated for the ability to induce orientation of endothelial cells without disrupting confluence.

Finally, adhesion of the endothelial cells to the topographically modified materials was evaluated

using a parallel plate flow chamber.

Specific aim 1. Wettability is correlated with biological settlement on engineered

topographies. Contact angles of water, methyliodide, isopropanol, and dimethylformamide are

measured using the sessile drop technique on smooth and micropatterned PDMSe. Measured

angles are correlated with values predicted by classical wetting theories. The wettability results

are correlated with previously published results of algae settlement and nuclear alignment of

endothelial cells. One-way analysis of variance (ANOVA) with Tukey's multiple comparisons

test are (a = 0.5) used to compare groups.

Specific aim 2. Measure the mechanical stability of gelatin films crosslinked with

glutaraldehyde and genipin. Gelatin films (10% w/v in water) are crosslinked by genipin (1.4,

2.9, and 4.3% MEQ) and glutaraldehyde (2.9, 4.3 and 5.9% MEQ). Samples are evaluated for

the ability to increase modulus, elongation at break and ultimate tensile strength while reducing

the percent swelling with water. One way ANOVA with Tukey's multiple comparisons test ( =

0.5) are used to determine significant differences in mechanical properties as a result of varying

crosslinking reagent and concentration.

Specific aim 3. Demonstrate that cells will align on topographies with 2 [im lateral

dimensions while maintaining confluence equal to that of smooth PDMSe. Percentage of









covered surface area, cell density, and alignment of focal adhesions are determined. At least

90% surface coverage must be achieved to be considered confluent. Alignment of focal

adhesions is indicated when the average offset angle to topography approaches zero. One way

ANOVA followed by pair-wise t-tests (a = 0.5) were used to determine significant differences

between surface types.

Specific aim 4. Demonstrate that cell alignment induced by micropattems increases cell

retention when subjected to flow generating 2 Pa of shear stress on the sample substrate.

Percentage of covered surface area and cell area before and after exposure to flow are

determined. Endothelial cell retention is calculated by comparing cell densities before and after

flow. One way ANOVA followed by pair-wise t-tests (a = 0.5) are used to determine if the

topographies increase cellular retention.









CHAPTER 2
BACKGROUND

Introduction

This work proposes the use of microscale topographies to improve endothelial cell

retention to biomaterials with the long-term goal of the development of a successful small

diameter vascular graft. In order to fully understand the rationale for this research, normal vessel

anatomy and the role of the endothelium must first be considered alongside other attempts to

improve endothelial cell adhesion to graft materials. These are discussed below as well as the

extensive collection of research on cellular responses to microscale topography and efforts to

model wettability of textured surfaces.

Vessel Anatomy and the Endothelial Layer

Three distinct layers make up the walls of blood vessels. The innermost layer is referred to

as the tunica intima (or tunica internal) and includes the endothelial lining with an underlying

layer of connective tissue. In arteries, an internal elastic membrane exists in the outer region of

this innermost layer. Beneath the tunica intima lays the tunica media. Sheets of smooth muscle

cells are supported by a framework of loose connective tissues which bind the tunica media to

the outermost layer, the tunica adventitia (or tunica externa). The tunica media of arteries is

thicker compared to veins and contains an external elastic membrane. Collagen (dominant in

veins) and elastin (dominant in arteries) fibers provide support.

The endothelium is a made up of a confluent layer of simple squamous cells which

regulate cardiovascular physiology. It provides a continuous, selectively permeable barrier

between the arterial wall and circulating blood. Additionally, the endothelium controls platelet

activation, adhesion, and aggregation and smooth muscle cell (SMC) proliferation and migration.









If allowed to occur uncontrolled, aggregation and proliferation of platelets and SMCs would lead

to vessel occlusion.

Endothelial Seeding of Graft Surface

As early as the 1970s, researchers sought to improve the antithrombogenic nature and

hence the patency of artificial vascular grafts though the incorporation of endothelial cells onto

the inner lumen of the graft surface [2, 3, 6-33]. They have shown that endothelial cells grow to

confluence on a wide variety of substrates, but are removed easily when exposed to shear

stresses equivalent to those present in natural human arteries.

Attempts to improve endothelial cell adhesion have included surface modifications of the

graft materials. Substrates have been coated with adhesion proteins (e.g. albumin, extracellular

matrix, gelatin and fibronectin), which have led to improved short term (<3 hours) cellular

retention during exposures to flow-induced shear stress [15, 18, 28-30, 32, 33]. Longer

exposures, however, show a rapid decrease in cellular attachment on many of these surfaces

which has been attributed to desorption of adhesion proteins from the substrate [29, 32, 33].

Additionally, exposed adhesion proteins have been found to promote platelet adhesion and

activation [32, 34]. Consequently, gaps resulting from insufficient initial cell seeding or removal

due to shear promote the formation of fibrin and fibrous encapsulation of the graft which leads to

occlusion.

Other attempts to improve endothelial adhesion have included carbon deposition, photo

discharge, and plasma discharge treatments. These treatments were aimed at either increasing

the density of reactive surface groups or influencing the adsorption of proteins. Success of these

techniques has been limited due to their unspecific interaction with cells and poor control over

protein orientation [33]. A more promising treatment has involved the use of a peptide sequence

found in fibronectin. Covalent binding of synthetic versions of the arginine-glycine-aspartate









(RGD) sequence has been shown to overcome late term removal of cells and resist platelet

adhesion and activation [13, 15, 32].

Shear-Induced Changes in Endothelial Cells

Endothelial cells located in vascular regions of relatively high shear stress (e.g., arteries

larger than 0.5 mm in diameter) tend to be elongated in the direction of flow. In these cells, actin

filament bundles which terminate at focal adhesions are aligned parallel to flow in an apparent

attempt to compensate for the stress [35]. Endothelial cells grown in static culture do not exhibit

these same qualities. Instead, the cells tend to be polygonal in morphology with only a small

number of stress fibers confined to the cell periphery [36]. These fibers are assumed to be

responsible for maintaining cell spreading and preventing contraction.

When cultured cells are exposed to flow-induced shear stresses, cytoskeletal and

sometimes morphological changes are induced. These changes are accompanied by a stiffening

of the cell which is related to an increase in stress fiber density [37]. These responses have been

found to be dependent on both the magnitude of the shear stress and the duration of the exposure.

At 0.2 Pa of wall shear stress, an increase in the density of actin filament bundles occurs between

2 and 3 hours of exposure, but cellular alignment does not occur and the stress fibers do not

exhibit preferential orientation in the direction of flow [36]. After 7 hours of exposure to 1 Pa of

wall shear stress, cells elongate and stress fibers align with the flow direction accompanied by

the coalescence of focal contacts so that they are fewer in number but greater in size [38]. For a

cell grown in static culture to align itself in this manner, it must break many of the focal

adhesions it created with the surface and form new ones [38, 39]. It is hypothesized here that

during this transitional period cells are removed from a surface. If this is the case, then a method

that would cause the cells to align prior to implantation would be advantageous.









In the 1990s Ballermann et al. of Johns Hopkins University significantly improved in vivo

endothelial cell retention by preconditioning cell-seeded grafts with in vitro shear stress [2, 3,

40]. They found that exposing cell seeded grafts to 0.1 Pa wall shear stress for 3 days and then

2.5 Pa for a subsequent 3 day period significantly improved cell retention and reduced

neointimal thickness on aortic interposition grafts in rats. This supports the hypothesis that

alignment of the actin cytoskeleton prior to implantation is advantageous.

Micropatterning of Cells

It has long been known that cells respond to the shape of the substrate on which they grow.

The earliest known report of this was made by Harrison in 1914 in which he observed that

fibroblasts found in embryonic nervous tissue from frogs take on a polygonal shape when

cultured against smooth glass, but become drawn-out when grown on spider silk [41]. The term

'contact guidance' was later used to describe the phenomenon when Weiss performed a similar

experiment with nerve cells grown on glass fibers [4].

In the past several decades, literature on cellular responses to topography has expanded

rapidly. Researchers have investigated numerous combinations of cell types and topographical

geometries and dimensions. Appendix A provides a table summarizing the pertinent literature.

The degree of contact guidance varies with topographical dimensions and geometry as well as

the cell type studied. As an example, Clark et al. examined chicken embryonic cerebral neurons

and two epithelial cell types: baby hamster kidney (BHK) and Madin Darby canine kidney

(MDCK) [42]. All three cell types were grown on grooved polymethylmethacrylate (Perspex)

with channel widths and spaces ranging from 4 to 24 [m and depths ranging from 0.2 to 1.9 nm.

In all cases, alignment increased with decreased spacing and increased depth. Depth had a

greater influence than spacing for the dimensions studied and the MDCK cells were more

sensitive to topography than BHK cells.









Many researchers have investigated the potential use of topography for preventing fibrous

encapsulation and subsequent contraction and stiffening of implants [43, 44]. This goal is of

particular interest for maintaining the aesthetic appearance and mechanical integrity of breast

implants. Campbell and von Recum examined the influence of pore size of Versapor filter

materials (acrylic copolymers on nylon supports) implanted subcutaneously in canines on fibrous

tissue growth [43]. Nonadherent, contracting capsules were formed on materials with pore sizes

less than 0.5 im. Thin, tightly adhered capsules were formed on implants with pore sizes

ranging from 1.4 to 1.9 [im and inflammatory tissue infiltrated pores sizes greater than 3.3 im.

Schmidt and von Recum later performed a similar experiment in which 2, 5 and 8 [m

wide/spaced grooves were patterned into silicone elastomer and implanted into rabbits [44]. In

general it was found that the 2 and 5 im grooves (depths ranging from 0.4 to 0.6 im) resulted in

fewer attached cells and thinner fibrous capsules than the smooth control and 8 [im grooves.

Additionally the surfaces were tested in vitro with murine peritoneal macrophages and cellular

alignment was observed.

In a more recent study, van Kooten and von Recum have shown that in addition to altering

cellular morphology, topography can increase the density of focal adhesions [45]. Fibroblasts

grown on 2 rm wide/spaced grooves were shown to have a greater density of focal adhesions

than the same cells grown on 8 [m wide/spaced grooves and smooth fibronectin-coated silicone

substrates. A 0.5 im groove depth was used for the study.

Walboomers et al. have shown that topography can be tailored to increase cell density

relative to a smooth substrate [46]. Fibroblasts were grown on both smooth and microgrooved

(1-10 rm wide and 0.5-5.4 [m deep) polystyrene. It was shown that for samples of equal

projected planar areas, the number of adhered fibroblasts was increased on all topographies









relative to the smooth control. When the numbers were corrected to account for the surface area

added by roughness, however, the 1.5 [m deep grooves reduced cell density as did the 1 and

2 rm wide grooves.

Fibroblasts are the most common cell type used in studies with microscale topographies.

Literature specific to endothelial cell responses to topography is considerably more limited.

Matsuda and Sugawara bovine aortic endothelial cell attachment on 20 to 130 [m wide channels

and found that alignment increased with decreased channel width [47]. Mrksich et al. grew

bovine capillary endothelial cells on polyurethane coated with gold and patterned with alkane

thiols and adsorbed fibronectin [48]. They examined V-shaped channels that were 25 and 50 [m

wide and had equal spacing. They showed that endothelial cells adhere to ridges or valleys,

depending on which was patterned with the alkane thiol and therefore adsorbed fibronectin.

Palmaz et al. examined migration of human aortic endothelial cells on nitinol containing 1, 3, 15

and 22 [m channels [49]. Cells aligned with all channels and migration was greater on larger

channels. Uttayarat et al. studied smaller channel topographies in fibronectin treated

polydimethylsiloxane elastomer (PDMSe). Channel widths ranged from 2.7 to 5.5 [m and

depths ranged from 0.2 to 4.6 [im [50]. Cell proliferation was similar on all substrates and cell

elongation and alignment increased with channel depth. Focal adhesions formed in channels for

all topographies except the 4.6 [m deep channels. Barbucci and Magnani investigated the

influence of the combination of topography and chemical patterning on cell behavior [31, 51].

They observed that endothelial cells increasingly align themselves on ridges as the topographical

spacing is reduced from 100 to 10 |tm. Similar results were found by Wilkerson et al during the

study of endothelial cell growth on ridges ranging in spacing from 20 to 5 [m [52, 53]. Feinberg

et al has also examined endothelial cell growth on topography and chemical patterns [53, 54].









They found that cells are disrupted by topographies with profile heights greater than 1 [m and

that focal adhesions form almost exclusively on fibronectin (FN) regions of FN-treated PDMSe.

Furthermore, the area of individual focal adhesions does not vary with surface treatment and is

approximately 2 im2.

Topography and Wettability

Although topography is clearly a significant factor in determining cell confluence and

shape, the mechanism for this response has not been elucidated. The change in wettability of a

surface that results from surface roughness, i.e., topography, is likely a contributing factor.

Wettability is often characterized in terms of the three phase contact angle which relies on the

relative interfacial tensions according to Young's equation given below [55]:

YG = YSL + LV Cos (2-1)

Young's equation assumes the surface is chemically and topographically homogeneous

and does not take into account the dynamic nature of wetting. Many groups have demonstrated

the bidirectional nature of surface wetting and therefore, one must consider dewetting as well

[56-58]. It has been hypothesized that the hydrophobicity and hence the force required to affect

spreading is a function of the hysteresis between advancing and receding angles.

Numerous groups have studied the wetting characteristics of rough surfaces. The earliest

report to correlates wetting with topography was made by Wenzel, who assumed the contours of

the topography are fully wetted [59]. The apparent contact angle on the textured surface (cos 0 )

was related to the Young's contact angle on a smooth surface of the same material as follows:

cos O* = r cos (2-2)

Wenzel defined the roughness ratio (r) as the actual surface area divided by the area of the

surface when projected onto a two dimensional plane.









A more detailed approach by Cassie and Baxter proposes an alternative to the Wenzel

equation [60]. They evaluated the wetting by water of waxy surfaces which where not just

rough, but also porous. Under this condition, water did not follow the contours of the

topography and instead rested upon a composite structure of wax and air. The ratios of the areas

of liquid beneath the drop in contact with solid and air relative to the planar surface area were

termedfi andf2 respectively. The resulting contact angle (OD) for the porous surface was then

thermodynamically determined to be the following:

cos OD = f cos 0 f2. (2-3)

Marmur[57] and Quere et al.[61] have independently examined the thermodynamics of the

wetting regimes to determine when air entrapment will be energetically favored. Quere et al.

defined the variable 4s as the fraction of liquid beneath the drop in contact with solid. It is

equivalent to thefi term in Cassie and Baxter's relationship. Air entrapment would be favored

for liquids of sufficiently high surface tensions (O>cos-1[(Vf-l)/(r-fi)]) and would be metastable

for liquids satisfying the condition of 90o<0
Wenzel would be favored for liquids of intermediate surface tensions (i.e.

cos-'[(1-fj)/(r-fj)<0
the drop would occur for liquids of sufficiently low surface tension (O
apparent contact angles (0') for cases of air entrapment and wicking are given by the following:

f -1
Air entrapment: cos 0* = -1 + f (cos 0 +1) for 0 > cos 1 1 (2-4)
r-f

0* 1-f (2-5)
Wicking: cos O6 = 1 + f(cos O -1) for 0 < cos (2-5)
r-f1









Application to this Work

Based on the background work presented here, studies were developed to examine the

potential of microscale topographies to induce endothelial cell morphologies that increase

resistance to shear removal. In the following chapters, studies are presented that examine the

influence of microscale topographies on cellular attachment. In the first study, wettability is

correlated with the biological response of algae spores (Ulva) and porcine endothelial cells.

Later, genipin and glutaraldehyde crosslinked gelatin is investigated for its ability to replicate

and maintain fidelity of microscale features. Then the gelatin topographies are evaluated

alongside fibronectin-treated PDMSe topographies for potential to support endothelial cell

growth. Cell density, confluence and orientation are measured in relation to the microscale

features. Finally, endothelial cell retention to microscale topographies is measured after

exposure to flow-induced shear stress in a parallel plate flow chamber.









CHAPTER 3
INFLUENCE OF TOPOGRAPHY ON WETTABILITY AND BIOADHESION

Notice of Previous Publication

The contents of this chapter were originally published in Biofouling 2006 by Taylor and

Francis [62]. It is reprinted here in accordance with the copyright agreement.

Introduction

Reports on cellular responses to topographical cues on both nanometer and micrometer

scales have increased in the past few decades [63-66]. Appropriately scaled nanotopographies

have been shown to prevent cell attachment by prohibiting formation of focal contacts [66, 67].

Alternatively, cells can respond to microscale features by altering their shape such as elongating

along grooves [45, 63]. In the area of marine fouling, topography has been shown to alter

settlement of algae [68, 69], barnacles [70] and bacteria [71]. The change in wettability of a

surface that results from surface roughness (i.e., topography) is likely a contributing factor to

these responses.

Wettability is often characterized in terms of the three phase contact angle which relies on

the relative interfacial tensions according to Young's equation [55]:

YSG =YSL + LV COs0. (3-1)

Young's equation assumes that the surface is both chemically and topographically

homogeneous and does not take into account the dynamic nature of wetting. Many groups have

demonstrated the bidirectional nature of surface wetting and therefore, one must consider

dewetting as well [56, 58, 72].

Numerous groups have studied the wetting characteristics of topographically rough

surfaces. The earliest report that correlates wetting with topography was made by Wenzel [59],

who assumed the contours of the topography become fully wet and the change in contact angle is









due to an increase in surface area that topography provides. Wenzel defined a roughness ratio (r)

as the actual surface area divided by the area of the surface when projected onto a two

dimensional plane to account for the change in wetting in terms of contact angle as follows:

cosO = rcosO (3-2)

A more detailed approach by Cassie and Baxter [60] proposed an alternative to the Wenzel

equation. They evaluated the wetting by water of waxy surfaces which where not just rough, but

also porous. Under this condition, water did not follow the contours of the topography and

instead rested upon a composite structure of wax and air. The ratios of the areas of liquid

beneath the drop in contact with solid and air relative to the planar surface area were termedfi

andf2 respectively. The resulting contact angle (OD) for the porous surface was then

thermodynamically determined to be the following:

cos 0D = f cos 0 f2. (3-3)

More recently, Quere et al. [61, 73-75] demonstrated that for a given surface, regimes of

both Wenzel and Cassie-Baxter behavior exist across a range of liquid surface tensions. They

defined the variable 4s as the fraction of liquid beneath the drop in contact with solid. It is

equivalent to thefi term in Cassie and Baxter's relationship. Air entrapment, fully wetted, and

wicking occurs for liquids of sufficiently high, moderate, or low surface tensions respectively.

In the case of wicking, the liquid is drawn into the topography at the advancing edge so that the

drop rests on a composite surface of liquid and solid. The corresponding relationships and

criteria for each case are given below.

f -1
Air entrapment: cos 0* = -1 + f (cos 0 +1) for 0 > cos 1 1 (3-4)
r-f

1- f f -1
Fully wetted: cos 0 = r cos 0 for cos1 < r-f r-f










Wicking: cos 0* = 1 + f(cos 0 -1) for 0 < cos1 (3-6)
r-fA

Quere et al. also indicated that the air entrapment state would be metastable for the

following condition:

f -1
90 < 0 < cos 1 (3-7)
r-f

In the present study, a series of engineered microtopographies was created in a

polydimethylsiloxane elastomer (PDMSe). The engineered patterns include a biomimetic

inspired design that is based upon the configuration of placoids of fast moving sharks. Changes

in wettability were measured and compared against the values predicted by the Wenzel and

Cassie-Baxter relationships. Some of these surfaces were then selected to test the hypothesis that

wettability influences the contact-sensing processes used by living cells. For this purpose we

selected two well-characterized but contrasting model systems to represent both marine and

biomedical fouling, viz. the motile spores of the marine alga Ulva (syn. Enteromorpha), and

porcine vascular endothelial cells (PVECs) which form the inner lining of arteries.

The green algal genus Ulva (formerly Enteromorpha) is the most common macroalga

contributing to 'soft' fouling of man-made surfaces throughout the world [76] and has been

developed extensively as a model system for experimental studies [77-80]. Fouling is initiated

by the settlement and subsequent adhesion of motile spores, a process which is influenced by a

variety of surface-associated cues. We have previously used engineered microtopographies in

PDMSe to identify surface features thatpromote settlement [68, 69]. Our hypothesis in the

present study was that topographic patterns that mimic a natural antifouling surface, viz. the

placoid structure of shark skin, may provide a surface with low settlement properties.









Endothelial cells are widely used as models in which to study the influence of substratum

morphology on adhesion and contact-mediated growth of animal cells [31, 51, 52, 81-84]. We

have chosen to use porcine vascular endothelial cells (PVECs) for our study because of the local

availability of a well characterized cell line [85]. The pig has been shown to be an ideal

preclinical model for vascular research, as in vitro tests have concluded that the coagulation and

fibrinolytic systems of swine closely resemble that of humans [86, 87]. In the present study

PVECs were used to investigate the affect of feature spacing (5 to 20 [tm) on cellular orientation.

This response was then correlated with the influence of topography on wettability.

Materials and Methods

Material

A platinum catalyzed PDMSe (Dow Coming Corporation's Silastic T-2) was chosen for

this study due to its high transparency and reproducibility. The PDMSe is filled with micron and

sub-micron silica particles. In the unmodified state, the polymer is known to promote minimal

bioadhesion because of its combination of low surface energy and low modulus [69]. The

PDMSe was prepared by mixing ten parts by weight of resin with one part by weight curing

agent. The PDMSe was typically cured at 220C for 24 hours.

Pattern Designs

The features studied included channels, ridges, pillars, pits and ribs (Fig. 3-1). Channels,

ridges, pillars, and pits were 5 [tm wide and spaced 5, 10, and 20 [tm apart. The rib designs are a

reduction of the scales of fast moving sharks. We refer to this biologically inspired pattern as the

'Sharklet AFTM' because it is an antifouling topography that was inspired by, but does not

reproduce, the skin of the shark. The ribs are 2 atm wide, spaced 2 atm apart, and have lengths

ranging from 4-16 m. Both 1.5 and 5 um high channel and pillar features were investigated,

whereas the ribs of the Sharklet AFTM were 4 um high.









Silicon Wafer Processing

Patterns were etched into silicon wafers using standard photolithography techniques as

described previously [88]. Wafers were then critically cleaned using a piranha etch (50:1 H2S04

and H202) at 1200C for 10 min followed by subsequent rinsing in acetone and ethanol prior to

each replication with PDMSe. Hexamethyldisilazane was used to methylate the surfaces in order

to prevent adhesion.

Pattern Transfer and Die Production

Patterns are transferred to PDMSe in either negative (channels, pits and Sharklet AFTM) or

positive (pillars and ridges) form (Table 3-1). Negatives were replicated directly from the etched

wafer so that the PDMSe topography is inverted compared to the silicon wafer. For example,

pillars in the wafer would transfer as pits into the PDMSe. Positives were generated by first

solution casting polystyrene (0.15 g/mL in chloroform) against the wafer followed by curing the

PDMSe against the polystyrene. Epoxy dies (Epon 828 with Jeffamine D230, 9.7:2.73 by

weight) were then made from both positively and negatively patterned PDMSe.

Sample Production

Samples included PDMSe films that were either free standing or adhered to glass slides.

In both cases, the PDMSe was cured in a glass mold (Fig. 3-2) as described previously [68].

Smooth samples were cast directly off the glass, while patterned samples were produced by

casting against epoxy or silicon dies. For glass-backed samples, slides were first pretreated with

0.5% allyltrimethoxysilane (ATS) in a 95% ethanol/water solution to improve adhesion. Three

replicates of each pattern type were produced. Fidelity of the surface features was verified with

the aid of light microscopy and SEM.









Contact Angle Measurements

Wettability was evaluated on free-standing PDMSe films containing 5 [im high channels,

pits, and Sharklet AFTM and 1.5 rm high ridges by the Sessile drop method with 2 [tL drops.

This method looks at advancing contact angles measured in the first few seconds of contact.

Video capture goniometry was used and angles were measured with ImageTool software.

Liquids included in the study were nanopure water (17 MQ*cm resistivity), methylene iodide,

and dimethylformamide. Surfaces were rinsed with ethanol and dried at 800C prior to testing

with each liquid. One drop was placed on three replicates of each pattern and two angle

measurements, one from the left and one from the right, were taken per drop. In this manner, six

measurements were taken per pattern. Drops were viewed down the lengths of channels and

ribs.

Comparison with Model

Wettability data were compared against values predicted by Quere's combined model of

Wenzel and Cassie-Baxter relations. Predicted contacted angles were calculated from the model

using the roughness ratios and solid surface fractions of each topography. For both relations, the

contact angles on rough surfaces are related to the contact angle on the smooth surface. In order

to account for this, data were normalized by dividing the contact angles for each liquid on

textured surfaces by the angle the same liquid makes on smooth PDMSe. Normalized values

predicted by the models were then plotted against normalized values measured on the surfaces.

Linear regression was performed to test the validity of the models.

Predicted Wetting on Novel Topographies

Once the model was determined to give a good approximation of the wetting across

engineered topographies, it was used to predict the effectiveness of proposed topographies.









Topographies considered included circular pillars, square pillars, star-shaped pillars, ring-shaped

pillars, a combination of triangular and circular pillars, a gradient array of circular pillars, and

hexagonal pillars (Fig. 3-3 and Table 3-2). Both 1 and 3 [m features heights were evaluated.

The Sharklet AFTM topography was also considered at these depths to determine effectiveness in

altering wettability.

Ulva Zoospore Assay

PDMSe samples containing 5 tm wide ridges and pillars spaced 5, 10 and 20 tm apart at

1.5 and 5 am heights in addition to the 5 am deep Sharklet AFTM topography were evaluated for

settlement of Ulva spores. Three replicates of each type were tested and all samples were

backed by glass slides. Settlement data for the pillars and ridges have been published previously

[68, 69]. The 5 am Sharklet AFTM topography was evaluated using the same protocol except for

one slight deviation. Due to the ultrahydrophobic nature of the Sharklet AFTM topography,

samples for this study were shipped to the bioassay site in nanopure water to ensure air was

displaced from the features and the samples were fully wetted during the assay.

Ulva zoospores were obtained from fertile plants of Ulva linza collected from Wembury

Beach, UK (5018' N; 4002' W) and prepared for experiments as previously described [79].

Briefly, 10 mL of spore suspension (adjusted to 2x106 mL-1) is added to each sample and

incubation is carried out for 60 min in the dark followed by fixation with 2% glutaraldehyde in

artificial seawater (Instant OceanTM). Settled spore counts were taken using a 10X objective with

a Zeiss Kontron 3000 image analysis system attached to a Zeiss epifluorescence microscope and

video camera as described by Callow et al. [68]. Thirty images were taken of each of the 3

replicate samples to quantify the number of attached spores. For smooth samples and both flat

and textured areas of patterned samples, images were taken at 1 mm intervals along the axis of









the slide. Spore settlement data are reported as the mean number of adhered spores (x = 90) with

95% confidence limits.

Porcine Vascular Endothelial Cell (PVEC) Assay

Free-standing PDMSe samples containing 5, 10, and 20 [tm spaced, 5 tm wide ridges at

both 1.5 and 5 tm heights were evaluated. In order to promote cell attachment, surfaces were

coated with fibronectin (FN) using the method of Ostuni and Whitesides [89]. This protein

adsorption makes the surface hydrophilic and improves cellular adhesion. Topography coupled

with fibronectin should induce cellular alignment within channels to maximize contact area

while minimizing tension on the cell membrane. Briefly, lyophilized bovine plasma FN (Sigma)

was dissolved in 2 mL of 0.22 ptm filtered water at 370C for 45 minutes and then diluted to

50 [g/mL in Hanks Balanced Salt Solution (HBSS). Sterilized samples were placed in

individual wells of a 24-well plate, and FN was added in 0.5 mL aliquots to each sample. During

exposure to vacuum (100 kPa) to remove trapped air, samples were left to incubate for 1 h at

room temperature. Air must be removed to prevent denaturizing of the protein which would

affect adhesion [90]. The FN solution was aspirated out and then the samples were washed 3

times with HBSS.

PVECs obtained from the main pulmonary artery of 6 to 7-month-old pigs were supplied

by Dr. Edward Block's lab between passages 2 and 5 [91]. Cells were seeded at a density of

2x105 cells per sample in 1 mL of serum-free media. Serum-free media was selected because

the adhesion protein was already adsorbed on sample surfaces. The cells were incubated at 370C

and 5% CO2 for 48 to 72 hours.

Samples were fixed with cold 10% n-buffered formalin for 20 minutes. The cell bodies

were then stained for 20 minutes in 1% crystal violet solution. Hematoxylin (Richard Allan









Scientific) was used to stain cell nuclei so that nuclear elongation could be used to quantify

contact guidance. Cells were stained in hematoxylin for 2 minutes

Cells were imaged on the surface at 200X magnification using a Nikon Optiphot

microscope and Matrox image capturing system. Multiple images were taken at each feature

width that included at least 5 nuclei. Images were analyzed to measure the nuclear form factor

(NFF) of each nucleus. The NFF is the log of the ratio of the cell length to width (Fig. 3-4). This

measurement was adapted from a procedure introduced by Dunn and Heath which requires the

measurement of the length and width of nucleus at its widest point [92]. A 5x5 grid was

superimposed on the images, and 5 nuclei were chosen per image, each from a separate square of

the grid. Using this method, at least 20 nuclei per topography type were quantified.

Statistical Methods

Results are reported using mean values and 95% confidence intervals. One-way analysis

of variance (ANOVA) and multiple comparison tests (Tukey, 95% confidence interval) were

used to compare groups.

Results

Contact Angle Measurements

As expected, topography increased water contact angles and decreased both methylene

iodide and dimethyl formamide contact angles (Table 3-3). The most effective topography at

altering wettability was the Sharklet AFTM, whereas the 20 [tm spaced ridges and pits behaved

the most like smooth PDMSe. The water contact angle on PDMSe was increased by as much as

20%, while the contact angles of methylene iodide and dimethyl formamide were reduced by as

much as 35% and 33%, respectively. Droplets on the pits and Sharklet AFTM maintained a

circular contact area, whereas droplets on the channels and ridges elongated along the features.









Because of their cylindrical, rather than spherical, geometry the contact angles on the channels

and ridges must be treated as estimates.

Receding contact angles were not evaluated by the captive air method because of difficulty

in bubble placement. For example, the channel patterns were only 0.33 cm wide (Fig. 3-5) and

the topographies became invisible when wetted. Consequently, it was impossible to be certain

the air bubble remained in the desired region. In addition, the Wilhelmy plate technique was

also deemed unsuitable because of the inability to prepare a proper sample (same topography on

all sides).

The effect of topography height was examined by comparing the 5 [tm spaced, 5 [tm deep

channels against the 5 [tm spaced and 1.5 [tm deep ridges. Because droplets were placed away

from the pattern boundaries, the feature height was the only difference sensed by the spreading

drops. The water contact angle was significantly higher on the 5 [tm deep channel compared to

the 1.5 [tm deep ridge. The effect of feature spacing was examined by looking at trends within

the pits, channels, and ridge topographies. Increased feature spacing and decreased pattern depth

resulted in diminished contact angle changes.

Comparison with Model

The sessile drop contact angle data were compared against the model to determine its

viability for use in prescreening ideas for new patterns. The 1.5 [am high ridges have relatively

low roughness factors (1.1 to 1.3), and solid fractions (0.2 to 0.5). On these surfaces water

droplets appear to follow the situation proposed by Wenzel (Fig. 3-6) rather than the metastable

state described by Quere. The Sharklet AFTM topography, on the other hand, has a high

roughness factor (5.0) and moderate solid fraction (0.47), and all three test liquids followed the

situations described by Cassie (air pockets or wicking).









Quere proposed that air entrapment would be favored for liquids of sufficiently high

surface tensions (O>cos-l[(fi-l)/(r- fi)]) and would be metastable for liquids satisfying the

condition of 90o
metastable air pocket state is not favored, and that instead the Wenzel regime is followed.

Consequently, the model has been adapted to eliminate the metastable state. In order to test the

reliability of the adapted model, data were normalized with respect to the smooth contact angles.

The model fits the data well (y = 0.99x) with a coefficient of determination of 0.89 (Fig. 3-7).

Predicted Wettability on Novel Topographies

The model predicts that the hexagons would be the least effective at increasing the

hydrophobic nature of PDMSe (Table 3-4) relative to the various designs (i.e., circular pillars,

square pillars, star-shaped pillars, combination of triangular and circular pillars, and gradient

array of circular pillars). The results reveal a minimal increase of only 1 and 3 in the water

contact angle for the 1 and 3 [tm high hexagonal features in PDMSe, respectively. These

changes would likely be insignificant when considered with the typical standard error in contact

angle measurements of 3. The model predicts that the 3 tm tall mixed star pattern will be the

most effective and lead to an increase in water contact angle of approximately 31 relative to

smooth PDMSe. A maximum height of 3 [tm was chosen for this investigation because 2 [tm

diameter pillars in PDMSe tend to collapse at higher aspect ratios (Fig. 3-8).

All pattern types were predicted to be fully wetted by water when feature heights are held

to 1 am. Increasing the height to 3 am, however, should promote the trapping of air in all

topographies except the hexagons, circular pillars, and gradient pillars.









Ulva Zoospore Assay

The ridge topographies enhanced spore settlement (Fig. 3-9). The most significant effect

was observed on the 5 am spaced, 5 am high ridges, which increased settlement by 150%

relative to the smooth surface. This dimension is roughly equivalent to the diameter of the pear-

shaped swimming spore at its widest point and the diameter of the settled spore (Fig. 3-10). As

spacing increased, the density of settled spores approached that of smooth PDMSe. Settlement

density decreased on the shorter 1.5 pm high ridges compared to the 5 pm high ridges, but still

remained at least as high as the density on smooth PDMSe. Settlement occurred almost entirely

in valley regions for all ridge topographies.

The Sharklet AFTM topography, which has feature dimensions smaller than the spore

body, significantly reduced settlement density by -86% relative to smooth PDMSe (Fig. 3-9).

Spores avoided the 2 pm wide channels and were largely confined to defects and slightly wider

spaces (-3 pm) located between adjacent Sharklet AFTM diamonds (Fig. 3-10).

PVEC Assay

Cell growth on smooth PDMSe was random with respect to orientation. Consequently,

NFFs were not significantly different from zero (Fig. 3-11). Cells attached to ridge patterned

substrata became aligned with the topographies. The PVECs settled almost entirely in the

valleys formed by adjacent ridges similar to the Ulva spores (Fig. 3-12). Cell orientation was

most strongly directed by the 5 pm deep, 5 pm spaced ridges. NFFs varied directly with feature

height and inversely with feature spacing for 5 pm deep features (Fig. 3-11).

The NFFs are significantly different for all widths at the 5 pm depth. At the 1.5 pm

depth only the 5 pm wide ridges and smooth surfaces showed a significant difference. One of

the difficulties in the analysis is the fact that the nuclei are not round, but rather elongated in









random directions on the smooth FN treated PDMSe samples. Consequently, the mean nuclear

form factors for smooth areas are near zero, but with a large standard deviation.

Discussion

Researchers have independently shown that topography alters wettability [59, 60] and

surface energy influences bioadhesion [93-97]. This study investigated the interrelationship of

all three factors (topography, wettability and adhesion) simultaneously using two different cell

types viz. motile algal spore which 'choose' where to settle and adhere and cultured animal cells

(PVECs) which are known to adapt to underlying substrate morphology. The wetting response

was well described by the Wenzel and Cassie-Baxter equations for the topographies investigated,

but ideally one would also like to predict the effect topography has on bioadhesion. It is

interesting to note that both biological models responded to the channel topographies by trying to

fill the valleys either through settlement packing (Ulva) or by elongation (PVECs). This

suggests that their responses are governed by the same underlying thermodynamic principles as

wettability. Consider a cell settling on a textured surface. If the cell is too large to rest between

or on top of the features, it must bridge, align, or conform to their shape. Bridging is similar to

the air pocket state and alignment is similar to the wicking states described by the Cassie-Baxter

relation. Alternatively, conforming resembles Wenzel behavior.

Consider a surface that an organism will settle on but for which it has a relatively low

affinity (e.g., PDMSe for Ulva spores). If the topography of this surface is engineered to expand

the Cassie-Baxter regime, then the organism may be induced to bridge the features. This would

increase tension along the unsupported regions of the organism's membrane. Additionally,

bridging would reduce the area of contact between the organism and surface, which would

decrease the adhesion strength. Thus, bridging reduces the potential for settlement by creating

unfavorable energy barriers. This would be very useful, particularly in limiting marine fouling.









Using this hypothesis, the Sharklet AFTM topography was engineered to enhance

hydrophobicity with dimensions smaller than the Ulva spore so that bridging would be necessary

for settlement to occur. It is important to note that the Sharklet AFTM design is biomimetically

inspired rather than a true biomimic. Although the basic pattern of the placoids has been

maintained, the dimensions have been reduced and the tips of the ribs have been flattened. As

designed, spores avoided the topography which resulted in an 86% reduction in settlement

density. This result provides the first demonstration that engineered microtopographies can

inhibit the settlement of spores of marine algae.

In addition to preventing settlement, topographies can also be engineered to promote it.

Consider an organism settling on a surface which it prefers (e.g., PVECs on fibronectin-coated

PDMSe). If the topography of this surface is engineered to expand the Cassie regime, then the

organism may be induced to align with topographies. This was evident in the PVEC study

presented here. For the topographies studied, the Cassie-Baxter regime increased with increased

spacing and increased depth. Similarly, PVECs showed increased alignment with increased

spacing and depth as demonstrated with NFFs. This result is consistent with research by von

Recum et al. demonstrating rat dermal fibroblasts become increasingly oriented on 0.5 [tm deep

microgrooves as the width is reduced from 10 to 2 tm [44, 45, 98, 99].

Microbubbles on surfaces are reported to denature surface adsorbed proteins, which

increases cell adhesion [90]. Fibronectin pretreatment of PDMSe was used in the PVEC assay to

convert the surface to a hydrophilic surface needed for initial cell attachment. To minimize any

potential artifacts in the PVEC assay that could be caused by differential fibronectin adsorption

through the presence of microbubbles, all PDMSe surfaces were degassed during fibronectin

adsorption to eliminate surface-adsorbed air bubbles from topographies. It can therefore be









concluded that it is unlikely that the results on PVEC alignment can be ascribed to artifacts

caused by microbubbles. In the case of Ulva spore settlement, it can also be reported that

hydrophilic modification of the topographies (which would eliminate microbubble formation)

did not alter the inhibition of zoospore settlement by the Sharklet AFTM topography [100]. Thus,

it is concluded that Ulva zoospores were contact sensing the topography and were not influenced

by the presence of microbubbles [101].

These results suggest that wettability models can be useful in predicting cellular contact

guidance for engineered topographies. It is important to note that bioadhesion is complex and

does not only rely on surface energetic but is also species-specific [102]. The material modulus

and surface elasticity of the cell membrane are also important to consider. The process is further

compounded by the variety of adhesive proteins that an organism may secrete. Additionally, the

use of wettability models is limited by their assumption that the droplet is much larger than the

topographical features. This allows for line tension effects to be neglected. Investigation of

wetting of these topographies by picoliter-sized drops may provide greater insight. Although

further investigation is needed, these relationships may eventually be used to develop models

capable of predicting the contact guidance of cells and microorganisms. Such a model would be

of value in the biomedical device and marine coating industries.
















Figure 3-1. SEM images ofPDMSe microtopographies. A) 5 [tm diameter, 5 itm diameter,
5 [tm spaced pits. B) 5 itm wide, 20 itm spaced channels. C) 5 [tm wide and 20 [tm
spaced ridges. D) Sharklet AFTM topography. Pillars, pits, channels, and Sharklet
AFTM are all 5 [tm deep, while ridges are 1.5 [tm high. Scale bars represent 20 im.


1x3" Glass
Slide
(optional)


Glass
Plates

w)


Metal
Spacer
---_PD MS
^^ PDMSe

Epoxy or
SSilicon Die


Figure 3-2. Glass mold used to make PDMSe samples. The epoxy and silicon dies were used
only for patterned samples.





























Figure 3-3. AutoCad sketches of proposed topographies. A) 2 pm diameter, 2um spaced pillars.
B) triangles and 2 pm pillars. C) 4 pm wide, 2 pm spaced stars. D) 2 pm wide, 1 pm
spaced square pillars. E) Rings with 2 pm inner diameter and 6 pm outer diameter,
spaced 2 pm apart. F) 4 and 2 pm wide stars. G) 2 pm diameter pillars spaced 1, 2
and 4 pm apart in a gradient array (repeat unit designated by triangle). H) hexagons
with 12 pm long sides and spaced 2 pm apart. I) 2 pm wide, 2 pm spaced channels.
Scale bars represent 20 pm.


- m UI


-


I I

I I



W I
I


Figure 3-4. Measurements taken to calculate nuclear form factor where L is the length of the
nucleus parallel to the ridges and Wis the width of the nucleus orthogonal to the
ridges.









1cm


1cm I


w


Figure 3-5. Layout of channel topographies. Each channel width (5, 10 and 20 kim) is contained
within a 1 cm by 0.33 cm area.

1.0





U0


.5



cos 0

Figure 3-6. Change in wettability induced by the 20 [m spaced ridges (grey squares) and
Sharklet AFTM (black triangles) topographies compared to smooth PDMSe. Both
measured data and model predictions are given. Dashed lines are used to indicate the
metastable air pocket state proposed by Quere.










1.3


1.1

0.9


0.7


0.5 -
0.5


y = 0.99x
R2 = 0.89


.t .


0.7 0.9 1.1


Normalized Measured Angle
Theta* / Theta

Figure 3-7. Comparison of contact angles predicted by the model to contact angles measured on
the surfaces. Data were normalized with respect to contact angles on smooth
PDMSe. Linear regression indicates a near 1:1 relationship (slope-0.99) with high
correlation (R2 = 0.89) to the data.


Figure 3-8. SEM images of 2 tm diameter pillars in PDMSe that are A) 5 tm high and 4 tm
spaced and B) 3 tm high and 2 tm spaced. Increased height causes pillars to bend.
Scale bars represent 15 am.









2000

1600

1200

800

400

0


0 en ( 10 0 0
CM LO C) CM
C\1 C\1
cr r c cC/)


5 pm deep


1.5 pm deep


Figure 3-9. Ulva settlement on smooth (SM) and textured PDMSe. Topographies studied
included the Sharklet AFTM (Shark) in addition to 5 tm wide ridges that were 5, 10,
and 20 tm spaced (5R, 10R, and 20R) and either 1.5 or 5 tm high. The Sharklet
AFTM topography was evaluated in a separate experiment as indicated by the darker
bars. Error bars indicate +2 standard errors of the mean. For all surfaces, counts are
based on the mean of 90 counts, 30 from each of 3 replicates.


Figure 3-10. Ulva settled on smooth and textured PDMSe. A) Smooth. B) 5 tm wide, 5 tm
spaced, and 5um deep channels. C) 5 tm deep Sharklet AFTM in PDMSe. Images
were taken via light microscopy. Scale bars represent 25 am.


HHinTm


Cl) IC)


01
o
CD









0.50

0.40

0.30

0.20

0.10

0.00

-0.10


5 pm deep


1.5 pm deep


Figure 3-11. PVEC alignment on smooth (SM) and textured PDMSe. Topographies studied
included 5, 10, and 20 tm spaced, 5 tm wide ridges (5R, 10R, and 20R) that are both
1.5 and 5 tm high. Error bars indicate +2 standard errors of the mean.


Figure 3-12. Endothelial cells grown on smooth and textured PDMSe. A) Smooth. B) 5 [m
wide, 5 rm spaced, and 5 [im tall ridges. C) 5 .im wide, 5 .im spaced, and 1.5 [im tall
ridges. Images have been processed to improve contrast. Scale bars represent 50 im.


SM 5R 10R 20R SM 5R 10R 20R


rs--




I I


i __









Table 3-1. Dimensions
Height
Feature Heig
(1m)
1.5
1.5
Pillar 15
5.0
5.0
5.0
5.0
Pit 5.0
5.0
5.0
Channel 5.0
5.0
1.5
1.5
1.5
Ridge 5.0
5.0
5.0
5.0
Sharklet
AFT 4.0
AFTM


2 Negative


of Topographies Used in Wettability and Bioadhesion Studies
Width Spacing Replication
(pm) (pm) Type
5 5
5 10
5 20
Positive
5 5
5 10
5 20
5 5
5 10 Negative
5 20
5 5
10 5 Negative
20 5
5 5
5 10
5 20
Positive
5 5
5 10
5 20









Table 3-2. Dimensions of Novel Theoretical Topographies
t Height Spacing(s) Width(s)
(Gtm) (Gtm) (pm)
1 2 2
Circular Pillars 2 2
3 2 2
1 2
10
Triangle/Circles 2
3 2
10
1 2 4
Star Pillars 2 4
3 2 4
1 1 2
Square Pillars 3 1 2
3 1 2
1 2 2
Ring Pillars
3 2 2
2
1 2
Mixed Star 4
Pillars 2

1
3 2

1 2 2
3
4
Gradient Pillars
1
3 2 2
3
4
1 2 20
Hexagon Pillars 2 20
3 2 20
1 2 2
Channels
3 2 2
1 2 2
Sharklet AFTM
3 2 2









Table 3-3. Measured Contact Angles on Microtopographies
Features Spacing Contact Angles ()
(pm) Water Mel DMF
Smooth 1.00 1.0 108 4 71 6 55 8
5 0.80 1.8 115 2 65 2 50 + 8
Pits 10 0.91 1.4 112 2 69 4 52 4
20 0.97 1.1 110 6 65 6 56 6
5 0.50 2.0 133 8 51 2 39 6
Channels 10 0.67 1.7 121 6 62 4 49 8
20 0.80 1.4 116 6 68 12 48 6
5 0.50 1.3 116 8 63 8 46 4
Ridges 10 0.33 1.2 115 8 63 6 46 8
20 0.20 1.1 111 6 66 4 52 8
Sharklet
market 2 0.47 5.0 135 3a 46 8 35 2
AF TM
aIndicates droplet would not settle on the surface and had to be captured with video









CHAPTER 4
CHARACERIZATION OF GLUTARALDEHYDE AND GENIPIN CROSSLINKED
GELATIN FILMS

Introduction

Collagen is the primary protein component in bone, cartilage, skin and connective tissue.

Collagen has been investigated for use as a biomaterial, but due to its antigenicity extensive

further research would be needed to determine the impact on the immune system. Alternatively,

gelatin has relatively low antigenicity. Gelatin is formed from denatured collagen, through

heating or physical and chemical degradation of the protein to destroy the triple-helix structure.

In the biomedical sector, gelatin is most commonly used for drug delivery capsules, wound

dressings, adsorbent surgical pads, and vascular graft sealants.

Gelatin has the advantage of being a natural, biodegradable biopolymer. As such, upon

implantation no cytotoxicity is evident and over time it can be resorbed and replaced with native

collagen. At temperatures above 400C, aqueous gelatin solutions exist in the solvated state

allowing them to be cast into a variety of forms. At these elevated temperatures, the polypeptide

exists as flexible single coils. Upon cooling, gelation takes place in which the triple helical

structure of collagen is partially recovered. Gelation requires the concentration of gelatin in

water to be above a certain critical minimum point, typically accepted to be between 0.4 and 1%

by weight [103]. A hydrogel results, which allows for the transport of water and nutrients

through the bulk.

The primary drawback of gelatin lies in poor mechanically stability. In creating a gelatin

hydrogel, dry gelatin powder is mixed with water that has typically been heated to above 40C

which is the denaturation point for native soluble collagen. Upon allowing the solution to cool,

gelation occurs as there is a partial recovery of the collagen triple helix occurs along segments of

the polymer chains. At the triple helix regions, three chains are combined to form a type of









crosslink. However, if gelatin is submerged in an aqueous environment long enough it will

eventually dissolve completely. Consequently, crosslinking must be employed to improve

mechanical stability. This is typically accomplished through chemical crosslinking with

bifunctional aldehydes, diiosocyanates, carbodiimides, epoxy compounds and acyl azide

methods [104]. Physical methods such as dehydrothermal treatment and ultraviolet and

gamma irradiation have also been used [105-107]. The improvement of mechanical properties

depends on the crosslink density and can be modeled using rubber elasticity theory. Briefly, as a

rubber is stretched a retractive force is generated due to the decrease in entropy that occurs as the

polymer chains become stretched. The basic equation that relates the retractive stress (o) to the

extension ratio (a) is given below:


Ua= P RTa- (4-1)


In this equation, p represents density and Me denotes the molecular weight between crosslinks.

Although the stress-strain relationship is not linear, it is clear that increasing the crosslink density

(which decreases M,) results in higher modulus values.

Glutaraldehyde (GTA) is the most common crosslinker for gelatin systems and it primarily

reacts with lysine and hydroxylysine amino acid residues. Although it reacts heterogeneously

with gelatin (Fig. 4-1), it only requires one GTA molecule to form a crosslink. GTA efficiently

stabilizes the biopolymer, but is known to exhibit localized cytotoxic effects as it is released

during material degradation [5].

Genipin (GEN) provides an alternative to dialdehyde crosslinking. GEN is a natural

crosslinker that is obtained from an extract (geniposide) of gardenia fruits. Two GEN molecules

combine to form a single crosslink between primary amino groups (Fig. 4-2). The mechanism

begins with a ring-opening condensation reaction with a primary amine [108] and is completed









with a dimerization reaction which may involve free radicals [109]. GEN has been shown to be

nearly as efficient as GTA at stabilizing collagen-based biomaterials, but with a much lower

associated cytotoxicity [110]. In comparison to GTA, GEN fixation of cell-free xenogenic

vascular grafts results in the formation of a more consistent endothelial layer in vivo [111].

Attempts to generate microscale topographies in gelatin films have been limited. Yang et

al produced gelatin ridges adhered to a glass substrate by modification of conventional

photolithography techniques [112]. Briefly, gelatin was first spin coated onto glass and then

photoresist was coated on top. UV photolithography was then used to generate the desired

pattern in the photoresist and then the three-layer structure was immersed in GTA to crosslink

the exposed regions of gelatin. The gelatin microtopgraphy was revealed after rinsing in acetone

to strip away photoresist and immersion in hot water to remove uncrosslinked gelatin. The

fidelity of the resulting patterns depended on both the line width (at least 5 .im) of the gelatin

and spacing between features (at least 10 .im). Yu et al. produced parallel grooves (5-500 .im

wide) in a chitosan-collagen-gelatin composite via photolithography and replication from a

silicone intermediate [112]. Good reproducibility was achieved for dimensions greater than

10 im.

In the present study, gelatin films were crosslinked with varying concentrations of GTA

and GEN. These films were evaluated for their potential at producing stable microtextured cell

culture substrates. Mechanical properties (tensile and swelling) were determined in addition to

the ability to produce fine (-2 ism wide) microscale features.

Materials and Methods

PDMSe Mold Preparation

Smooth polydimethylsiloxane elastomer (PDMSe) wells were produced using Dow

Corning Corporation's Silastic T-2. The resin and curing agent were mixed in a 10:1 ratio,









degassed and poured over two 3 inch by 2 inch microscope slides that were adhered to one

another. Large top and bottom glass plates separated by 5 mm spacers were used to ensure a flat

surface on the back of the wells (Figure 4-3). The PDMSe was allowed to cure for 24 h at room

temperature and then removed from the mold. Excess silicone was cut away, leaving about a

1cm border around the well

Topographically patterned PDMSe molds were prepared in a two-step curing process

(Fig. 4-4). First, PDMSe (10:1 ratio of base resin to curing agent) was cast directly from

topographically modified silicon wafers. The wafers were prepared using standard

photolithography techniques (processing performed by James Schumacher). Wafers were

prepared using deep reactive ion etching (DRIE) as previously described in Chapter 3. The target

dimensions of the topography were 2 [m wide channels separated by 2 [m wide ridges that are

1 [m tall. For the wafers, the channels are etched into the surface and are therefore considered

to be negative features. When PDMSe is cast against the wafer, channels replicate as ridges

protruding out from the surface and so the topography is inverted.

In an initial cure step, PDMSe was then cast against clean wafers within a glass mold with

spacers to give -0.5 mm thick PDMSe film. Cured films were then removed from the wafers

and the desired pattern portion cut out leaving -2 mm thick border of smooth PDMSe around the

edges. Two 2 in x 3 in glass microscope slides were adhered to one another and then treated

with hexamethyldisilazane via vapor deposition. The textured film was then suctioned pattern-

side down to the center of the microscope slides. This was then placed on a clean HMDS-treated

glass plate with 3 mm spacers at the corners of the plate. Approximately 120 g of PDMSE was

mixed, degassed and poured over the film and slides and a second clean, HMDS treated glass

plate was laid over top. The PDMSe was allowed to cure overnight at room temperature before









being removed from the mold. Excess silicone was then cut away, leaving -1 cm border of

smooth PDMSe around the well.

Gelatin Film preparation

Gelatin derived from bovine calf skin was supplied as a dry powder (Sigma).

Uncrosslinked gelatin films were prepared by first dissolving the powder in 500C nanopure water

at a 10% (wt/v). For each film, 8 mL of the heated gelatin solution was poured into a 50.8 mm

by 76.2 mm by 2 mm PDMSe well. A clean glass slide was then across the well in order to level

the solution. A desiccator lid was placed above the well in order to slow evaporation of the

water. The film was allowed to dry for 24 hours at room temperature.

GTA and GEN were used to stabilize smooth gelatin films. Four GTA concentrations

were investigated: 2.2, 3.2, 4.3 and 9.1 wt/wt% which correspond to 2.9, 4.3, 5.9 and 13% molar

equivalents (% MEQ), respectively. GEN crosslinking was carried out at 3 different crosslinker

concentrations: 4.8, 9.1 and 13 wt/wt% (1.4, 2.9 and 4.3% molar equivalents). Molar equivalents

are based on a molecular weight of 1.2x105 g/mol for gelatin as previously measured by Cuevas

[94]. The average molecular weight of repeat units was assumed to be 65 g/mol.

GTA crosslinking was performed on partially dried gelatin films. Five hours after initial

casting of uncrosslinked gelatin, 4 mL of an appropriate GTA solution was pipetted on top of the

film (Figure 4-5). The desiccator lid was then placed above the well and the GTA was allowed

to react overnight at room temperature.

GEN crosslinking was carried out by bulk mixing. GEN was first dissolved in nanopure

water and heated to 500C. Gelatin was then slowly added while mixing to a final concentration

of 10% (wt/v). The solution was then placed in a 500C oven for 5 minutes to ensure the gelatin

completely dissolved. For each film, 8 mL of the heated gelatin-GEN solution was poured into a

50.8 mm by 76.2 mm by 2 mm PDMSe well. A clean glass slide was then across the well in









order to level the solution. A desiccator lid was placed above the well in order to slow

evaporation of the water. The film was allowed to react and dry for 24 hours at room

temperature. In addition to the smooth films, a topographically patterned GEN crosslinked

gelatin film (2.9 % MEQ) was also prepared. The topography included 2 [m wide channels

formed between 2 .im spaced ridges that were 1 .im tall.

Soxhlet Extraction of Gelatin

For some studies, gelatin films were Soxhlet extracted in nanopure water for 72 hours to

remove residual unreacted GEN. Samples were then immersed in fresh nanopure water and

allowed to equilibrate for 24 hours before mechanical tests were performed.

Postcuring of Gelatin

In order to evaluate the thermal stability of GEN crosslinked gelatin exposed to moderate

heat, two films of each GEN concentration were immersed in water and placed in a 500C oven

for 3 hours. Samples were then immersed in fresh nanopure water and allowed to equilibrate for

24 hours before mechanical tests were performed.

Mechanical Testing

Samples for tensile testing were cut using an ASTM D1822-68 type L dog bone die (1 in

gauge length and 3.1 mm cross-sectional width). Three specimens were cut from each gelatin

film. Samples were tested using an Instron 4301 with Series IX software. All samples were

tested at a crosshead speed of 50.8 mm/min (2 in/min). Additionally GEN-crosslinked samples

without post-processing (Soxhlet extraction or postcuring) were tested at a second, slower

crosshead speed of 5 mm/min to determine the effect of strain rate. Pneumatic grips were used

with the pressure set to 24 psi.









Swelling Study

Swelling studies were carried out in order to further characterize the abilities of GTA and

GEN to stabilize gelatin films. A 12 mm diameter circular punch was used to cut out samples

from smooth, hydrated films. Four samples of each type were punched. Samples were placed in

the oven at 500C for 3 hours to dry and the initial weight of each sample disc was measured.

Samples were placed in individual wells of 12-well culture plates. Then 3 mL of nanopure water

was added to each well. At ten time points (5, 10, 15, 20, 30, 60, 120, 240, 1200 and 10000 min)

the water uptake in the gelatin samples was measured. For each time point, samples were

removed from water and their surfaces blotted dry with a task wipe. Each sample was then

immediately weighed to determine the hydrated weight. Percent swelling was then calculated by

comparing the hydrated (Wt) and initial dry weights (Wo) according to the following equation:

W -W
%Swelling 100% (4-2)
Wo

After the swelling study was complete, samples were dried for 3 hours at 500C and the

final dry weights (Wf) taken. The mass change of the samples throughout the study was then

calculated by substituting Wf for Wo in Equation 4-2.

Evaluation of Microscale Gelatin Features

The fidelity of the topographically patterned gelatin film was evaluated using white light

interference profilometry. A Wyko model NT1000 profilometer coupled with Vision 32

software was used for all measurements. The microscope has 2 lenses: the exterior objective that

was available in three magnifications namely 5X, 20X and 50X and the internal 'field of view'

(FOV) lens which was available in 0.5X, 1X and 2X magnifications. High resolution was

needed for the present study and so the 50X external objective and 2X FOV were used for a

combined magnification of 100X.









Profilometry was performed on both dry (ambient conditions for 24 h) and rehydrated

(immersed in nanopure water for 24 hours) gelatin films. The surface of the rehydrated film was

blown dry with nitrogen gas immediately preceding testing. The Vision 32 software was used to

measure the channel dimensions (width, spacing and height). Six measurements per dimensions

were taken at random across the sample.

Statistical Analysis

Results are reported as mean values with 95% confidence intervals. For each study, one

way analysis of variance (a = 0.05) was used to determine if any significant differences among

the treatment means existed. As appropriate, Tukey's multiple comparisons test was used to

determine which treatments were significantly different.

Results and Discussion

Mechanical Testing of Gelatin Films

Tensile testing of GEN and GTA crosslinked gelatin films indicated that the mechanical

properties are influenced by the concentration of crosslinker. As crosslinker concentration is

increased, the stress-strain curves become steeper (Fig. 4-6), indicating stiffness is increased.

For both GEN and GTA, the initial Young's modulus of the gelatin increased significantly with

increased crosslinker concentrations (Fig. 4-7). Interestingly, both chemistries were equally

efficient at stabilizing the gelatin in terms of modulus. No significant differences existed

between GEN and GTA at concentrations of 2.9 and 4.3% MEQ. Mean moduli values were

-120 and -155 kPa, respectively, for these two concentrations. This contradicts results

published by Cuevas which state GEN is significantly more efficient at crosslinking gelatin

[113]. The reason for the difference is not immediately clear, but might be attributed to

differences in the length of storage of GEN. Increasing the GTA concentration to 5.9% MEQ led

to a significant increase in modulus with a value of 280 + 14 kPa. A direct comparison with









GEN could not be made at this concentration. A 5.9% MEQ GEN crosslinked gelatin film could

not be produced due to GEN's low solubility in water (slightly less than 0.02 g/mL).

Elongation at break for gelatin was also affected by crosslinker concentration. An increase

in crosslinker amount tended to decrease the elongation at break (Fig. 4-8). Significant

differences for the mean elongation values were observed for the highest and lowest

concentrations of both GEN and GTA. Elongation values across all treatments ranged from

42 9% to 82 16%. As with modulus, elongation did not differ significantly among the GEN

and GTA samples at either 2.9 or 4.3 % MEQ.

Ultimate tensile strength (also known as break stress) did not vary significantly with

crosslinker amount or chemistry (Fig. 4-9). GTA tended to increase the ultimate tensile strength

compared to GEN, particularly at a concentration of 2.9 % MEQ. However, high variability in

the data prevented definitive conclusions.

Mechanical testing of GEN crosslinked gelatin was carried out at both 5 mm/min and

50.8 mm/min (2 in/min) strain rates in order to determine how the time scale of perturbations

affects the mechanical stability of the gelatin. No obvious differences were observed between

the characteristic stress-strain curves for the two strain rates at any of the crosslinker

concentrations (Fig. 4-10). Similarly, the initial Young's modulus did not vary significantly with

strain rate either (Fig. 4-11).

The thermal stability of GEN crosslinked gelatin was evaluated by processing samples by

two methods. The 50C postcure for 3 hours did not significantly affect the gelatin at GEN

concentrations of 2.9 and 4.4% MEQ (Fig. 4-12). However, at 1.4% MEQ the initial Young's

modulus was decreased significantly by the heat, lowering the modulus from 98 19 to

67 3 kPa. Soxhlet extraction significantly reduced the initial modulus for all GEN









concentrations. In particular, the 1.4% MEQ GEN crosslinked gelatin samples were so degraded

that the films fell apart under their own weight and dog bone specimens could not prepared. At

2.9 and 4.3% MEQ of GEN, Soxhlet extraction of the samples resulted in -50% reduction of the

initial Young's modulus.

Swelling Studies of Gelatin Films

The goal of this chapter was to choose an optimum crosslinker for later cell studies with

topographically modified gelatin. With this in mind, it was necessary to limit the swelling of

gelatin over time when immersed in aqueous environments. Dried samples of GEN and GTA

crosslinked gelatin were immersed in nanopure water for up to 7 days and their percent swelling

was plotted with time (Fig. 4-13). The GEN crosslinked samples tended to level off more

quickly and at a lower swelling percentage than the GTA samples.

For both GEN and GTA, increasing the concentration of crosslinker resulted in a decrease

in swelling. By 1200 min (20 h), the only treatments that did not significantly differ in the

degree of swelling were 1.4% MEQ GEN and 5.9% MEQ GTA (Fig. 4-14). With the exception

of these two, all of the GTA samples swelled to a greater extent than all of the GEN samples.

The swelling of GEN crosslinked samples decreased from 360 20% to 100 10% as the

crosslinker concentration increased from 1.4 to 4.3% MEQ. Similarly, the swelling of GTA

crosslinked samples decreased from 520 10% to 370 20% across a range of 2.9 to 5.9%

MEQ of the crosslinker. These results suggest that GEN samples would be better than GTA

samples at maintaining the fidelity of microscale topographies during culture

The transport of water through each film was evaluated assuming Fickean diffusion. The

diffusion coefficient (D) was determined by the following equation which is based on Fick's 2nd

law [114, 115]:










;= (4-3)


In this equation, Wt and Wo represent the mass of water in the hydrogel at time t and

infinite time respectively. D is the diffusion coefficient and 1 is the average thickness of the gel

during the swelling study. The diffusion coefficient for each sample was calculated from the

slope of a linear regression to Wt/Wo versus the square root of time (Fig. 4-15). It is important

to note that the weight of the GTA crosslinked gelatin samples leveled off between 480 and

1200 minutes (4 and 20 h) but underwent a sharp weight increase between 1200 and

10000 minutes (roughly 1 and 7 days). This suggests that the network of the material underwent

degradation during this time period and so the infinite weight time point was selected to be

1200 minutes to exclude the effects of degradation. The diffusion coefficient of gelatin did not

significantly vary with type or concentration of crosslinker used (Table 4-1). The average

diffusion coefficient was 13 2 pm2/s.

To further evaluate the diffusion kinetics, a generalized rate equation was used to

determine whether Fickean diffusion was rate determining mode of mass transport. The equation

used was as follows [114, 116]:

W
t = kt". (4-4)


The term k is a characteristic constant of the gel and n is a characteristic exponent which

depends on the primary mode of transport of the penetrant. Fickean kinetics defines n = 0.5.

Values of n between 0.5 and 1.0 indicate that the desorption process is non-Fickean. The

characteristic exponent, n was calculated from the slope of the linear regression of log (Wt/W,)

versus the log of time (Fig. 4-16). The values for n varied significantly with crosslinker type but

not concentration (Table 4-1). The value of n was 0.39 0.07 for GEN samples and 0.50 + 0.06









for GTA samples. This suggests the rate of diffusion of water through GTA crosslinked gelatin

is governed by Fickean diffusion, whereas other processes are significant for GEN crosslinked

gelatin. The value of n might have been decreased because of a desorption of unreacted genipin

In order to determine if GEN and GTA crosslinked gelatin degrades or leaches crosslinker

into the water, the weights of dried samples before and after the 7 day swelling study were

compared (Fig. 4-17). The mass loss of GTA crosslinked samples was 7.4 0.9% and it did not

vary significantly with crosslinker concentration. In contrast, the mass loss of GEN crosslinked

samples increased with increasing crosslinker concentration. The 4.3% GEN samples exhibited

a 20.2 2.3% mass loss, which was significantly greater than that of 1.4% and 2.9% GEN

samples which lost 11.9 5.4% and 14.7 1.2% respectively. Some of the mass loss is likely

due to the leaching of unreacted genipin from the gelatin into water. The total mass loss is

similar to the amount of GEN in the original film, which suggests that at least some of the

change in mass is due to a loss of gelatin.

Evaluation of Microscale Gelatin Features

GEN (2.9% MEQ) crosslinked gelatin was able to replicate the 2 .im wide microchannels

as shown in the profilometry images (Fig. 4-18). Feature widths did not vary significantly from

the silicon wafer master, with both substrates having channel widths of 2.3 to 2.4 rm and ridge

widths of 1.6 to 1.7 [im (Table 4-1). Feature depths were not as accurately replicated.

Compared to the silicon wafer, channel depth decreased from 0.90 + 0.02 [m to 0.77 0.02 [m

for dehydrated gelatin. The features in the rehydrated gelatin appeared even shallower with a

depth of 0.55 0.01 rm. However, the true rehydrated feature height may have been larger.

Because the profilometry measurements were taken in air, water from the bulk of the gelatin may

have diffused to the surface and partially filled the channels. The replication of the feature

depths could possibly be improved if the PDMSE mold were hydrophilically modified prior to









casting the gelatin. This could be accomplished either through plasma treatment or acid

immersion and would allow the gelatin to fully wet the contours of the PDMSe.

Conclusion

Both GEN and GTA stabilized the gelatin films. For equal crosslinker concentrations

(% MEQ), GTA provided a slight advantage at improving tensile properties (similar modulus but

longer elongation at break), but GEN was significantly better at stabilizing gelatin against

swelling in water. Although GEN is known to be less cytotoxic than GTA, some concern

remains about whether cells will grow on the GEN crosslinked gelatin surfaces. Swelling studies

indicate that unreacted genipin will likely be leached from the gelatin samples. Soxhlet

extraction in water was not found to be an acceptable method of removing residual unreacted

GEN from gelatin because the mechanical properties of the films deteriorated too much. As

such, the 2.9% MEQ GEN was chosen as the best crosslinker concentration to proceed with into

cell culture work because it should provide the best trade-off of enhancing tensile properties and

resistance to swelling while minimizing the amount of unreacted GEN. Topographically

modified gelatin films crosslinked with 2.9% MEQ GEN were found to replicate 2 [m wide

features adequately, although the PDMSe mold would need to be hydrophilically modified in

order for feature depth to be replicated accurately.


























R-NH2
GELATIN

+


O 0
II 2 H 2 II
HC-C -C -C -CH
GLUTARALDEHYDE


/ 0

R-N=C-C -C -C -CH
H

H2 H2 H2
R-N=--C -C -C -0=N-R

CHO CHO
-H H2 I| / H2 |
R-N=-C -C -CC-C -C -CFC-CI-b-CHIr-CHO
H n


H2 H H2 H2
R-N=C-C -C -C -C
H 0O OH



R -N

CHO CHO
H2C-Hl H2--CH2

S \ H2 HH // NR
R-N- --C -C -C N-R


H2C-CH2 H2-C-CI-
CHO CHO
CHO CHO


Figure 4-1. Chemical Reactions between gelatin and glutaraldehyde [117].



















H2O

0 OCH3



H2 -R
S GELATIN

HOH2C OH

GENIPIN


0 OCH3

H
C-N-R
H


CH2UH


O OCH3






HOH2C OH


0. QCH"
-^ ^-' 3


H20


;H2OH


H,0OH


Figure 4-2. Crosslinking mechanism ofgenipin [108, 109, 118].













-U


Metal Spacer

PDMSe
a-i----------

S 2x3" Glass Slide



C


Figure 4-3. Mold design for creating smooth PDMSe wells for casting gelatin. A) Pour PDMSe
over 2 in by 3 in glass slides and allow ot cure in the glass mold shown. B) Remove
PDMSe and slide from the mold. C) Peel away slide an cut out well.


Spacer
--- __ PDMSe
Si- tterned
icon Wafer


Spacer

-----PDMSe
O~ 2x3" Glass
Slide


Figure 4-4. Process for preparing topographically patterned PDMSe wells. A) Cure PDMSe
against patterned wafer. B) Suction patterned PDMSe to glass slides. C) Pour fresh
PDMSe on top and allow to cure. D) Remove PDMSe from mold and cut out the
well.


Glass
Plates




B


Glass/
Plates
\


Glass
Plates\








Uncrosslinked Gelatin
in PDMSe Mold


Add GTA


Dry 5hrs


React
Overnight


Remove from
Mold


Figure 4-5. Process for crosslinking gelatin films with glutaraldehyde.












0.25

0.20
(0
: 0.15

1 0.10
C/0
0.05


0.2 0.4 0.6 0.8
Strain (mm/mm)


-2.9% MEQ
-4.4% MEQ
-5.8% MEQ


0.2 0.4 0.6
Strain (mm/mm)


Figure 4-6. Representative stress-strain curves for GEN and GTA crosslinked gelatin. A) GEN
crosslinked gelatin. B) GTA crosslinked gelatin. Tensile testing was carried out at a
crosshead speed of 50.8 mm/min (2 in/min).


-1.4% MEQ
-2.9% MEQ
-4.3% MEQ


0.00 0-
0.0


0.25

0.20

0.15

0.10

0.05

0.00











SGEN
* GTA


*** **


2.9


Crosslinker Concentration (%MEQ)

Figure 4-7. Initial Young's modulus versus crosslinker concentration for GEN and GTA
crosslinked gelatin. Tensile testing was performed at a crosshead speed of
50.8 mm/min (2 in/min). Asterisks denote groups with means that are not statistically
different (Tukey's Test, a = 0.05).


SGEN
* GTA


f *


.4 2.9 4.3 5.
Crosslinker Concentration (%MEQ)


Figure 4-8. Elongation at break versus crosslinker concentration for GEN and GTA crosslinked
gelatin. Tensile testing was performed at a crosshead speed of 50.8 mm/min
(2 in/min). Asterisks denote groups with means that are not statistically different
(Tukey's Test, a= 0.05).


300


, 200

100
o 100


120

100

80

60

40

20

0


** **


-









350
300
250
200
150
100
50
0-


SGEN
* GTA


.4 2.9 4.3 5.9
Crosslinker Concentration (%MEQ)


Figure 4-9. Ultimate tensile strength versus crosslinker concentration for GEN and GTA
crosslinked gelatin. Tensile testing was performed at a crosshead speed of
50.8 mm/min (2 in/min). Significant differences did not exist among any of the
means (Tukey's Test, a = 0.05).


- 1.4%, 2 in/min
-2.9%, 2 in/min
- 4.3%, 2 in/min


- 1.4%, 5 mm/min
--2.9%, 5 mm/min
--4.3%, 5 mm/min


0.2 0.4 0.6 0.8
Strain (mm/mm)


Figure 4-10. Stress-strain curves of GEN and GTA crosslinked gelatin. Testing was performed
at 2 in/min (solid lines) and 5 mm/min (dashed lines).


0.25

0.20

t 0.15
U,
a 0.10
0.05
0.05


0.00 r^
0.0










200

150

100

50


* 5 mm/min
U 50.8 mm/min

*


2.9 4.3


Genipin Concentration (%MEQ)


Figure 4-11. Effect of strain rate on the initial modulus of GEN crosslinked gelatin. Asterisks
denote groups with means that are not statistically different (Tukey's Test, a = 0.05).


200

150

100

50


* Fresh U 50C Postcure U Extracted


1.4


2.9


4.3


Genipin Concentration (%MEQ)


Figure 4-12. Effects of post-processing on the initial modulus of GEN crosslinked gelatin.
Samples were tested without post-processing (Fresh), after heating for 3 h in a 50C
oven while immersed in water (50C Postcure), or after being Soxhlet extracted in
water for 72 h (Extracted). Mechanical testing was performed at a crosshead speed of
50.8 mm/min (2 in/min). Asterisks denote groups with means that are not statistically
different (Tukey's Test, a = 0.05).


**** ****


***
***
T












-GEN-1.4%
- GEN-2.9%
- GEN-4.3%


Time (min)


- GTA-2.9%
- GTA-4.3%
- GTA-5.9%


Time (min)

Figure 4-13. Swelling of GEN and GTA crosslinked gelatin for 7 days in water. A) GEN
crosslinked gelatin. B) GTA crosslinked gelatin. Samples were initially dried
overnight in a 50C oven. Error bars represent +2 standard errors.


1000

800

600

400

200


100


1000


10000


1000

800

600

400

200


100


1000


10000









600
SGEN
500 GTA
S400

.- 300 -

200-

100 -


1.4 2.9 4.3
Crosslinker Concentration (% MEQ)


Figure 4-14. Swelling at 20 h of GEN and GTA crosslinked gelatin films. Swelling was carried
out in nanopure water for 20 h. All sample means are significantly different from one
another (Tukey's Test, a = 0.05).


0.80


0.60

0.40


0.20

0.00


y = 0.14x
R2 = 0.98


y = 0.13x
R2 = 0.99


y=0.15x y = 0.14x
R2 = 1.00 R2 = 1.00


* Sample 1
* Sample 2
A Sample 3
* Sample 4


0 1 2 3 4 5


Time/2 (min)

Figure 4-15. Representative plot used to calculate diffusion coefficients. Fractional water
uptake (Wt/Woo) is plotted against the square root of time. The diffusion coefficient
is calculated from the slope of this plot according to Equation 4-3.











y = 0.44x- 0.73
R2 = 0.98


-0.2 = 0.53x- 0.90
R2 = 0.97
-0.3
y = 0.46x- 0.79
-0.4 R2 = 0.97

-0.5 y = 0.46x- 0.79
R2 = 0.99
-0.6
0.0 0.5


* Sample 1
* Sample 2
A Sample 3
* Sample 4

1.5


Log Time (min)


Figure 4-16. Representative plot used to calculate the time constant for diffusion kinetics. The
log of fractional water uptake (Wt/Woo) is plotted against the log of time. The time
exponent (n) for the welling kinetics is calculated from the slope of this plot
according to Equation 4-4


-25.0

-20.0


*GEN
* GTA


-15.0

-10.0

-5.0

n n


2.9 4.3 5.9


Crosslinker Concentration (% MEQ)


Figure 4-17. Mass loss of GEN and GTA crosslinked gelatin samples after swelling for 7 days in
water. Samples were dried for 3 h at 500C.


0.0

-0.1


0)
-j
























rim
756
600

400

200

0

-200
-354


+


m-
a-- 441


nm
-- 6S8


Figure 4-18. Profilometry images of channel topographies replicated in different materials. A)
Silicon wafer. B) PDMSe mold. C) Dry gelatin. D) Rehydrated gelatin. Gelatin
samples were crosslinked with 2.9% MEQ genipin. Images are of 48x76 im2 sample
areas.


Table 4-1. Diffusion Parameters Determined for GEN and GTA Crosslinked Gelatin
Crosslinker Diffusion
Crosslinker Concentration Coefficient, D
(% MEQ) (im2/s) n
1.4 13 2 0.46 0.08
GEN 2.9 13 + 1 0.39 + 0.05
4.3 13 2 0.36 0.03
2.9 15 2 0.54 0.06
GTA 4.3 12 + 2 0.49 + 0.07
5.9 14 2 0.47 0.04


rim
-- 498


~mr~n~









Table 4-2. Feature Dimensions of Topographically Modified Substrates Measured by WLIP
Material Ridge Width Channel Width Feature Depth
(ltm) (ltm) (ltm)
Silicone Wafer 2.3 + 0.1 1.7 + 0.1 0.90 + 0.02
PDMSe 1.6 0.1 2.4 0.1 0.90 + 0.01
Dehydrated Gelatin 2.7 + 0.1 1.3 0.1 0.77 0.02
Rehydrated Gelatin 2.4 0.1 1.6 0.1 0.55 0.01









CHAPTER 5
ENDOTHELIAL CELL GROWTH ON TOPOGRAPHICALLY PATTERNED
SUBSTRATES

Introduction

Researchers have sought to improve the antithombogenic nature and hence the

patency of artificial vascular grafts though the incorporation of endothelial cells onto the

inner lumen of the graft surface [2, 3, 6-33]. They have shown that endothelial cells

grow to confluence on a wide variety of substrates, but are removed when exposed to

shear stresses equivalent to those present in natural human arteries. Attempts to improve

endothelial cell adhesion have included surface modifications of the graft materials by

coating with adhesion proteins such as albumin, extra cellular matrix, gelatin and

fibronectin [15, 18, 28-30, 32, 33] as well as non-specific treatments such as carbon

deposition and plasma discharge [33]. These methods have found some success at

improving short term adhesion, but cellular retention is not maintained for longer

exposures (>3 h). A more newly developed treatment has involved the use of a peptide

sequence found in fibronectin. Covalent binding of synthetic versions of the arginine-

glycine-aspartate (RGD) sequence has been shown to overcome late term removal of

cells and to resist platelet adhesion and activation [13, 15, 32].

Another approach to improving endothelial resistance to shear looks at cell

morphology in natural arteries. Endothelial cells located in vascular regions of relatively

high shear stress tend to be elongated in the direction of flow and have actin filament

bundles which terminate at focal adhesions that are aligned parallel to flow [35].

Alternatively, endothelial cells grown in static tend to be polygonal in morphology with

only a small number of stress fibers confined to the cell periphery [36]. When statically

cultured cells are exposed to shear, cytoskeletal and sometimes morphological changes









are induced. These changes are accompanied by a stiffening of the cell which is related

to an increase in stress fiber density [37]. Depending upon the magnitude and duration of

shear exposure, cells elongate and stress fibers align with the flow direction accompanied

by the coalescence of focal contacts so that they are fewer in number but greater in size

[36, 38]. In order for a cell grown in static culture to align itself in this manner, it must

break many of the focal adhesions it created with the surface and form new ones. It is

likely that during this transitional period cells are removed from a surface. If this is the

case, then a method which would cause the cells to align prior to implantation would be

advantageous.

It has long been known that cells respond to the shape of the substrate on which

they grow. [41]. In the past several decades, literature on cellular responses to

topography has expanded rapidly. Researchers have investigated numerous combinations

of cell types and topographical geometries and dimensions as listed In Appendix A.

Confining the discussion to only the area of endothelial cells, Barbucci and Magnani

investigated the influence of the combination of topography and chemical patterning on

cell behavior [31, 51]. They observed that endothelial cells increasingly align themselves

to ridges as the topographical spacing is reduced from 100 to 10 |tm. Similar results

were found by Wilkerson during the study of endothelial cell growth on ridges ranging in

spacing from 20 to 5 [m [52]. Additionally, Feinberg has shown that cells confluence is

disrupted by topographies will profile heights greater than 1 .im and that focal adhesions

form almost exclusively on fibronectin (FN) regions of FN-patterned

polydimethylsiloxane elastomer (PDMSe) [54]. Additionally, he showed that the area of









individual focal adhesions does not vary with surface treatment and is approximately

2 m2.

This work proposes that microscale topographies can be used to orient the

cytoskeletal components of endothelial cells. In the following studies, microscale

channel and topographies were generated in PDMSe and gelatin. The height of the

topographical features was maintained at -1 [im so as not to disrupt endothelial cell

spreading. Because this height was significantly shorter than for Sharklet AFTM (3 .im)

which was introduced in Chapter 3, the new name of Sharklet CETM was developed to

indicate it is cell enhancing. The topographies of primary interest had lateral dimensions

of 3 rm so that focal adhesions could be supported. Additionally, the height of the

primary topographical features was maintained at -1 .im so as not to disrupt endothelial

cell spreading. Porcine endothelial cells were cultured on the topographies and cell

density, confluence, density and spreading were examined. Additionally the ability of

topographies to align focal adhesions and nuclei was investigated

Materials and Methods

Engineered Topographies

Silicon wafer masters were prepared using standard photolithography techniques

(processing performed by James Schumacher). Both channel and Sharklet CETM patterns

were included with feature heights of -1 im for most topographies. Negatives of these

topographies were generated in the silicon wafers, so that positives (channels are defined

by ridges protruding out of the surface) would be formed once a material is cast against

them. For example, a channel in the wafer becomes a ridge in the replicating material.

For convenience, the following naming scheme was developed by the Brennan group:

[depth]_[topography type]_[width]x[spacing]









All feature dimensions are given in micrometers. The depth was identified as '-' if the

features were etched below the surrounding planar surface as in the case of the silicon

wafer master and "+" if the features were raised above the surrounding material as in the

initial replicating material (Fig. 5-1). The topography type was classified as either "CH"

for channels or "SK" for Sharklet CETM. Although, the target lateral dimensions were

2x2 for most of the topographies in the following studies, the true dimensions for some of

the topographies were closer to 3x1 (see Results section). For the purpose of naming

topographies, the actual dimensions were used. As an example, +1_CHlx3 refers to

positive channel features that are 1 .im tall, 1 .im wide and 3 .im spaced. The names and

descriptions of all topographies used are given in Table 5-1.

Two-dimensional representations were first created using AutoCAD and then

electronically transferred in chrome onto quartz optical photomasks using e-bream

lithography. Clean silicon wafers were then coated with positive photoresist via spin

coating. Two techniques for generating topography were then used: deep reactive ion

etching (DRIE) and photoresist (PR) exposure. In the PR process, first the photoresist

layer was exposed to UV light long enough to fully penetrate the photoresist. In areas of

the mask where no chrome is present, the UV light is transmitted and chemically alters

the photoresist to make it more soluble. Then wafers were exposed to a developer

solution to remove all regions exposed to UV. In this manner, the pattern was

reproduced in the photoresist.

In the DRIE process, wafers were then exposed to reactive ion etching so that the

features are transferred into the underlying silicon. Etched wafers were cleaned via a

piranha etch (50:1 of H2S4 andH202) at 1200C for 10 minutes followed by subsequent









rinsing in acetone and ethanol prior to each replication with PDMSe. Clean wafers were

treated with hexamethyldisilazane (HMDS) to prevent adhesion by generating unreactive

methyl groups on the surface. The HMDS was applied by vapor deposition under

28 inHg (95 kPa) vacuum.

PDMSe Mold Preparation

Smooth and patterned PDMSe wells were formed using Dow Corning

Corporation's Silastic T-2 as previously described in Chapter 4.

Preparation of Gelatin Films

Gelatin derived from bovine calf skin was supplied as a dry powder (Sigma).

Genipin crosslinked (10 w/w%) gelatin films were prepared as previously described in

Chapter 4. Briefly, the appropriate amount of genipin was dissolved in nanopure water

and heated to 500C. Then gelatin was added to create a 10 wt/v% aqueous solution. The

mixture was then cast into the PDMSe mold (smooth or with topography) and allowed to

react for 24 h at room temperature. In order to minimize the amount of residual

unreacted GEN in the films, samples were immersed in nanopure water for 3 days at

room temperature. The nanopure water was exchanged every 24 h.

Prior to cell seeding, all samples were sterilized in 70% ethanol and rinsed 3 times

in phosphate buffered saline (PBS). They were then equilibrated in PBS at 370C in an

incubator for 4 h.

Preparation of PDMSe Films

PDMSe films were cast directly against silicon wafers. The base resin and curing

agent were mixed (10:1 by weight), degassed and poured over top of the silicon wafer.

Curing was carried out at room temperature for 24 h in a glass mold with spacers to

generate -1 mm thick films.









Characterization of Topographically Modified PDMSe and Gelatin Films

Dimensions of PDMSe and gelatin topographies were analyzed using scanning

electron microscopy (SEM) and white light interference profilometry (WLIP).

Fibronectin Adsorption to Samples

Lyophilized bovine plasma fibronectin (Biomedical Technologies, Inc.) was

dissolved (1 mg/mL) in 0.22 [m filtered water at 370C for 45 minutes as described by the

manufacturer. The solution was then diluted to 50 [g/mL in PBS. Samples were first

sterilized in 70% ethanol for 30 minutes. Samples were rinsed 3 times with PBS and

placed into the bottoms of the wells of culture plates. Gelatin samples were held in place

with PDMSe washers placed above the samples. Enough fibronectin solution was added

to just cover the surfaces (same volume for each sample) and allowed to react for 30

minutes (preliminary assay) or 1 h (all other studies) at room temperature. Samples were

rinsed 3 times with PBS prior to cell seeding.

Cell Culture, Imaging and Processing

Porcine vascular endothelial cells (PVECs) were supplied by Dr. Edward Block

(Veteran's Administration Hospital, Gainesville, FL) between passages 2 and 3. Cells

were previously harvested from the pulmonary artery of 6 to 9 month old pigs [85, 91].

Cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum

and 1% antibiotic-antimycotic solution. They were incubated at 370C and 5% CO2.

Unless used immediately, cells were passage every 3-4 days when they were -90%

confluent. Cells were not typically used beyond passage 5. Prior to seeding cells on test

surfaces, they were detached from the culture flasks by incubation with 1 mL of 0.05%

trypsin for 10 minutes. Then 2 mL of media was added to stop the enzymatic reaction of









the trypsin. Cell concentration was measured using a hemocytometer and 1% crystal

violet stain. Then the solution was diluted to achieve the desired seeding density.

Preliminary assay

The preliminary cell culture assay was carried out for 4 days using 12-well culture

plates and two replicates of each sample described in Table 5-2. Cells (passage 6) were

seeded at a density of 3.5 x 104 cells/mL with 1 mL being applied to each sample. Due to

a lack of availability, cells could not be obtained between passages 2 and 5 at the time of

the preliminary study. For the samples indicated, fibronectin treatment was carried out

for 45 minutes.

On day 4, cells were washed twice with PBS and stained with 0.1% Mayer's

hematoxylin (Sigma) for 8 minutes. They were then washed twice with water and stained

with Eosin Y (Sigma) for approximately 30s. Cells were then washed twice with 95%

ethanol. Cells were covered in PBS until imaged (less than 4 h). Preliminary cultures

were imaged on a Zeiss Axioplan 2 microscope at 400X magnification. PDMSe and

gelatin discs were removed from the well plates prior to imaging. A needle was used to

minimize bending of the sample substrates. When necessary, the backside of the samples

(the side without cells) was rinsed with isopropanol to remove residual H&E stains. Cell

densities (cells/mm2) were determined manually for each image.

Immunofluorescent assay

The immunofluorescence assay was carried out until confluence was reached on

controls (4 days) using 6-well culture plates. Cells (passage 5) were seeded at a density

of 1.0 x 104 cells/mL and 2.5 mL per well. Two replicates of each sample described in

Table 5-3 were included. All samples were fibronectin treated for 1 h.









On day 4, cells were rinsed twice with PBS and then fixed in 4% formaldehyde for

5 minutes. Then permeability of the cell membranes was increased using 0.3% Triton X-

100 (prepared with PBS) for 5 minutes. In order to stain for focal contact adhesions, cells

were treated with mouse anti-vinculin primary antibody (1:400 in PBS) for 1 h at 37C.

Cells were then rinsed 5 times in PBS and treated with goat anti-mouse conjugated to

Alexa Fluor 488 (Molecular Probes, 1:400 in PBS)) for 1.5 h at 37C. After 5 rinses

with PBS, cells were treated with 5 gm phalloidin-TRITC for 12 h at 37C in order to

stain the actin cytoskeleton. Before placement in the incubator, 4 gL DAPI per mL PBS

was added to stain the cell nuclei. Cells were then rinsed 5 times with PBS and covered

in PBS until being imaged.

Fluorescent imaging was carried out using a Zeiss Axioplan 2 microscope with

epifluorescence and digital capture system. Each sample was imaged at 2 locations each

for 200X and 400X magnifications. At each location, 4 separate images were acquired.

The first used white light and captured the topographical pattern. The remaining 3 used

UV light though appropriate filters to capture the nuclei (DAPI), actin (TRITC) and focal

adhesions (Alexa-Fluor 488) separately.

Images obtained for the fluorescent cell culture assay were processed using ImageJ

software to create composite images as well as determine cell density, cell area and

orientation of focal contact adhesions Cell density and orientation of nuclei were

calculated from images of DAPI at 200X magnification. The image was first rotated so

that the underlying topography (based on corresponding optical image) was oriented

vertically. Then the image was converted to an 8-bit black and white image and ellipses

were fitted to the cell nuclei using the "Analyze Particles" feature (Fig 5-2). ImageJ









generated a result file which includes a count of the ellipses, lengths of major and minor

axes, and angle formed between major axis and horizontal reference line. Cell density

was calculated by dividing the number of ellipses by the area of the field of view (0.095

mm2). Elongation of nuclei was found by dividing the length of the major axis by the

length of the minor axis. Orientation angles were found by subtracting the output angle

by 900, the angle formed between the topography and horizontal reference line. In this

manner, orientation angles ranged from -90 to +900. A negative sign in the orientation

angle indicates that the nuclei are offset in the counterclockwise direction as opposed to

the clockwise direction. The nuclear orientation within a given image was determined

using Hermans orientation function. Hermans parameter is typically used to describe the

degree of orientation of fibers within a composite. It is calculated from the following

equation:

f = 2 < cos2 0 >-1. (5-1)

In Equation 5-1, is the trigonometric average. Hermans parameter (f) ranges

from -1 (perpendicular orientation to topography) to +1 (parallel orientation to

topography). A value of zero indicates random orientation.

Cell area, elongation and orientation were determined from composite images of

actin (Phalloidin-TRITC) and nuclei (DAPI) at 400X magnification (Fig 5-3A).

Individual cells were traced in green (Fig 5-3B) and then these lines were separated from

the image using the "RGB split" feature. The green image was converted to black and

white and the interior of the cells filled with black (Fig 5-3C). The "Analyze Particles"

feature was used and ellipses were fitted to the cells. ImageJ generated result files

containing the area of each cell (based on number of pixels and not an elliptical fit) the









lengths of the major and minor axes, and the angles formed between the major axes and a

horizontal reference line. Elongation and orientation of cells were calculated using the

same methods as for nuclei.

Focal contact adhesions were analyzed from images of vinculin at 400X

magnification (Fig. 5-4A). Images were converted to black and white (Fig. 5-4B) and

then the "Analyze Particles" feature was used and ellipses were fitted to each focal

adhesion (Fig. 5-4C). Due to inherent noise in the vinculin imaging, it was necessary to

only include particles of the appropriate size (20 to 80 pixels) and shape (major axis at

least 25% longer than minor axis). Hermans orientation of the focal adhesions is

calculated using the same methods as for nuclei.

Cell culture assay 3

The third cell culture assay was carried out for 4 days using 6-well culture plates.

Cells (passage 5) were seeded at a density of 1.0 x 104 cells/mL and 2.5 mL per well.

Two replicates of each sample listed in Table 5-3 were included. All samples were

fibronectin treated for 1 h.

On day 4, cells were rinsed twice with PBS and then fixed in 4% formaldehyde for

5 minutes. Cells were then rinsed thee times with PBS before being stained with 1%

crystal violet for 2 minutes and rinsed with distilled water until water remained clear.

Cells were immediately imaged using a Zeiss Axioplan 2 microscope at 50X and 200X.

Five images per sample were taken at each magnification.

Cultures were imaged on a Zeiss Axioplan 2 microscope at 200X magnification. A

manual count of cells in each image was made and densities (cells/mm2) for each image

reported. Images were then processed using ImageJ software to determine cell coverage

area (i.e. confluence). The brightness and contrast were first adjusted and then the image









was converted to an 8-bit black and white image. Under "Set Measurements" only "area"

and "area fraction" were selected and then the "Analyze Particles" tool was used to find

the area fraction (ratio of black pixels to total pixels) for all cell groups. The average area

per cell for each image was then calculated by multiplying the area fraction by the area of

the field of view (0.38 mm2) and then dividing by the number of cells.

Cell culture assay 4

The fourth cell culture assay was carried out until confluence was achieved on

controls (6 days) using 6-well culture plates. Cells (passage 5) were seeded at a density

of 1.0 x 104 cells/mL and 2.5 mL per well. Two replicates of each sample listed in Table

5-4 were included. All samples were fibronectin treated for 1 h.

On day 6, cells were rinsed twice with PBS and then fixed in 4% formaldehyde for

5 minutes. Cells were then rinsed thee times with PBS before being stained with 1%

crystal violet for 2 minutes and rinsed with distilled water until water remained clear.

Cells were immediately imaged using a Zeiss Axioplan 2 microscope at 200X and 400X.

Thee images per sample were taken at each magnification.

Cultures were imaged on a Zeiss Axioplan 2 microscope at 200X and 400X

magnifications. Cell confluence was determined from the 200X images using ImageJ

software. The brightness and contrast were first adjusted and then the image was

converted to an 8-bit black and white image. Under "Set Measurements" only "area" and

"area fraction" were selected and then the "Analyze Particles" tool was used to find the

area fraction (ratio of black pixels to total pixels) for all cell groups.

Cell density, area, elongation and orientation were determined from 400X images.

A manual count of cells in each image was made and densities (cells/mm2) for each

image reported. Coverage area was determined as mentioned above and the average area









per cell for each image was then calculated by multiplying the area fraction by the area of

the field of view (0.092 mm2) and then dividing by the number of cells. In order to

determine cell elongation and orientation, 12 cells from each image were traced and filled

in black using ImageJ software. Cells were selected randomly with the only criteria

being that their full outline could be observed. ImageJ software was then used to fit

ellipses to the black-colored cells. Elongation was measured as the length of the major

axis to minor axis of the cell. Orientations were measured according to Hermans

orientation parameter as outlined in the immunofluorescence assay above.

Statistical Methods

All results are reported as mean values +2 standard errors. One way analysis of

variance (ANOVA, a = 0.05) was performed for each dataset to determine if any

statistical differences exist among the groups. Pair-wise t-tests (a = 0.05) were

performed as appropriate to determine which groups were statistically different.

Results

Characterization of Topographically Modified PDMSe and Gelatin

PDMSe samples replicated both the PR and DRIE wafers with high fidelity.

Differences in fidelity between PR and DRIE replicates were evaluated using SEM

images (Fig. 5-5). The DRIE process led to thinner ridges (wider channels) that more

closely matched the target dimensions. WLIP was used to determine the dimensions of

the features (Figs. 5-6 though 5-8). The profilometry data verified that the lateral

dimensions of the topographies varied based on the wafer processing type, but there was

not a significant difference in feature height (Table 5-5).









Preliminary Cell Culture Assay

As expected, PVECs did not grow on unmodified PDMSe substrates, but grew to

confluence on fibronectin-adsorbed PDMSe (Fig. 5-9). Additionally, PVECS grew on all

topographically modified PDMSe surfaces. Cells appeared slightly more elongated on

the +1_SK_2x2 and +3_SK_2x2 topographies compared to smooth and channel-modified

PDMSe. Cell densities did not vary significantly among smooth PDMSe and

topographically modified PDMSe surfaces pretreated with fibronectin (Fig. 5-10).

Cells were not observed on gelatin surfaces. This may be due at least in part to the

opacity of the gelatin samples. Because light could not be transmitted though the sample,

the Zeiss Axioplan 2 microscope could not be used and instead samples were viewed

using a Nikon scope lit though the objective. Even in this configuration, no cells were

observed.

Immunofluorescent Cell Culture Assay

PVECs grew to confluence on smooth PS and all PDMSe substrates (Fig. 5-11).

Cell densities were similar, ranging between 450 and 650 cells/mm2 for all surfaces tested

(Fig. 5-12). The +1_SK_3xl (PR) patterned PDMSe yielded a significantly higher

density of cells compared to all other surfaces except the +1_CH_2x2 (DRIE) patterned

PDMSe. No significant differences existed among the remaining groups.

The average surface area of each cell showed greater variability than cell density

(Fig. 5-13). Cells tended to be largest on the smooth PS and PDMSe surfaces with

average areas of -2000 and -1800 [im2 respectively. The +1_SK_3xl (PR) pattern

resulted in significantly smaller cells (-1300 itm2) compared to all other surfaces.

Additionally, cells on the +1_CHlx3 (PR) pattern were significantly smaller (-1500

inm2) than those on the PS controls. Cells on all surfaces tended to be elongated by -80%









(Fig 5-14). No significant differences in elongation of cells were detected between

smooth and topographically modified samples, although the orientation of the elongation

appeared more random on the smooth surfaces.

The topographies were found to enhance alignment of cells, nuclei, and focal

adhesions parallel to topographical features (Figs. 5-15, 5-16 and 5-17). Orientation of

these elements were essentially random on smooth polystyrene and PDMSe surfaces, as

the Hermans orientation parameters did not significantly differ from zero. Nuclei became

partially aligned parallel to the topographical features as indicated by an increase in

Hermans parameter. Although Hermans parameter for the +1_CHlx3 (PR) topography

was significantly different from zero, it was not significantly different from f for smooth

PDMSe. The Sharklet CETM topographies generated the highest degree of orientation

(f-0.3) and orientation on these two surfaces were significantly different from smooth

PS, smooth PDMSe and the +1_CHlx3 (PR) topography.

Alignment of cells followed the same trend as the orientation of nuclei. All four

topographies significantly enhanced orientation relative to smooth PS and PDMSe. The

greatest degree of cell orientation was observed on the +1_SK_2x2 (DRIE) topography

which yielded a Hermans orientation of -0.45. Despite a trend for the Sharklet CETM

topographies to increase orientation of cells relative to channel topographies, no

significant differences were observed.

Analysis of focal contact adhesions indicated that all surfaces promote alignment

with the long axis of the underlying topography. Histograms of focal adhesion angles on

PS and smooth PDMSe surfaces indicated an even distribution across all angles, whereas

histograms for topographically modified PDMSe surfaces indicated a peak at or near the









topography angle (Fig. 5-18). Orientation was significantly enhanced on all four

topographies relative to smooth PS and PDMSe surfaces. The greatest degree of

orientation was observed on the two Sharklet CETM topographies. Hermans orientation

parameters for these two were -0.44. Although orientations on channels topographies

were not significantly different from that of the Sharklet CETM topographies, Hermans

parameter tended to be lower at a value of -0.31 for each. This is consistent with the

trends observed for cell bodies and nuclei.

Cell Culture Assay 3

Cells did not grow to confluence during the four culture days of assay 3 (Fig. 5-19)

in which fibronectin treatment was increased to 1 hour from 30 minutes in the

preliminary assay. Cell densities were highest on the two Sharklet CETM surfaces and

lowest on the two controls (PS and smooth PDMSe). All of the topographically modified

PDMSe surfaces induced significantly greater cell densities than the smooth PDMSe

control (Fig. 5-20). Additionally, all topographies with the exception of the +1_CHlx3

(PR) pattern yielded a greater density of cells than on the PS control. The +1_CH_2x2

(DRIE) pattern provided the greatest density of cells, significantly greater than on all

other surfaces except the +1_SK_3xl (PR) pattern. Cells were significantly more dense

on the +1_SK_3xl (PR) pattern than the remaining surfaces except +1_CH_2x2 (DRIE)

pattern.

Although the densities of cells on the Sharklet CETM topographies were greatest,

their degree of confluence did not vary significantly from the PS and smooth PDMSe

controls (Fig. 5-21). Additionally, these did not vary significantly with the confluence on









the +1_CH_2x2 (DRIE) surface. The +1_CHlx3 (PR) pattern, however, resulted in

significantly less surface coverage than all other surface types.

Cells tended to spread less (smaller area) on the topographically modified surfaces

than on the smooth PS and PDMSe controls (Fig. 5-22). The +1_CHlx3 (PR) pattern

did not vary significantly from the PS control, however. Cell areas did not vary

significantly among the four different patterns, although the two Sharklet CETM

topographies had the lowest mean call area values.

Cell Culture Assay 4

Cells proliferated and were nearly confluent on all surfaces (Fig. 5-23). Cells on

the smooth polystyrene and PDMSe controls varied in shape and had no apparent long

range orientation. Most cells were more elongated than typical endothelial cells, and

their appearance may be evidence of a mixed cell population. It is possible that either

fibroblast or smooth muscle cells contaminated the primary culture.

In contrast to the cells on the smooth controls, cells appeared to orient with the long

axis of features for all topographies studied. The topographies increased cell density

relative to the smooth polystyrene and PDMSe controls (Fig. 5-24). This affect was

greatest on the -3_CH_5x5 (DRIE) surface and became less apparent as the spacing of

the channels increased to 10 and 20 im. There was no significant difference in cell

density among the two Sharklet CETM topographies and these led to 55 and 22%

increased in cell density compared to the smooth polystyrene and PDMSe controls

respectively.

Quantitative analysis of the images indicated that the cells were most confluent on

the +1_CH_2x2 (DRIE) topography (Fig. 5-25). Confluence was not statistically

different on this surface compared to smooth polystyrene, smooth PDMSe, +1_CH_2x2









(DRIE) and -3_CH_5x20 (DRIE). Between 95 and 97% of these surfaces were covered

with cells. Increasing the depth of the Sharklet CETM topography led to a small but

significant reduction in confluence as the surface coverage dropped from 97 to 93%. The

-3_CH_5x5 (DRIE) topography resulted in the most disrupted confluence with only 89%

of the surface are covered.

As was observed in the earlier assays, the topographies tended to reduce the

average area occupied per cell to a value closer to that observed in vivo (Fig. 5-26). The

largest cells (-1000 im2) were observed on tissue culture polystyrene and the smallest

cells (-500 rm2) were grown on the -3_CH_5x20 (DRIE) topography. Increasing the

spacing between 5 irm wide channels to 20 irm led the cell area to approach that for

smooth PDMSe (-800 im2). The areas of cells on +1_CH_2x2 (DRIE), +1_SK_2x2

(DRIE), and +3_SK_2x2 (DRIE) were similar at -600 im2.

Cells elongated and oriented with features for all topographies studied. The affect

of topography was more significant in this assay than any of the previous. The possibility

of a mixed cell population might account for the difference among the assays. Cell

elongation was similar on the +1_CH_2x2 (DRIE), +1_SK_2x2 (DRIE), +3_SK_2x2

(DRIE) and -3_CH_5x5 (DRIE) topographies which had mean values ranging from 3.6 to

4.0 (Fig. 5-27). For the 5 im wide channels, elongation decreased with increased spacing

between channels. Cell elongation on smooth polystyrene and PDMSe controls were not

significantly different and were 1.7 and 1.8, respectively.

Hermans orientation parameter ranged from 0.94 to 0.98 for cells on all

topographies with the +1_CH_2x2 (DRIE) topography resulting in the largest mean value

(Fig. 5-28). It is unclear what caused the dramatic results. An older supply of fibronectin









was used in this study, but the increased alignment may have more to do with the

biological variability in the cell source. Hermans orientation parameter for the smooth

controls was not significantly different from zero, indicating random alignment of cells.

Discussion

Endothelial cells are not able to thive on the GEN crosslinked gelatin samples.

This is most likely due to cytotoxic effects of residual unreacted GEN. This might be

overcome with a more robust wash process, possibly involving longer leach times and

immersion in PBS at 370C to more closely simulate the cell culture conditions.

When combined with fibronectin treated PDMSe, all four topographies with 2 rm

lateral dimensions and 1 [m heights support endothelial cell growth. The subtle

differences in topography generated by the photoresist and deep reactive ion etch

processes did not result in significant differences in cellular response. The only

exception was a slight increase in cell density and decrease in average cell area observed

for the +1_SK_3xl (PR) topography relative to its DRIE counterpart during the

immunofluorescence assay. This difference was not observed in either the preliminary

assay or assay 3, suggesting it may have been an outlying occurrence.

The microscale topographies tend to increase endothelial cell density relative to

smooth FN-treated PDMSe, although the differences were not always significant for all

assays. Additionally, cells on these topographies tend to spread across a smaller area and

more closely approach the size observed in porcine arteries. Cell spreading, nuclei and

focal adhesions are found to orient with the underlying topographies. In comparison to

the channel topography of the same dimensions, the Sharklet CETM topography tends to

increase endothelial cell density and orientation of cytoskeletal components.









An increase in height of the Sharklet CETM topography from 1 to 3 [m results in a

slight (-3%) but significant decrease in the confluence of endothelial cells. Cell density

and orientation are not significantly affected by the change in height. Cellular

elongation and density decreased significantly as spacing increased from 5 to 20 [im on 3

lm deep channels, This is consistent with results observed by Wilkerson et al [52, 62].

Although cellular orientation on this topography is similar to that on +1_CH_2x2 (DRIE)

and +1_SK_2x2 (DRIE) topographies, cell confluence was decreased significantly (-6%)

indicating it is not a good candidate for cell seeding.

It is important to note that all of the PDMSe topographies were treated with

fibronectin prior to cell culture. It is unclear what if any affect the protein adsorption had

on the shape of the topography. It is conceivable that the protein may have filled in the

topography to some degree so that the cells were presented with a somewhat smoother

surface than what is indicated by the reported feature dimensions. If this work is carried

forward in the future, the fibronectin adsorption to the topographies should be

characterized. This might be accomplished with the use of immunofluorescently labeled

fibronectin. A confocal microscope could be used to analyze the dimensions of the

hydrated, labeled protein layer. Alternatively, the samples could be freeze dried after

fluorescent labeling to lock in the structure. Then a microtome could be used to section

the samples so that the Zeiss microscope with epifluorescence could be used to determine

the thickness of the fibronectin layer in relation to the underlying topography.

Conclusion

Fibronectin treated PDMSe is a better substrate for culturing endothelial cells than

fibronectin treated genipin crosslinked gelatin. In the PDMSe substrate, the +1_CH_2x2

(DRIE) and +1_SK_2x2 (DRIE) topographies are effective at orienting cytoskeletal









components while maintaining cellular confluence. These topographies will be evaluated

for their ability to improve endothelial cell retention to shear in Chapter 6.


Figure 5-1. Example of convention used for naming topographies. A) Positive (+)
features are raised above the plane of the surrounding material and B)
negative (-) features are formed below the plane of the surrounding material.


S ~ B

0 0

rsP 41
04* **


I~I
~ 00
*r


Figure 5-2. Processing of DAPI images to measure cell density and nuclear orientation.
A) Image of nuclei on +1_SK_2x2 (DRIE) at 200X. B) Conversion to black
and white. C) Ellipses fitted to nuclei and counted. D) Magnification of
ellipses to demonstrate measurement of orientation angle and elongation.
Blue arrows indicate topography direction. Scale bars represent 50 rnm.

































Figure 5-3. Processing of images to measure cell area, elongation and orientation. A)
Portion of overlaid mage of nuclei (DAPI) and actin (phalloidin-TRITC for
+1_CH_2x2 (DRIE) at 400X. B) Cells outlined in green. C) Cells filled with
black for area calculation. D) Ellipses fitted to cells for measurement of
orientation angle and elongation. Blue arrows indicate topography direction.
Scale bars represent 25 jim.














































Figure 5-4. Processing of Alexa Fuor 488 images to measure alignment of focal
adhesions. A) Image of focal adhesion (400X). B) Image converted to black
and white. C) Image of ellipses fitted to adhesions and measured for
alignment. Scale bars represent 25 pm.


C

p
a,


Figure 5-5. SEM images of PDMSe replicates of silicon wafers patterned by different
processing methods. A) Photoresist. B) Deep reactive ion etching. Target
dimensions of the channel topographies were 2 pm wide, 2 pm spaced and 1
pm depth. Scale bars represent 10 nm.


' ^ ;^' "~ *"* :. \ ~ ^ ^ ...' *


.:.'


*~ ~ ':IqL r, .



P F S.

*i^ 1- > ."^ ^ 'f;b I \ ':
it'' '


*; : 1-.. 1 V
^^* t:' \ -^ '-. *'. .


0 c,


'B 'I

o ~ ~ % LI 0a
fto'
ft LIa~~

II 0 C

C, 00 P
I)~~ LIa a


i






E




xr
1
a












A B48
350 3W


Aim


-3 100
so .100



-250
-500
--462 -840


C r 3 D -_.

-200 -




-400 .1

-600
-713 -41
Figure 5-6. WLIP images of topographies formed by the DRIE process (100X). A)-
1_CH_2x2 silicon wafer. B) +1_CH_2x2 PDMSe replicate of (A). C) -
1 SK 2x2 silicon wafer. D) +1_SK_2x2 PDMSe replicate of (C). Images
are of 48x76 mn2 sample areas.


rm
A 20 B
150




I11 IE, M
250
-50sa
-450
-567


26


00




00


rm
49M

-360

200

5so

-100

-250

1,43


Figure 5-7. WLIP images of PDMSe topographies formed by the photoresist process
(100X). A) +1_CHlx3 PR. B) +1_SK_3xl PR. Images were taken at 100X
magnification and are of 48x76 [m sample areas.









rwn rnm
A K75 B M
F em F 65


200
0
-50

- -2004 -


Figure 5-8. WLIP images of gelatin channels (100X). Gelatin topographies were
replicated from the -1CH3xl DRIE silicon wafer via a PDMSe intermediate.
A) Dehydrated gelatin. B) Hydrated gelatin. Images are of 48x76 tm2
sample areas.


811111~












































-i









Figure 5-9. PVECs grown on PDMSe topographies in the preliminary assay. A) Smooth
without fibronectin. B) Smooth. C) +1_CHlx3 (PR). D) +1_CH_2x2
(DRIE). E) +1_SK_3xl (PR). F) +1_SK_2x2 (DRIE). Fibronectin was
adsorbed to all surfaces unless otherwise indicated. Topography directions are
indicated by arrows. Images were captured at 400X. Scale bars represent
25 rnm.









400
350
E 300 -
.L 250
o- 200
150

o 100
50 -
0
+1 SK SM +1CH +1CH +1 SK SM-
2x2 1x3 2x2 3x1 no FN

Figure 5-10. Density of PVECs on PDMSe topographies in the preliminary assay. All
samples were pretreated with fibronectin (FN) unless stated otherwise. Data was
obtained though manual count of images at 400X magnification. Groups of statistically
indistinct means are indicated by tie bars (t-test, a = 0.05).












































SFigure 5-11. Fluorescent images of PVECs grown on A) TCP, B) smooth PDMSe and
C-F) topographically modified PDMSe. All PDMSe samples were pretreated
with adsorbed fibronectin. The light microscope views at the left of images C
though F show the underlying topography to the fluorescently labeled PVECs
to the right of the images. Images were captured at 400X magnification and
scale bars represent 25 nm.









800


E 600


400


0 200 -


0
+1 CH PS +1 SK SM +1 CH +1 SK
1x3 2x2 2x2 3x1

Figure 5-12. Density of PVECs on topographies in the fluorescent assay. Cells were grown on
fibronectin-adsorbed PDMSe surfaces and PS. Data was obtained though processing
of fluorescent DAPI images at 200X magnification. Tie bars connect groups with
means that are not statistically different (t-test, a = 0.05).


2500

2000
E
S1500

1000

500

0
PS +1CH +1SK SM +1CH +1SK
2x2 2x2 1x3 3x1

Figure 5-13. Mean cell area for PVECs on topographies in the fluorescent assay. Cells were
grown on fibronectin-adsorbed PDMSe surfaces and TCP. Values were generated
though the processing of overlaid images of cell nuclei (DAPI) and actin (phalloidin-
TRITC). Tie bars connect groups with means that are not statistically different (t-test,
a = 0.05).






















1.0


SM +1 CH
1x3


+1 CH
2x2


+1 SK
3x1


+1 SK
2x2


Figure 5-14. PVEC elongation on topographies in the fluorescent assay. Cells were grown on
fibronectin-adsorbed PDMSe surfaces and TCP. Tie bar connects group with means
that are not statistically different (a = 0.05).


0.8

0.6


0.4

0.2

0.0

-0.2

-0.4


SM +1CH +1CH +1SK +1SK
1x3 2x2 3x1 2x2


Figure 5-15. PVEC orientation on topographies in the fluorescent assay. Cells were grown on
fibronectin-adsorbed PDMSe surfaces and PS. Smooth PS and PDMSe do not
significantly alter orientation away from random (Parameter=0). All topographies
significantly increase orientation. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05)












E
S0.3


0.2


C

0.2

I
-0.3
PS SM +1 CH +1 CH +1SK +1SK
1x3 2x2 3x1 2x2


Figure 5-16. Orientation of PVEC nuclei on topographies in the fluorescent assay. Cells were
grown on fibronectin-adsorbed PDMSe surfaces and PS. Smooth PS and PDMSe do
not significantly alter orientation away from random (Orientation Parameter=0). All
topographies significantly increase orientation relative to smooth PS. Tie bars
connect groups with means that are not statistically different (t-test, a = 0.05).



S0.8
a,
E


S0.4 -
0.6


S0.0 -
CU


0.0. -

S-0.2
PS SM +1 CH +1 CH +1 SK +1 SK
1x3 2x2 3x1 2x2

Figure 5-17. PVEC focal adhesion orientation on topographies in the fluorescent assay. Cells
were grown on fibronectin-adsorbed PDMSe surfaces as well as PS. Error bars
indicate 2 standard errors. Orientation on smooth PS and PDMSe surfaces are not
significantly different from random (parameter=0). All topographies significantly
increase orientation. Tie bars connect groups with means that are not statistically
different (t-test, a = 0.05)







































F 18%
15%

> 12%
C
, 9%

_ 6%

3%

0%
Alignment Index


Figure 5-18. Histograms of alignment indices for focal adhesions on A) TCP, B) smooth
PDMSe, C) +1_CHlx3 (PR) PDMSe, D) +1_CH_2x2 (DRIE) PDMSe, E)
+1_SK_3xl (PR) PDMSe, and F) +1_SK_2x2 (DRIE) PDMSe. An alignment index
of zero indicated the adhesion is perfectly aligned with the topography, while indices
of-1 and 1 indicate adhesions are off-angle from topography by 90 o clockwise and
900 counter-clockwise respectively.


C 18%0
15%
> 12%

Alignment Index


3%
0%
-1 Alignment Index 1


















































Figure 5-19. Light microscope images of PVECs grown on topographies for Assay 3. A) PS, B)
smooth PDMSe, C) +1_CHlx3 (PR) PDMSe, D) +1_CH_2x2 (DRIE) PDMSe, E)
+1_SK_3xl (PR) PDMSe, and F) +1_SK_2x2 (DRIE) PDMSe. All surfaces were
pretreated with 50 pg/mL fibronectin for 1 h. Topographies are oriented from left to
right as indicated by arrows. Scale bars represent 50 rm.









500

400

300

200

100

0


PS +1CH +1CH +1SK +1SK
1x3 2x2 3x1 2x2


Figure 5-20. PVEC density on topographies in Assay 3. All surfaces were pretreated with 50
[g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).


SM +1 SK +1 SK
2x2 3x1


+1 CH +1 CH
2x2 1x3


Figure 5-21. PVEC coverage on topographies in Assay 3. All surfaces were pretreated with
50 [g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).









1200

1000

800

2 600
0-
( 400 -

200

0
SM PS +1CH +1CH +1 SK +1 SK
1x3 2x2 3x1 2x2

Figure 5-22. PVEC area on topographies in Assay 3. All surfaces were pretreated with 50
[g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).





















































Figure 5-23. Light microscope images of PVECs grown on topographies for assay 4. A) TCP.
B) smooth PDMSe. C) +1_SK_2x2 PDMSe. D) +3_SK_2x2 PDMSe. E)
+1_CH_2x2 PDMSe. F) -3_CH_5x5 PDMSe. G) -3_CH_5x10 PDMSe. H) -
3_CH_5x20 PDMSe. All topographies were replicated from deep-reactive-ion-
etched wafers. All surfaces were pretreated with 50 [g/mL fibronectin for 1 h.
Topographies are oriented vertically in all images. Scale bars represent 100 im.



115









2500


2000

E
E- 1500


1000


500


0
PS SM -3 CH -3 CH +1 SK +3 SK +1 CH -3 CH
5x20 5x10 2x2 2x2 2x2 5x5


Figure 5-24. PVEC density on topographies in Assay 4. All surfaces were pretreated with 50
[g/mL fibronectin for 1 h. Tie bar connect group with means that are not statistically
different (t-test, a = 0.05).


100


+1 SK SM PS +1 CH -3CH +3SK -3CH
2x2 2x2 5x20 2x2 5x10


-3CH
5x5


Figure 5-25. PVEC confluence on topographies in Assay 4. All surfaces were pretreated with
50 [g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).









1200

1000

800

600

400


200

0
PS SM -3CH -3CH +1 SK +1 CH +3 SK -3 CH
5x20 5x10 2x2 2x2 2x2 5x5

Figure 5-26. PVEC spreading on topographies in Assay 4. All surfaces were pretreated with
50 [g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).



51


PS SM -3CH -3CH
5x10 5x20


+3 SK -3 CH
2x2 5x5


Figure 5-27. PVEC elongation on topographies in Assay 4. All surfaces were pretreated with
50 [g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).


+1 CH
2x2


+1 SK
2x2










1.00


L._
( 0.75
E

- 0.50
C-

" 0.25
a,_
O
m 0.00

r
aU -0.25
I


-0.50
SM PS -3CH -3CH +1CH-3CH +3SK+1SK
5x10 5x20 2x2 5x5 2x2 2x2


Figure 5-28. PVEC orientation on topographies in Assay 4. All surfaces were pretreated with
50 [g/mL fibronectin for 1 h. Tie bars connect groups with means that are not
statistically different (t-test, a = 0.05).


Table 5-1. Names of Topographies
Topography Description
1 [m wide channels formed between 3 [m wide ridges
that protrude out of the surface by 1 [m
3 [m wide and 1 [m spaced channels formed below the
surface to a depth of 1 [im
2 [m wide channels formed between 2 [m wide ridges
that protrude out of the surface by 1 [m
3 [m wide and 1 [m spaced ribs arranged in the Sharklet
AFTM pattern. Ribs protrude out of the surface by 1 [m
2 [m wide and 2 [m spaced ribs arranged in the Sharklet
AFTM pattern. Ribs protrude out of the surface by 1 [m
2 [m wide and 2 [m spaced ribs arranged in the Sharklet
AFTM pattern. Ribs protrude out of the surface by 3 [m
5 rm wide and 5 [m spaced channels formed below the
surface to a depth of 3 [im
5 rm wide and 10 rm spaced channels formed below the
surface to a depth of 3 [im
5 rm wide and 20 rm spaced channels formed below the
surface to a depth of 3 mrn


Name


+1 CH lx3

-1 CH 3xl

+1 CH 2x2

+1 SK 3xl

+1 SK 2x2

+3 SK 2x2

-3 CH 5x5

-3 CH 5x5

-3 CH 5x5









Table 5-2. Samples for Preliminary Assay
Materials Topography
Smooth
Smooth
PDMSe +1 CH 2x2(DRIE)
PDMSe -
+1_CH_3xl (PR)
+1_SK_2x2 (DRIE)
+1_SK_3xl (PR)


Fibronectin (Y/N)


# of Replicates


Genipin
Crosslinked
Gelatin


Smooth
Smooth
CH_2x2 (DRIE)
CH_2x2 (DRIE)


Table 5-3. Samples for Immunofluorescence Assay and Assay 3
Material Topography Fibronectin (Y/N) # of Replicates
TCP Smooth Y 2
Smooth Y 2
+1_CH_2x2 (DRIE) Y 2
PDMSe +1_CH_3xl (PR) Y 2
+1_SK_2x2 (DRIE) Y 2
+1_SK_3xl (PR) Y 2

Table 5-4. Samples for Assay 4
Material Topography Fibronectin (Y/N) # of Replicates
PS Smooth Y 2
Smooth Y 2
+1_CH_2x2 (DRIE) Y 2
+1_SK_2x2 (DRIE) Y 2
PDMSe +3_SK_2x2 (DRIE) Y 2
3_CH_5x5 (DRIE)
-3_CH_5x10 (DRIE) Y 2
-3 CH_5x20 (DRIE)









Table 5-5. Feature Dimensions of Topographies Determined by WLIP
Width Spacing Depth
Material Topography Name (in
(Itm) (Gtm) (Gtm)
+1_CH_3xl (PR) 2.8 0.1 1.2 0.1 0.66 0.02
+1 CH 2x2 (DRIE) 2.3 0.1 1.7 0.1 0.86 0.03
PDMSe -
+1_SK_3xl (PR) 2.5 + 0.1 1.5 + 0.1 0.70 + 0.02
+1_SK_2x2 (DRIE) 1.8 0.1 2.2 0.1 0.89 0.02
Hydrated Gelatin -1_CH_2x2 (DRIE) 2.4 0.1 1.6 0.1 0.55 0.01
Dehydrated Gelatin -1_CH_2x2 (DRIE) 2.7 0.1 1.3 0.1 0.77 0.02









CHAPTER 6
INFLUENCE OF TOPOGRAPHY ON SHEAR STABILITY OF ENDOTHELIAL CELLS

Introduction

The overall goal of this research was to investigate the potential for microscale

topographies to improve endothelial cell resistance to flow induced shear stresses. This would

be a significant advance in the development of a successful small diameter vascular graft. As

shown in Chapter 5, microscale topographies can be used to orient endothelial cells along with

their nuclei and focal adhesions. It is proposed that this will stabilize the cells against removal

by shear stresses applied parallel to the orientation direction.

Parallel plate flow chambers often have been used to evaluate cellular adhesion [71] as

well as flow induced changes to cell structure [119, 120] and physiological functions [121].

These chambers generate constant shear stresses across the test substrates as defined by the

following equation:


6r Q (6-1)
h2w

In this relationship Tw refers to the shear stress at the wall (ie. sample substrate), [t is the viscosity

of the fluid medium, Q is the volumetric flow rate, and h and w define the chamber height and

width respectively. A derivation of Equation 6-1 can be found in Appendix B.

In this study, porcine vascular endothelial cells were cultured on microscale topographies.

Both channels and the Sharklet CETM topography were produced in fibronectin-treated

polydimethylsiloxane (PDMSe) elastomer. For both microtopographies, the protruding features

(ridges and ribs) were 3 .im wide, 1 .im spaced and 1 .im tall. Porcine vascular endothelial cells

(PVECs) were cultured on these substrates and then exposed to a physiological shear stress

(2 Pa). Cellular retention and morphological changes were then evaluated.









Materials and Methods

Design of Parallel Plate Flow Chamber

The top and bottom plates of the chamber were generously supplied by Dr. Roger

Tran-Son-Tay's group. They are made of LexanTM polycarbonate and the upper plate contains

inlet and outlet ports (Fig. 6-1). The lower plate has an indentation and cut-out designed to fit a

1 inch by 3 inch glass slide. Preliminary tests of the flow chamber with a plain glass slide

proved that it has a tendency to leak. Consequently, the chamber design was modified to create a

better seal (Fig. 6-2). A glass slide was placed in the indentation to create a level surface and

then the height and width of the chamber were defined by the use of specially prepared PDMSe

gaskets.

The gaskets were prepared so that the chamber dimensions (height x width) were

0.4x17.0 mm for the preliminary study and 0.5x12.5 mm for the final study. A Harvard compact

infusion pump (Model 975) was used to provide continuous and steady flow. The pump held

two 60 mL syringes and these were connected though a Y-shaped adapter. The second highest

flow setting was used to deliver fluid at 60 mL/min. This arrangement resulted in shear stress of

2 Pa on the test substrate for both studies based on an approximate viscosity of PBS and media

(-1 cP = 0.001 Pa*s),

Production of PDMSe Topographies

Topographically modified PDMSe films were prepared by direct casting against etched

silicone wafers. Two topographies were included: 2 [m channels separated by 2 [m wide

ridges that are 1 [m tall (+1_CH 2x2) and the Sharklet CETM pattern with 2 [m wide and 2 [m

spaced ribs that are 1 .im tall (+1_SK_2x2). The topographically modified wafers were prepared

by James Schumacher using a 19 second DRIE cycle. Prior to replication with PDMSe, wafers

were rinsed with ethanol and then treated with hexamethyldizilazane (HMDS) via vapor phase









deposition for approximately 5 min. During this time, polyester sheets were taped to one side of

two clean glass plates. HMDS treated wafer were placed on top of one of the plates and 1 mm

thick spacers were placed at the covers of the plate. Silastic T2 was mixed (10:1 ratio of base

resin to curing agent), degassed and poured over the wafers. The top place was lowered onto the

PDMSe until it rested on the spacers. The PDMSe was allowed to cure for 24 h at room

temperature before being removed from the mold.

Preliminary Shear Study

Sample gasket preparation

A razor blade was used to cut 16 mm wide samples from each topographically modified

PDMSe film. The +1_CH_2x2 topography was 2 in long and the +1_SK_2x2 topography was 1

in long. The samples were cut to leave a 3 mm border at the ends of each length.

In order to prepare each sample gasket, transparency films were cut to create thee 17 mm

by 77 mm strips. Three strips were taped together using double sided tape (a total of two tape

layers) and then the laminate was taped to a polyester-sheet-covered glass plate. Next a

topographically modified PDMSe sample was suctioned pattern-side-down to the center of the

transparency laminate. Spacers (2 mm thick) were placed in the corners of the plate. PDMSe

was mixed, degassed and poured over top. A second polyester sheet covered glass plate was

then lowered on top until it rested on the spacers. The PDMSe was allowed to cure for 24 h at

room temperature and then the sample gasket was cut from the film.

Samples were sterilized by soaking in 70% ethanol for 1 h. Then the depression of the

sample gasket was treated with fibronectin (Biomedical Technologies, Inc.). It was supplied as a

1 mg/mL solution of 0.03 M Tris Cl buffer (pH 7.8) in 30% glycerol. It should have been diluted

to 50 tg/mL in phosphate buffered saline (PBS), but it was mistakenly diluted using nanopure

water. The indentation of each sample gasket was filled with 0.5 mL of the fibronectin solution









and it was allowed to adsorb for 1 h at room temperature. Samples were then rinsed 2 times each

with PBS and covered with PBS until cell seeding.

Cell culture

Porcine vascular endothelial cells were generously supplied by Dr. Block (Veteran's

Administration Hospital, Gainesville, FL). PDMSe sample gaskets were placed in the bottom of

150 mm diameter Petri dishes. Four samples of each type (smooth, +1_CH_2x2 and

+1_SK_2x2) were included. PVECs (passage 3) were diluted to 100,000 cells/mL in 10% FBS

supplemented RPMI media. Then the indentations of each sample gasket were filled with the

cell suspension (-0.5 mL per sample) to give a seeding density of 4,000 cells/cm2. Cells were

allowed to settle for 4 h and then more media was added so that the final level was

approximately 2mm above the gasket. Samples were incubated at 37C and 5% CO2 and cells

were allowed to grow for 7 days.

Shear treatment

Two samples of each type were exposed to a wall shear stress of 2 Pa using the parallel

plate flow chamber. Phosphate buffered saline (PBS, IX)) was used as the fluid medium and

flow was applied at 60 mL/min for 2 minutes which represents the longest time frame possible

for the 2 Pa wall shear stress on the substrates based on the syringe pump used.

Staining and imaging

Samples (both static and shear treated) were first fixed in 4% formaldehyde for 10 minutes.

They were subsequently rinsed 3 times with PBS and then stained with 1% crystal violet for 5

minutes. Slides were gently rinsed in water until water remained clear. Imaging was performed

using a Zeiss Axioplan 2 microscope at 400X magnifications. Eight images per sample were

taken at each magnification for a total of 16 images per surface treatment and shear combination.









Images were taken from the central 1 cm wide by 2.5 cm long area of the shear samples to ensure

fully developed flow had been applied. Cell density was measured by processing of the images

using ImageJ software.

Final Shear Study

Gasket preparation

Samples were prepared separately from the gasket for the final shear study. The change

was made so that smaller volumes of media would be needed during cell culture and so that all

samples could be seeded with the same suspension of cells. Spacers (2 mm thick) were placed in

the covers of a clean glass plate. PDMSe (50 g) was mixed, degassed and poured over top. A

second clean glass plate was then lowered on top until it rested on the spacers. The PDMSe was

allowed to cure for 24 h at room temperature and then the outline of the gasket was cut from the

film. A 12.7 mm (0.5 in) wide and 76.2 mm (3 in) long section was cut from the gasket center to

define the chamber dimensions. Note that all although the gasket was 2 mm thick, it compressed

to 1.6 mm thick when clamped into the flow chamber. As such, the gasket provided a chamber

height of 0.5 mm when combined with a 1.1 mm thick sample.

PDMSe culture well preparation

During a second preliminary study (not presented here) it was found that the PDMSe

samples tended to float in the culture media. This was problematic because the endothelial cells

settled on the Petri dish instead of the samples. To prevent this, a PDMSe culture well was

produced which provides better adhesion to the samples. To produce the culture well, two stacks

containing five 2 inch by 3 inch glass slides each were taped together using double-sided tape.

The two stacks were taped side-by-side to a clean glass plate to create a 4 inch by 3 inch mold.

Spacers (6 mm thick) were placed around the mold and then PDMSe (160 g) was mixed,

degassed and poured over top. A second glass plate was pressed above the mold until it rested









on the spacers. The PDMSe was allowed to cure for 24 h at room temperature and then it was

removed from the mold. Excess PDMSe was cut away, leaving -5 mm thick border around the 4

inch by 3 inch well. The well was then placed in a 15 mm diameter Petri dish to provide support.

Sample preparation

Samples for the final shear study were 12.7 mm (0.5 in) wide, 76.2 mm (3 in) long and 1.1

mm thick. Each sample contained regions of smooth, +1_CH_2x2 and +1_SK_2x2 (Fig. 6-3).

A razor blade was used to cut 25.4 mm wide and 12.7 mm long sections from each

topographically modified PDMSe film. For each sample, one section of each of the topographies

was suctioned patterned-side down to a clean glass plate with a half inch (12.7 mm) long gap

between them. A fresh batch of uncured PDMSe (30 g per samples) was then mixed, degassed

and poured over top. Spacers (1.1 mm thick) were used and a second glass plate was then

pressed over top. The PDMSe was allowed to cure for 24 h at room temperature and then the 1

inch by 3 inch samples were cut from the film.

Four samples were suctioned to the bottom of the PDMSE culture well and then the well

and samples were immersed in 70% ethanol for 1 h to sterilize. After that time, they were rinsed

thee times with PBS and then 20 mL of 50 [g/mL fibronectin was pipetted over the samples in

the well. The fibronectin solution was allowed to adsorb for 1 h and then the samples were

rinsed thee times with PBS just prior to cell seeding.

Cell culture

Porcine vascular endothelial cells (passage 2) were generously supplied by Dr. Block

(Veteran's Administration Hospital, Gainesville, FL). PVEC density was determined using a

hemocytometer and then the suspension was diluted to 30,000 cells/mL in 10% FBS

supplemented RPMI 1640 media. Twenty-five milliliters were placed above the samples in the









PDMSE culture well to give a seeding density of 10,000 cells/cm2. Samples were incubated at

37C and 5% CO2 and cells were allowed to grow for 3 days.

Shear treatment

A razor blade was used to cut each sample in half down its long axis. One half was

immediately fixed and stained as described below and the other half was exposed to 2 Pa of wall

shear stress using the parallel plate flow chamber. In this manner, four samples were shear tested

and four were not. The samples were suctioned into the indentation of the gasket using a

minimal amount of vacuum grease. Serum-free RPMI 1640 media was used as the fluid medium

and shear was applied for 2 minutes which represents the longest time frame possible for the 2

Pa wall shear stress based on the syringe pump used.

Staining and imaging

Samples (both static and shear treated) were first fixed in 4% formaldehyde for 10 minutes.

They were subsequently rinsed 3 times with PBS and then stained with 1% crystal violet for 5

min. Slides were gently rinsed in water until water remained clear. Imaging was performed

using a Zeiss Axioplan 2 microscope at 400X magnifications. Twelve images were taken per

surface treatment and shear combination. Images were taken from the central 1 cm wide and 38

mm long area to ensure fully developed flow had been applied. Cell density, elongation and

orientation were measured by processing the images using ImageJ software as described in

Chapter 5.

Statistical Methods

Two way analysis of variance (ANOVA, a = 0.05) was used to determine if any significant

differences exist among the topography and shear treatments. Mean values +2 standard errors

are reported.









Results


Preliminary Shear Study

Endothelial cells did not grow well for the preliminary shear study. The cells grew

sporadically across the samples, with areas of good cell growth surrounded by regions nearly

devoid of healthy cells (Fig. 6-4). The poor proliferation was attributed to the error in

fibronectin treatment mentioned in the description of methods. The protein was dissolved in

water rather than PBS which would have disrupted the conformation of the protein and disrupted

its ability to adsorb evenly to the PDMSe surfaces.

Because of the wide discrepancies in cell proliferation within a sample, the variability in

cell density was too great for any definitive conclusions to be drawn. In general, the density of

cells grown in static culture did not vary among the smooth and topographically modified

PDMSe samples (Fig. 6-5). The mean cell density on all samples was -300 cells/mm2. After

exposure to 2 Pa of shear stress on the test substrate, greater differences in mean cell density

were observed among the surfaces. Although statistical differences do not exist, the +1_SK_2x2

topography tended to improve cellular retention whereas the +1_CH_2x2 topography tended to

decrease cellular retention relative to smooth PDMSe.

Final Shear Study

The floor of the PDMSe sample well for the final shear study must not have been level.

After 3 days of cell culture, it was discovered that nearly all of the cells settled and proliferated

on one sample located against a far edge of the well. Only this sample was used in the analysis

of results. Cell density before exposure to flow on the other 3 samples was far too low

(<1 cell/mm2) to evaluate cell retention.

Cells were nearly confluent on the smooth and topographically modified sections of the

remaining sample prior to exposure to flow (Fig. 6-6). Cell growth was much more consistent









than observed in the preliminary study. Cell density did not significantly vary with surface

topography and the average cell density before flow among all surfaces was 520 60 cells/mm2

(Fig. 6-7).

The topographies induced morphological changes in the PVECs consistent with the results

of Chapter 5. Cell elongation increased from 2.3 0.3 on smooth fibronectin-treated PDMSe to

3.7 0.3 and 3.8 0.3 on the +!_SK_2x2 and +1_CH_2x2 topographies respectively (Fig. 6-8).

Orientation was also increased by the topographies. Hermans parameter for the smooth control

was not significantly different from zero, indicating random orientation (Fig. 6-9). The

+!_SK_2x2 and +1_CH_2x2 topographies induced orientation to a similar degree, with Hermans

parameter values of 0.90 + 0.04 and 0.92 0.01 respectively.

Cell density decreased significantly on all topographies after exposure to 2 Pa of wall

shear stress in the flow chamber for 2 minutes (Fig. 6-7). The average settlement density after

flow among all surfaces was 220 40 cells/mm2 and it did not significantly vary with

topography. Likewise, cell retention was statistically equivalent on all surfaces with 43 20%

of cells retained (Fig. 6-10).

Closer inspection of the after flow images revealed that PVECs on both the +1_SK_2x2

and +1_CH_2x2 topographies appeared to maintain better spreading than cells on smooth

fibronectin-treated PDMSe (Fig. 6-6). The cells on the topographically modified sections were

spread relatively evenly across the surface, whereas the cells on the smooth section tended to be

isolated to small, dense clusters. Evaluation of mean cell areas before and after flow confirmed

these results (Fig. 6-11). Cell spreading on smooth fibronectin-treated PDMSe was reduced

from 1,100 100 [im2 to 470 + 100 [im2. Alternatively, PVEC spreading on both topographies









was -1000 im2 and did not significantly change after exposure to 2 Pa of shear stress on the test

substrate.

Discussion

In the preliminary shear study, the +1_SK_2x2 topography tended to improve cell

retention after exposure to shear whereas the +1_CH_2x2 topography tended to decrease it.

Initially these results seemed promising and were consistent with the trend seen with algae

spores (Ulva) for similar topographies with 3 rm feature heights [122]. Unfortunately, no

definitive conclusions regarding cell retention can be drawn from the preliminary study because

the PVECs response was too variable due to inadequate pretreatment of the PDMSe surfaces

with fibronectin. The results of this study clearly demonstrate that surface chemistry is more

important that topography when attempting to support endothelial cell growth.

Unfortunately, the trends in endothelial cell retention suggested by the preliminary study

could not be confirmed. The final shear study indicates cell retention is not significantly

improved by either topography, at least not in terms of cell density. Alternatively, both the

+I_SK_2x2 and +1_CH_2x2 topographies enhance the ability of PVECs to maintain cell

spreading during exposure to flow. This is consistent with the hypothesis outlined in Chapter 1

which states that cells are removed when exposed to flow because they must break focal

adhesions to adapt their morphology. These results suggest that endothelial cell retention might

be improved by further optimization of material chemistry and topographical dimensions.

The discussion from Chapter 5 on the possible infilling of the topographical recesses with

fibronectin needs to be revisited here. It is unclear what if any affect the protein adsorption had

on the shape of the topography. It is conceivable that the protein may have filled in the

topography to some degree so that the cells were presented with a smoother surface than what is

indicated by the reported feature dimensions.









In the most extreme scenario of infilling, fibronectin may be wicked away from the ridge

tops and completely fill the topographical recesses. In such a case, the resulting surface is

smooth but still has the potential to direct cellular growth based on patterned chemical and

mechanical (due to phase contrast) cues. The fibronectin in the recesses would be thicker than a

monolayer and therefore more susceptible to being partially desorbed by flowing media. This

would help explain why so many cells were removed from the topographically modified surfaces

despite the fact that the PVECs were aligned in the flow direction. As such, combining the

topographies with a different material or switching to two-dimensional chemical patterns might

yield better results.

Conclusion

This study investigated endothelial cell adhesion to topographies with 1 .im tall features

having 2 [im lateral dimensions. Such topographies incorporated into fibronectin-treated

PDMSe help PVECs maintain cell spreading but do not improve cell retention after exposure to

2 Pa of shear stress on the substrate. The adsorbed fibronectin layer should be characterized in

order to gain further insight into the nature of contact guidance observed in this study.

Endothelial cell retention may be improved by combining the topographies with a different

material or by switching to two-dimensional chemical patterns.








Outlet Flow


Polycarbonate


PDMSe Gasket-


Polycarbonate-


r r
.I11111111111111111111111111111111111


e-


rb


..... Cell-seeded
Sample
Yei


Figure 6-1. Original design of flow chamber.


Outlet Flow


Polycarbonate .......


PDMSe Sample-o


Polycarbonate


Figure 6-2. Modified design of flow chamber.


.................................................................. A O


Inlet Flow


Glass
support


Inlet Flow


















3 in
(76.2 mm)


1 in
(25.4 mm)
SL



+SK 2x2


0.75 in
(38.1 mm)

0.5 in
(12.7 mm)
0.5 in
(12.7 mm)
0.5 in
(12.7 mm)

0.75 in
(38.1 mm)


Figure 6-3. Layout of samples for the final shear study. White regions depict smooth areas of
the sample.











Smooth +1 SK 2x2 +1 CH 2x2




1












.I
-" =























Figure 6-4. PVECs before exposure to flow in the preliminary shear study. Images were taken
at 400X. Thee images (low, middle and high density) are given per surface type to
indicate the high variability observed. Scale bars represent 100 gm









Preliminary Shear Study


500 U Before Flow
E After Flow
E 400

300

S200

100

0
SM +1 SK 2x2 +1 CH 2x2

Figure 6-5. Density of PVECs before and after flow in the preliminary shear study. Smooth and
textured PDMSe samples were fibronectin treated. Cells were grown for 7 days and
then exposed to 2 Pa wall shear stress for 2 min. There are no significant differences
between any of the treatments (ANOVA, a = 0.05).








Before Flow


c _.-' *SLI -" *



SMK d

i.dll'










+ 1 -_











+1
CH -
2x2 -





Figure 6-6. PVECs grown on topographies in the final shear study. Cells were grown on
smooth, +1 _SK_2x2 and +ICH_2x2 fibronectin treated PDMSe. Images were
taken at 400X before and after exposure to flow. Topographical features are
oriented horizontally in the images. Scale bars represent 100 gm


After Flow










* Before Flow
* After Flow


+1 SK 2x2


+1 CH 2x2


Figure 6-7. Density of PVECs on topographies before and after flow in the final shear study.
Cells were grown on smooth and topographically modified PDMSe for 3 days and
then exposed to 2 Pa wall shear stress for 2 min. Asterisks denote statistically
indistinct groups (t-test, a = 0.05).


5


0


+1 SK 2x2


+1 CH 2x2


Figure 6-8. Elongation of PVECs on topographies before flow in the final shear study. Cells
were grown on smooth and topographically modified PDMSe for 3 days. Tie bars
denote statistically indistinct groups (t-test, a = 0.05).


800

700


600
E
E 500
()
S400

S300

200

100

0


SM


























+1 SK2x2


+1 CH 2x2


Figure 6-9. Orientation of PVECs on topographies before flow in the final shear study. Cells
were grown on smooth and topographically modified PDMSe for 3 days. Tie bars
denote statistically indistinct groups (t-test, a = 0.05).


100
90
80
70
60
50
40
30
20
10
0


+1 SK2x2


+1 CH 2x2


Figure 6-10. Retention of PVECs based on topography in the final shear study. Cells were
grown on smooth and topographically modified PDMSe for 3 days and then exposed
to 2 Pa wall shear stress for 2 min. Retention does not vary significantly among the
surfaces (t-test, a = 0.05).


0.8

0.6

0.4

0.2

0.0

-0.2

-0.4


SM


SM









1600
Before Flow D After Flow
1400

E 1200 -** **

1000

S800 -

600

400 -

200

0
SM +1 SK 2x2 +1 CH 2x2

Figure 6-11. Average area for PVECs on topographies before and after flow in the final shear
study. Cells were grown on smooth and topographically modified PDMSe for 3 days
and then exposed to 2 Pa wall shear stress for 2 min. Asterisks denote statistically
indistinct groups (t-test, a = 0.05).









CHAPTER 7
CONCLUSIONS AND FUTURE WORK

Conclusions

The ability to improve endothelial cell retention to biomaterial surfaces would be a

significant advancement in the search for a suitable synthetic small diameter vascular graft. This

work is the first known attempt to evaluate the influence of engineered microscale topographies

on endothelial cell retention during exposure to flow. The engineered microtopographies were

designed to orient endothelial cells in static culture with the goal of increasing their resistance to

shear.

Engineered topographies significantly influence both wettability and biological adhesion to

biomaterials. The topographies were developed though insights gained from classical wetting

theories. It was shown that the water contact angle of hydrophobic PDMSe can be increased

from 108 4 to 135 3 though properly scaled topographies. Channel topographies with

widths ranging from 5 to 20 tm and a depth of 5 tm increase alignment of PVECs relative to

smooth PDMSe. Endothelial cells settle within the channels and do not migrate over ridges,

preventing confluence from being reached.

Based on this earlier work with cell adhesion to fibronectin-treated PDMSe

microtopographies, it was decided that smaller topographical features would be necessary to

achieve confluence. A feature width and spacing of 2 [im was selected based on the size of focal

adhesions in endothelial cells. Additionally, gelatin was investigated for its ability to be

micropatterned and maintain shape. Because gelatin dissolves readily in water under

physiological temperatures, two crosslinking systems were investigated for their ability to

stabilize the film. Genipin and glutaraldehyde were found to be equally efficient at improving

modulus, but genipin is far better at decreasing the swelling of gelatin in water. The results









indicate that 10% (w/w) genipin in gelatin (2.9% MEQ) is the most efficient choice for

stabilizing the films. It was shown that 2 .im features were sufficiently replicated in gelatin films

of this composition.

Porcine vascular endothelial cells (PVECs) were cultured on genipin crosslinked gelatin

and PDMSe substrates. Unfortunately, the cells would not grow on the gelatin substrates even

after extending leaching of genipin into water and fibronectin adsorption to the gelatin surface.

It is suspected that the toxicity of residual unreacted genipin is too high for the cells to remain

viable. Cells grew to confluence on smooth and micropatterned PDMSe samples with adsorbed

fibronectin and the topographies resulted in a significant increase in focal adhesion and nuclear

alignment. Additionally PVECs on some of the topographies spread so that the average area

occupied per cell more closely approximated that of endothelial cells found in vivo.

Endothelial cell adhesion to topographies with 1 .im tall features having 2 .im lateral

dimensions was investigated in shear studies. Such topographies incorporated into fibronectin-

treated PDMSe help PVECs maintain cell spreading but do not improve cell retention after

exposure to flow generating 2 Pa shear stress on the test substrate. The adsorbed fibronectin

layer should be characterized in order to gain further insight into the nature of contact guidance

observed in this study. Endothelial cell retention may be improved by combining the

topographies with a different material or by switching to two-dimensional chemical patterns. The

ability to improved endothelial cell retention would be a great step forward in the development

of viable synthetic small diameter vascular grafts.

Future Work

Future directions of this project could include further examination of the alignment of

cytoskeletal components to microtopographies as well as the examination of the physiological

state of the endothelial cells as a function of the topography. In particular, the alignment of actin









filaments with flow has been discussed in the literature and it would be a nice advance if this

could be evaluated on the microtopographies presented here as well. This was one of the goals

of the fluorescent cell culture assay in Chapter 5, but the resolution of the images was not high

enough to achieve this. It would be recommended that a higher aperture objective be used to

improve this so that individual actin filament bundles could be distinguished from each other.

Even though cells grew to confluence on the textured substrates that does not necessarily

indicate that they are performing normal biological functions. Endothelial cells are responsible

for maintaining the homeostasis of the vasculature. Endothelial cells accomplish this though the

release and expression of factors which affect coagulation state, cellular proliferation and

leukocyte trafficking. One indicator of endothelial dysfunction is the impairment of endothelial

nitrous oxide formation which modulates vessel tone [123]. This results in stiffening of the

vessel wall which is associated with atherosclerosis. Additionally, it has been shown that

endothelial production of nitrous oxide modulates platelet adhesion which is a hallmark of

inflammation [124]. Release of nitrous oxide into the culture media by cells grown on the

textured substrates should be examined as an indicator of the physiological state of the cells.

Another indication of endothelial dysfunction is the induction of adhesive glycoproteins on

the cell surface. During inflammation, activated endothelial cells present selection molecules to

their surfaces which bind with lectin molecules on leukocytes in the blood. For instance, E-

selectin on endothelial cells (an inducible glycoprotein receptor) binds with Sialyl Lewis X of

various leukocytes. As demonstrated by Feinberg, atomic force microscopy (AFM) could be

used to probe the presence of E-selectin on endothelial cells cultured on the microtopographies

though the use of a tip modified with Sialyl Lewis X [54]. Additionally, leukocytes isolated









from blood could be exposed to endothelial cells cultured on the microtopographies within a

parallel plate flow chamber to examine their interaction under controlled shear.

It would be interesting to also investigate whether the engineered topographies can be used

to sort out robust cells from a population of cultured endothelial cells. Among any given cell

population, a distribution of cell viabilities exists. As shown in Chapter 6, the endothelial cells

that remain on the engineered topographies maintain spreading better than the cells remaining on

the smooth control. The cells that remain may have survived because of an inherent biological

advantage. Dr. Mark Segal (Department of Nephrology, University of Florida) suggests that it

would be beneficial to re-culture the remaining cells to confluence and then expose to flow again

to determine if shear resistance is improved in the second generation.

In addition to the proposed studies on actin alignment and endothelial functions, there is

also a need for further research into producing a microtextured hydrogel capable of supporting

endothelial cell growth. This would be especially important in the development of a tissue

engineered vascular graft because the supporting membrane would need to allow the transport of

water and nutrients. Genipin crosslinked gelatin, the hydrogel examined here, was capable of

replicating microscale topographic features with adequate mechanical stability but endothelial

cells did not proliferate on it in vitro. This might be able to be accomplished though a better

process of leaching unreacted genipin from the gelatin. Genipin is more soluble in ethanol than

water and so an extended (several days) soak in 70% ethanol may be sufficient. Gelatin would

remain swelled while unreacted genipin should diffuse to the surrounding ethanol. Additionally,

extended soaks under in vitro conditions (RPMI 1640 media at 370C) may also work. If work is

continued with this hydrogel, it would also be beneficial to examine its pore structure by

capturing scanning electron microscope (SEM) images of lyophilized samples.









The ability to improve endothelial resistance to shear removal while maintaining normal

cellular function would be a significant advance toward engineering an effective small diameter

vascular graft.











APPENDIX A
SUMMARY OF LITERATURE ON CELLULAR RESPONSES TO TOPOGRAPHY


Table A. Chronological Listing of Literature on Cellular Responses to Topography
Year Author(s) Material(s) Topography Cell Types(s) Results


1911 Harrison [125]


spider webs


spider webs and
1914 Harrison [41] spider webs mad
clotted plasma


Ohara and polystyrene
Black [126] epoxy


Wilkinson et al.
[127]


serum-coated glass


Brunette et al. titanium-coated
1983 28] silicon
[128] silicon


1986 Brunette [129]


Dunn and
Brown [130]







1986 Brunette [131]


titanium coated
silicon
epoxy replicates and
photoresist


quartz


titanium coated
silicon
epoxy replicates and
photoresist


cylindrical





cylindrical


channels 2 and 10 gm
wide with 5 to 30 gm
repeat spacing

depth not indicated

channels 2 gm wide
and 2 gm deep


channels (V shaped) -
70, 130 and 165 gm
wide with 80, 140 and
175 gm repeat
spacing


channels (square and V-
shaped) 0.5 to 60 gm
depth and 30 to 220
gm repeat spacing


channels 1.65 to 8.96
gm wide with 3.0 to
32.0 gm repeat
spacing and 0.69 gm
depth


major channels (square
and V-shaped)- 5 to
120 gm depth (width
not indicated)
minor channels 2 gm
deep on floor at 54 to
major channels


frog embryonic
neuronal cells

mesenchymal stem
cells from sea
urchin embryo
frog embryonic
neuronal cells
chicken embryonic
neuronal cells

chick heart
fibroblasts
murine epithelial
cells


human neutrophil
leukocytes


human gingivival
cells
porcine epithelial
cells


porcine periodontal
ligament
epithelial cells


chick heart
fibroblasts






human gingivival
fibroblasts


spindle shape cells
long projections align
with fibers



cells aligned to the
fibers



percent aligned cells
increased with
decreasing spacing
cells bridged 2 and
20 gm channels
cells were more
likely to migrate
along channels than
across them
cells aligned to long
axis of channels
epithelial cells didn't
bend around ridges
cells migrated along
channels
cells aligned to
channels
orientation increased
with decreased
spacing
some cells crossed
ridges and
extended into
channels
migration directed by
channels
deep depth enhanced
guidance of cells
alignment depended
greater on ridge
width than spacing
alignment increased
with decreasing
ridge width
cells aligned on
channels and flat
ridges
cells oriented
preferentially with
major channels
cells oriented with
minor channels if
no major channels











Table A. Continued
Year Authors Material(s) Topography Cell Types(s) Results


7 Clark et al.
[132]









Hoch et al.
1987 [133]













Chehroudi et al.
19 [133]


1988 Wood [134]


1989 Campbell et al.
[43]


Perspex










polystyrene


epoxy


quartz


steps 1, 3, 5, 10 and 18
[m heights








ridges 0.5 to 100 gm
wide and 0.5 to 62 gm
spacing with 0.03 to 5
[m heights


channels (v shaped) 17
gm wide and 10 gm
deep with 22 [m wide
ridges


channels .98 to 4.01
gm wide and spaced
with 1.12 to 1.17 gm
depths


Versapor filters
PDMS coated filters


Pore diameters 0.4 to
3.6 im


BHK cells
chick embryonic
neural cells
chick heart
fibroblasts
rabbit neutrophils






Uromyces
appendiculatus
fungus










porcine periodontal
ligament
epithelial cells
rat parietal implant
model


mesenchymal
tissue cells


subcutaneously
implanted into
dogs


as step height
increased, cells
oriented more and
were less likely to
cross the step
rabbit neutrophils
crossed 5 gm steps
twice as often as
other cell types
differentiation
maximized on 0.5
gm tall ridges
no differentiation on
ridges shorter than
0.25 gm or taller
than 1.0 gm
germ tubes highly
oriented on ridges
spaced 0.5 to 6.7
gm
channels increased
epithelial
attachment and
orientation
shorter length
epithelial
attachment and
longer connective
tissue attachment to
channel sections of
implant compared
to smooth parts
channels impeded
epithelial down
growth
cells migrated along
channels
highest alignment
seen on widest
repeat spacing
nonadherent,
contracting
capsules around
implants with pore
diameters < 0.5 gm
thin, tightly adhered
capsules on
implants with pores
from 1.4 to 1.9 gm
pores > 3.3 gm
infiltrated by
inflammatory tissue
little variation with
respect to surface
chemistry












Table A. Continued
Year Authors Material(s) Topography Cell Types(s) Results


19 Chehroudi et al.
[135]

















0 Clark et al.
[136]


Schmidt and
1991 von Recum
[44]


1 Clark et al.
[136]


titanium-coated
epoxy


Perspex


silicone


Quartz with poly-1-
lysine adsorption


channels (square and V-
shaped)- 30 [m repeat
spacing with 3,10 or
22 [m depth; 7 and 39
[m repeat spacing
with 3 or 20 [m depth


rat parietal implant
model


channels 4-24 [m
repeat with equal BHK and MDCK
width and spacing and
0.2 to 1.9 mm depths


dimples 2, 5 and 8 [m
wide/spaced; 0.38 to
0.46 [m high


channels -130 nm
wide and -130 nm
spaced at depths of
100, 210 and 400 nm


murine peritoneal
macrophages
in vivo study in
rabbits


BHK
MDCK
chick embryonic
neurons


endothelial cells
attached to smooth
and 3,10 [m
channels
endothelial cells
bridged 22 [m
channels
fibroblasts
encapsulated
smooth and 3,10
[m channels
fibroblasts inserted
into 22 Im
channels
epithelial down
growth greatest on
smooth and
channels oriented
parallel to implant
length
epithelial down
growth least on 10
and 22 [m
channels
perpendicular to
implant length
alignment inversely
proportional to
spacing and
directly related to
depth
alignment influence
by depth more than
spacing
2 and 5 [m textures
yielded less
mononuclear cells
and thinner fibrous
capsules
cells on 2 and 5 [m
textures were more
elongated and
contained more
pseudopods
BHK alignment
increased with
depth
single MDCK
aligned perfectly
on all and
elongation
increased with
depth
epithelial islands of
MDCK and all
embryonic neurons
unaffected by the
topography











Table A Continued
Year Authors Material(s) Topography Cell Types(s) Results


Clark et al.
1993
[137]






1993 Meyle et al.
[138]





1993 Meyle et al.
[139]






Oakley and
1993 Brunette
[140]





1994 Meyle [141]


Webb et al.
5 [142]


199 Chesmel and
Black [143]


Chou et al.
[144]


quartz treated with
hydrophobic silane
and laminin


epoxy





epoxy


chemical stripes -
2,3,6,12 and 25 [m
wide with equal
spacing and 2 gm
stripes spaced by 50


channels (square) 0.5
gm width and sapce
with 1 gm depth



channels (square) 0.5
gm width and space
with 1 Lm depth


titanium-coated
silicon




PDMS treated by
radio frequency
glow discharge


chrome-plated quartz
coated with poly-1-
lysine


polystyrene


titanium-coated
silicon treated with
radio frequency
glow discharge


channels (V-shaped) -
15 gm wide and
spaced with 3 gm
depth



channels (square) 1
gm wide, 4 gm
spaced and 1 gm deep


channels 0.13 to 4.01
gm wide and 0.13 to 8
gm spaced with 0.1 to
1.17 gm depths


channels radial arrays
of 5 gm long channels
at 1 intervals;, 0.5
[m wide and 0.5 or 5
gm deep

channels (V shaped) 3
gm wide with 6 to 10
rm repeat spacing


chick embryo
neurons
murine dorsal root
ganglia neurons





human gingivival
fibroblasts




human gingivival
fibroblasts






human gingivival
fibroblasts





human gingivival
fibroblasts



rat optic nerve
oligodendrocytes
(ONOs)
optic nerve
astrocytes
(ONAs)
hippocampal and
cerebellar
neurons (HCNs)


rat calavarial cells


human gingivival
fibroblasts


depth not indicated


alignment decreased
with decreased
width
growth cones bridged
narrow non
adhesive stripes
neurite branching
reduced by 15 [m
stripes
cells aligned with
channels and either
bridged or
conformed to
features
cellular prostheses
did not extend into
channel covers
cytoskeletal elements
oriented along
channels
microtubules oriented
at bottom of
channels at 20min
actin first observed at
wall-ridge edges
after 40 to 60min
most cells had
aligned focal
contacts by 3hrs
cells and focal
contacts aligned
with channels
ONOs and ONAs
aligned with
channels
HCNs did not align
ONAs showed
extensive network
of actin stress
fibers while ONOs
did not
ONOs alignment
maximized when
channel width same
as axon diameter

multi-layer protein
adsorption
confluence in 4 days
ECM in 7 days

cells aligned and
secreted more
fibronectin on
channels
twice as much ECM
on channels











Table A. Continues
Year Authors Material(s) Topography Cell Types(s) Results


PDMS treated with
ultraviolet (UV)
den Braber et light and
al. [145] radiofrequency
glow discharge
(RFGD)


Meyle et al.
[146]







Oakley and
1995 Brunette
[147]


silicon dioxide







titanium coated
silicon treated with
radio frequency
glow discharge


channels (square) 2, 5
and 10 gm wide with
equal spaces and 0.5
gm depth


channels 0.5 [m wide
and spaced and 1 gm
deep






channels (V shaped) -
15 gm wide and
spaced and 3 [m deep


rat dermal
fibroblasts


human fibroblasts
gengivival
keratinocytes
neutrophils,
monocytes
macrophages





porcine epithelial
cells


2 and 5 [m channels
induced stronger
orientation than 10
[m channels
growth lower on UV
treated surface than
RFGD treated
surface
100% of fibroblasts
and 20% of
macrophages
aligned
no orientation of
keratinocytes or
meutrophils
cells oriented along
channels
actin filaments and
microtubules
oriented along wall
and ridges
cell alignment less
variable within
single cells than
clusters of cells


Wojciak-
1995 Stothard et al.
[148]








Wojciak-
1995 Stothard et al.
[149]


den Braber et
6 al. [98]





den Braber et
6 al. [150]


quartz


quartz


channels 0.5, 5, 10 and
25 gm wide with
equal spacing and 0.5
and 5.0 gm depths


channels 5, 10 and 25
gm widths and 0.5, 1,
2 and 5 gm depths;
spacing not indicated


silicon treated with
radio frequency
glow discharge



PDMS treated with
radio frequency
glow discharge


channels 2, 5 and 10
gm wide/spaced and
0.5 gm tall


channels 1 to 10 gm
wide and 1 to 10 gm
spaced with 0.45 and
1.00 gm depths


cells spread faster on
shallower channels
cells elongated faster
marine P388D1 on deeper channels
mr cells elongated more
macrophages on wider channels
channels increased F-
actin during
initially attachment


BHK cells


rat dermal
fibroblasts




rat dermal
fibroblasts


F-actin condensation
at discontinuities in
topography
condensation
typically at right
angles to channel
edge with 0.6 gm
periodicity
vinculin orientation
similar to actin
microtubules formed
after 30min
proliferation did not
vary with texture
alignment of cells
increased with
decreasing channel
width
cells oriented on
ridge <4 gm wide
channel width and
depth did not affect
cellular alignment











Table A Continued
Year Authors Material(s) Topography Cell Types(s) Results


Britland et al.
1996
[151]















Matsuda and
1996 Sugawara
[47]


quartz


channels 100, 50, 25,
12 and 5 gm repeats
with 0.1, 0.5, 1, 3, and
6 [m depths
microcontact printing
(gCP) on smooth, in
channels, on ridges
and perpendicular to
topography


photoreactive
poly(N,N-
dimethylacrylamide
-co-3-azidostyrene
on tissue culture PS


polyurethane coated
with gold and
1996 Mrksich et al. patterned with
[48] alkane thiols and
adsorbed
fibronectin


Wokciak-
1996 Stothard et al.
[152]


fused silica


1997 Rajnicek et al. quartz coated with
[153] poly-l-lysine


channels 130, 80, 60,
40 and 20 mm wide
spaced 20 gm apart


V-sahped channels 25
and 50 [m wide with
equal spacing; depth
not indicated


square channels 2 and
10 [m widths with 30
to 282 nm depths







channels (square) 1, 2
and 4 [m wide and 14
nm to 1.1 gm deep


BHK21 C13















bovine aortic
endothelial cells


bovine capillary
endothelial cells


P388D1
macrophages
rat peritoneal
macrophages





embryonic
Xenopus spinal
cord neurons
rat hippocampal
neurons


cells aligned
strongest to 25 [m
wide gCP and 5
[m wide, 6 [m
deep channels
stress fibers and
vinculin aligned
with gCP and
channels
cell alignment
enhanced on
parallel channels
and gCP
cells aligned to
adhesive tracks on
channels with
matched pitch
strength of cues
became more
matched when
channels became
narrower and
deeper
cells avoided
photoreactive
regions & aligned
in channels
alignment increased
with decreased
channel width
cells only attached to
fibronectin regions
cells attached to
channels or ridges,
whichever
possessed the
alkane thiol and
therefore adsorbed
fibronectin
cells activated and
spread along
channels
increased membrane
protrusions
increased F-actin and
vinculin along
edges of single
steps or channels
Xenopus neurites
grew parallel to all
channels
hippocampal neurons
grew perpendicular
to shallow, narrow
channels and
parallel to deep,
wide channels











Table A Continued
Year Authors Material(s) Topography Cell Types(s) Results


Chehroudi et al. epoxy coated with
1997 titanium
[154] titanium


1997 Rajnicek and
McCaig [155]


Chou et al.
[156]


den Braber et
1998 l. [99]
al. [99]


quartz
polystyrene

both were coated with
poly-l-lysine


titanium-coated
silicon


silicone


1998 van Kooten et PDMS
al. [157]


1999 Palmaz et al.
[49]


nitinol


channels (V-shaped) -
35-165 [m wide at
30,60 and 120 [m
depths
pits (V-shaped) 35 to
270 [m wide and 30,
60 and 120 mm depths


channels (square) 1, 2
and 4 [m wide and 14
nm to 1.1 [m deep


channels (V shaped) 6
to 10 [m repeat
spacing with 3 [m
depth


channels (square) 2, 5
and 10 pm wide and
spaced with 0.5 pm
depths




channels (square) 2, 5
and 10 pm wide with
4, 10 and 20 pm
spaces and 0.5 pm
depths



channels 1, 3, 15 and
22 pm


rat parietal bone
implant model


embryonic
Xenopus spinal
cord neurons
rat hippocampal
neurons


human gingivival
fibroblasts


rat dermal
fibroblasts







human skin
fibroblasts


human aortic
endothelial cells


mineralization only
on topographies
bone-like foci
decreased as
channel depth
increased
more mineralization
as channel depth
increased
bone-like foci orient
on channels
cell orientation
unaffected by
cytochalasin B
taxol and nocodazole
disrupted
hippocampal
microtubules but
did not affect
orientation
alignment of neurites
affected by some
calcium channel, G
protein, protein
kinase and protein
tyrosine kinase
inhibitors
cells oriented along
channels by 16 hrs
channels altered the
expression and
levels of adhered
matrix
metalloproteinase-2
mRNA
microfilaments and
vinculin aggregates
aligned with 2 [m
channels only
vinculin located
mainly on ridges
fibronectin and
vinculin located in
channels
topography slowed
cell entrance to s-
phase of cell cycle
cells proliferated less
on 10 [m channels
than on 2 and 5 [m
channels
increasing channel
sizes increased
migration rate
cells aligned and
elongated with
channels











Table A Continued
Year Authors Material(s) Topography Cell Types(s) Results


van Kooten and
1999 von Recum
[45]












19 Walboomers et
al. [158]


silicone coated with
fibronectin (FN)









polystyrene (PS)
titanium-coated
polystyrene (Ti-PS)
silicone
poly-L-lactic acid
(PLLA)


ridges 2, 5 and 10 gm
wide and spaced 4, 10
and 20 gm apart
respectively with 0.5
gm heights










channels 1, 2, 5 and
10 gm widths and
spaces with depths of
0.5 gm


human skin
fibroblasts
human umbilical
vein endothelial
cells











rat dermal
fibroblasts


-all samples were
plasma treated


19 Walboomers et
al. [46]





Deutsch et al.
2000
2000 [159]




2000 Pins et al. [160]


Petersen et al.
2002 [161]
[161]


polystyrene


silicone


gelatin
collagen-
glycosaminoglycan
co-precipitate


agarose gel


channels 1-20 gm
wide and 0.5-5.4 gm
deep


pillars (rounded) 10
gm wide and 10 to 50
gm spaced
channels 10 gm wide
and spaced
all were 5 gm deep

channels 40-200 gm
wide and 40 to 200
gm deep


pits (rectangular) -
35x5, 35x10,45x5,
25x5,25x10, 55x5,
15x5, 15x10 and 55x5
em

depths were 18 to 40
tmn


rat dermal
fibroblasts


rat myocytes





keratinocyte


avian chondrocytes


topography
influenced initial
focal adhesion size
and density and
initial FN
deposition
no difference in FN
networks by day 6
PS and PLLA
reproduced better
than silicone or Ti-
PS
cell proliferation
greater on PLLA
and silicone
surfaces
alignment on PS,
PLLA and silicone
surfaces increased
with decreased
channel width
cells aligned on all
Ti-PS channels
equally
greatest alignment on
1 tm PLLA
channels
orientation increased
with channel depth
cells follow contours
of shallow and
wide channels but
bridge narrow and
deep features
myocytes attached
four times as often
to pillars than to
smooth PDMSe
cells oriented on
channels
cells differentiated to
form analogs of
basal lamina with
invaginations
cells isolated in wells
more cells isolated
per well as
dimension
increased from 15
to 25 gm
cells not retained in
wells >35 inm;
shallower cells
retained fewer cells











Table A Continued
Year Authors Material(s) Topography Cell Types(s) Results


Barbucci et al. sulfonated hyaluronic
Barbucci et al.
2002 [31acid (HyalS) on
glass


2003 Dalby et al.
[162]


quartz


hyaluronic acid
(Hyal) and
Magnam et al. (Hyal) and
2003 Magnani et al. sulfonated
hyaluronic acid
(HyalS) on glass


3 Scheideler et al.
[163]


titanium coated
silicon (channels)
and epoxy
replicates (ridges)


silicon oxide
Teixeira et al. siln
2003 [164] deposited on silicon
wafers


Tan and
2004 Saltzman
[165]





004 Wang and Ho
[166]







Recknor et al.
2005 [
[167]


hydroxyapatite on
silicon






chitosan and gelatin








polystyrene


chemical stripes 10,
25, 50 and 100 gm
wide & spaced HyalS
ridges in HyalS -step
height 300 nm 1 gm
channels 12.5 gm
wide, 2.5 [m spaced
with 2 gm depth

chemical stripes 10,
25, 50 and 100
gm;wide and spaced
HyalS
ridges in HyalS -step
height of less than 300
nm to 1 gm


channels 1 to 20 gm
wide and 0.4 to 2 gm
deep
ridges 1-20 [m wide
and 0.44 to 2 gm tall



channels (width x
space)) 70x330,
250x550,400x800,
650x950, 850x1150,
and 1900x2100 nm
with depths of 0.6 or
0.15 gm


micro 4 gm wide and
deep ridges spaced 10
[m apart and 4 gm
wide and tall pillars
spaced 4 gm apart;
nano randomly rough
channels 10, 20, 30
and 50 [m wide and
10 or 50 gm spaced
with 10 [m depth
protein resisistant tri-
block copolymer
applied to ridges


channels 10 gm wide,
20 [m spaced and 3
gm deeofor astrocytes
and 16 gm wide, 13
gm spaced and 4 gm
deep for AHPCs


HGTFN
endothelial cells


immortalized
primary human
fibroblasts
mouse fibroblasts
(3T3)
human primary
fibroblasts
bovine aortic
endothelial cells
human aortic
endothelial cells



human foreskin
fibroblasts
keratinocytes







human coreal
epithelial cells






human osteoblast-
like Saos-2 and
MG-63 cell lines




human
microvascular
endothelial cells




rat type-
astrocytes
adult rat
hippocampal
progenitor cells
(AHPCs)


decreasing stripe
dimensions
increases cell
locomotion and
orientation
cells and nuclei
aligned within
channels


decreasing stripe
dimensions
increased cell
locomotion and
orientation;


fibroblasts aligned to
topography
keratinocytes did not
align on ridges 2 to
10 gm wide
adsorbed fibronectin
enhanced cell
spreading
cells aligned with
ridges
percent aligned cells
independent of
lateral dimensions
alignment increased
with channel depth
actin filaments and
focal adhesions
aligned

cells aligned parallel
to ridges on both
micro and
micro/nano
structured surfaces

cells restricted to
channels by tri-
block copolymer
cell spreading
decreased as
channel width
decreased
greater than 85%
alignment of
astrocytes seen on
lamin coated
channels
AHPCs adhered and
extended processes
axially along
channels











Table A Continued
Year Authors Material(s) Topography Cell Types(s) Results


2005 Uttayarat et al.
[50]


PDMSe


g a O gelatin crosslinked
Yang and Ou .
2005 [112] with glutaraldehyde
and bound to glass


6 Charest et al.
[168]


2006 Pins et al. [169]









2006 Yu et al. [170]











Teixeira et al.
2006 [171]
[171]


polyimide


glass treated with
organosilane and
printed with
fibronectin (FN),
fibrinogen (FG) and
bovine serum
albumin (BSA)





chitosan-collagen-
gelatin blend


silicon


channels 2.7 to 3.9 gm
wide and 3.3 to 5.5
gm spaced with
depths of 0.2, 0.5, 1.0
and 4.6 gm





ridges at least 5 gm
wide and at least 10
gm spaced with 1.5
gm height


channels 8 gm wide, 4
gm deep, 16 gm
spaced
chemical stripes 10
gm wide lanes of FN
spaced by 10, 20 or
100 gm wide lanes pf
PEG


chemical stripes 600
nm wide and either 10
or 40 gm spaced and
20 gm long





channels 200 and 500
gm wide and spaced
with 80 gm depth and
10, 20 and 50 gm
wide and spaced with
20 gm depth


channels (width x
space)) 70x330,
250x550,400x800,
650x950, 850x1150,
and 1900x2100 nm

600 nm depth for all
features


Bovine aortic
endothelial cells







human
mesenchymal
stem cells






MC3T3-E1
osteoblast-like
cells


human dermal
fibroblasts








human
mesenchymal
stem cells










primary human
corneal epithelial
cells


cell elongation and
alignment
increased with
channel depth
focal adhesions
formed in channels
for all topographies
except 4.6 gm deep
cell proliferation was
similar on all
substrates


cells selectively
adhered to gelatin
and away from
glass


cells aligned to both
topography and
chemical patterns
separately
when presented with
both, cells align to
topography rather
than chemistry

cells on FN and FG
patterns had greater
tendency to spread
across adjacent
structures than on
BSA patterns

cells on 200 gm
channels initially
adhered in channels
and later migrated
to ridges
cells oriented parallel
to the 200 gm
channels
topographies smaller
than cells hindered
proliferation
cell alignment
switched from
perpendicular to
parallel when pitch
increased from 400
to 4000nm
between 800 and
1600nm pitches
both parallel and
perpendicular
alignment is
observed









APPENDIX B
CALCULATION OF SHEAR IN PARALLEL PLATE FLOW

The following is the calculation of shear stress at the plate for pressure flow through two

parallel plates. The important chamber dimensions are the width (w) of the plats and height (h)

of the separation (Fig. B). The calculation assumes laminar, fully developed flow with h<
Start with the equation of continuity assuming constant density as shown below:


+ -- + =0. (B-l)
ax ay az

In this equation, vi refers to flow in the i-direction. Under fully developed flow, fluid

movement only occurs in the x direction which gives rise to the following:


v = v, = 0 and = 0. (B-2)
9y az

Substituting these into Equation B-l gives the following:

x = 0. (B-3)
9x

Now apply the Navier-Stokes equation for the x-component as given below where p refers

to pressure and g refers to the gravitational force:

(9a+vv+Vv+Vv gP (B-4)
at ox 9y 9-+xz 2x ay 2 ax

Assume steady-state flow so that the following holds true:


at =0. (B-5)


For the case of the chamber height being much smaller than the chamber width, flow does

not vary in the z-direction so that the following holds true:

=- = 0. (B-6)
az az2









Substituting this into Equation B-4 gives the following:


0= x + -px. (B-7)
ay 2 x

Because flow direction is perpendicular to gravitational force, gx=0 and Equation B-7

becomes:

2 .V (B-8)


Because velocity is zero in all directions except x (Equation B-2) and velocity in the x-

direction is independent ofx (Equation B-3), solution of Navier-Stokes equations for y and z

components proves the following:

= =0. (B-9)
9y 8z

Therefore, pressure is only a function ofx (or a constant). Equations B-3, B-5 and B-6

indicate that Vx is only a function ofy (or a constant). In order for Equation B-8 to be true, the

derivative of P with respect to x must be equal to a constant as shown below:

= C, (B-10)
ox
a2 ap
S = C2. (B-11)
2y /2 x

Integrating Equation B-11 once gives

v -= x + C3. (B-12)
oy 'U Ox

Application of the following boundary condition based on symmetry in the y-direction to

Equation B-12









= 0 (B-13)


gives the equation below:

S=-1 )y (B-14)
cy 'U Cax

Integration of Equation B-14 results in the following:


vX= yf + 3. (B-15)


Apply the boundary condition that velocity at the plate (y=0.5h) is zero to Equation B-15

to get the equation below:


C2>JLY2 y- 21 (B-16)


Based on symmetry, the maximum velocity occurs at y=0 which gives the following:

1 @phi2
a V I.h2- (B-17)
xmax 2, cx > 2)

and


vx vxax 1- 2 (B-18)


Shear stress of a Newtonian fluid is given by the following:

ryX = -/ (B-19)
ay

Substitution of Equation B-18 into Equation B-19 and derivation gives the equation below:

y = 8pvx (B-20)

Shear stress at the plate becomes the following:









x = 4 /vxmax (B-21)
y Y05h h

The flowrate (Q) through the parallel plate chamber is the average velocity ()

multiplied by the cross-sectional area as shown below:

Q = (v)hw. (B-22)

The average velocity can be calculated by integrating Equation B-18 and dividing by the

chamber height as shown in the following equation:


05h V 2y
-0 5h hxmax h
v) = 3 2max. (B-23)
h 3

Substitution of Equation B-23 into Equation B-22 gives the following:


Q =2 V xa .hw (B-24)

Rearranging yields the equation below

3Q
Vxmax 2hw (B-25)
2hw

Substitution into Equation B-21 gives the final equation of shear stress in terms of the

volumetric flowrate and chamber dimensions as shown below:


T Y 6/Q (B-26)
y= 5h h2w











S z




x

' Flow
Direction


Figure B. Diagram of flow between parallel plates.









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BIOGRAPHICAL SKETCH

Michelle Carman, the daughter of Donna Fulford and Robert Carman, was born December

8, 1977 at Shands Hospital at the University of Florida where her father was attending dental

school. Michelle was born 10 weeks premature and spent the first month of her life under the

skillful watch of the NICU. She spent most of her childhood in Ocala, Florida where she

graduated salutatorian of Forest High School in 1996. From there, she returned to the University

of Florida and received her Bachelor of Science degree in chemical engineering in 2000 before

choosing to remain at UF to study biomedical engineering in graduate school.





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ENGINEERED MICROTOPOGRAPHIES TO INDUCE IN VITRO ENDOTHELIAL CELL MORPHOLOGIES STABLE TO SHEAR By MICHELLE LEE CARMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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Michelle Lee Carman 2

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To my family for their loving support. 3

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ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Tony Brennan, for the patience and guidance he showed me throughout my graduate studies. I would also like to thank Dr. Chris Batich, Dr. Roger Tran-Son-Tay, and Dr. Mark Segal for serving on my supervisory committee. I would like to acknowledge the support of my colleagues, both past and present. Their friendships as well as their technical expertise were invaluable. I am especially grateful for the efforts of Leslie Wilson, James Schumacher, Clay Bohn, Adam Feinberg and Thomas Estes. As fellow members of Dr. Brennans research group, Matthew Blackburn, Kenneth Chung, Amy Gibson, Dave Jackson, Chris Long, Chelsea Magin, Sara Mendelson, Sean Royston, Jim Seliga and Julian Sheets were also very helpful. I also thank the Goldberg and Batich group members for all of their assistance along the way. In particular, Olajompo Maloye was instrumental in helping me reassemble the cell culture lab so that my studies could be completed. This work would not have been completed without the financial support of the Office of Naval Research and the Alpha 1 Foundation. Finally, I would like to thank my family for their endless love and support through this experience. They managed to ground me even at the most chaotic moments. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................13 ABSTRACT ...................................................................................................................................15 CHAPTER 1. INTRODUCTION..................................................................................................................17 2. BACKGROUND....................................................................................................................22 Introduction.............................................................................................................................22 Vessel Anatomy and the Endothelial Layer...........................................................................22 Endothelial Seeding of Graft Surface.....................................................................................23 Shear-Induced Changes in Endothelial Cells.........................................................................24 Micropatterning of Cells.........................................................................................................25 Topography and Wettability...................................................................................................28 Application to this Work........................................................................................................30 3. INFLUENCE OF TOPOGRAPHY ON WETTABILITY AND BIOADHESION...............31 Notice of Previous Publication...............................................................................................31 Introduction.............................................................................................................................31 Materials and Methods...........................................................................................................34 Material............................................................................................................................34 Pattern Designs................................................................................................................34 Silicon Wafer Processing................................................................................................35 Pattern Transfer and Die Production...............................................................................35 Sample Production...........................................................................................................35 Contact Angle Measurements..........................................................................................36 Comparison with Model..................................................................................................36 Predicted Wetting on Novel Topographies.....................................................................36 Ulva Zoospore Assay......................................................................................................37 Porcine Vascular Endothelial Cell (PVEC) Assay..........................................................38 Statistical Methods..........................................................................................................39 Results.....................................................................................................................................39 Contact Angle Measurements..........................................................................................39 Comparison with Model..................................................................................................40 Predicted Wettability on Novel Topographies................................................................41 5

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Ulva Zoospore Assay......................................................................................................42 PVEC Assay....................................................................................................................42 Discussion...............................................................................................................................43 4. CHARACERIZATION OF GLUTARALDEHYDE AND GENIPIN CROSSLINKED GELATIN FILMS..................................................................................................................55 Introduction.............................................................................................................................55 Materials and Methods...........................................................................................................57 PDMSe Mold Preparation...............................................................................................57 Gelatin Film preparation..................................................................................................59 Soxhlet Extraction of Gelatin..........................................................................................60 Postcuring of Gelatin.......................................................................................................60 Mechanical Testing.........................................................................................................60 Swelling Study.................................................................................................................61 Evaluation of Microscale Gelatin Features.....................................................................61 Statistical Analysis..........................................................................................................62 Results and Discussion...........................................................................................................62 Mechanical Testing of Gelatin Films..............................................................................62 Swelling Studies of Gelatin Films...................................................................................64 Evaluation of Microscale Gelatin Features.....................................................................66 Conclusion..............................................................................................................................67 5. ENDOTHELIAL CELL GROWTH ON TOPOGRAPHICALLY PATTERNED SUBSTRATES.......................................................................................................................81 Introduction.............................................................................................................................81 Materials and Methods...........................................................................................................83 Engineered Topographies................................................................................................83 PDMSe Mold Preparation...............................................................................................85 Preparation of Gelatin Films...........................................................................................85 Preparation of PDMSe Films...........................................................................................85 Characterization of Topographically Modified PDMSe and Gelatin Films....................86 Fibronectin Adsorption to Samples.................................................................................86 Cell Culture, Imaging and Processing.............................................................................86 Preliminary assay.....................................................................................................87 Immunofluorescent assay.........................................................................................87 Cell culture assay 3..................................................................................................90 Cell culture assay 4..................................................................................................91 Statistical Methods..........................................................................................................92 Results.....................................................................................................................................92 Characterization of Topographically Modified PDMSe and Gelatin..............................92 Preliminary Cell Culture Assay.......................................................................................93 Immunofluorescent Cell Culture Assay..........................................................................93 Cell Culture Assay 3........................................................................................................95 Cell Culture Assay 4........................................................................................................96 6

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Discussion...............................................................................................................................98 Conclusion..............................................................................................................................99 6. INFLUENCE OF TOPOGRAPHY ON SHEAR STABILITY OF ENDOTHELIAL CELLS..................................................................................................................................121 Introduction...........................................................................................................................121 Materials and Methods.........................................................................................................122 Design of Parallel Plate Flow Chamber........................................................................122 Production of PDMSe Topographies.............................................................................122 Preliminary Shear Study................................................................................................123 Sample gasket preparation.....................................................................................123 Cell culture.............................................................................................................124 Shear treatment.......................................................................................................124 Staining and imaging..............................................................................................124 Final Shear Study..........................................................................................................125 Gasket preparation..................................................................................................125 PDMSe culture well preparation............................................................................125 Sample preparation.................................................................................................126 Cell culture.............................................................................................................126 Shear treatment.......................................................................................................127 Staining and imaging..............................................................................................127 Statistical Methods........................................................................................................127 Results...................................................................................................................................128 Preliminary Shear Study................................................................................................128 Final Shear Study..........................................................................................................128 Discussion.............................................................................................................................130 Conclusion............................................................................................................................131 CONCLUSIONS AND FUTURE WORK..................................................................................140 Conclusions...........................................................................................................................140 Future Work..........................................................................................................................141 APPENDIX A. SUMMARY OF LITERATURE ON CELLULAR RESPONSES TO TOPOGRAPHY....145 B. CALCULATION OF SHEAR IN PARALLEL PLATE FLOW.........................................155 LIST OF REFERENCES.............................................................................................................160 BIOGRAPHICAL SKETCH.......................................................................................................174 7

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LIST OF TABLES Table page 3-1 Dimensions of Topographies Used in Wettability and Bioadhesion Studies....................52 3-2 Dimensions of Novel Theoretical Topographies...............................................................53 3-3 Measured Contact Angles on Microtopographies..............................................................54 4-1 Diffusion Parameters Determined for GEN and GTA Crosslinked Gelatin......................79 4-2 Feature Dimensions of Topographically Modified Substrates Measured by WLIP..........80 5-1 Names of Topographies...................................................................................................118 5-2 Samples for Preliminary Assay........................................................................................119 5-3 Samples for Immunofluorescence Assay and Assay 3....................................................119 5-4 Samples for Assay 4.........................................................................................................119 5-5 Feature Dimensions of Topographies Determined by WLIP..........................................120 A Chronological Listing of Literature on Cellular Responses to Topography....................145 8

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LIST OF FIGURES Figure page 3-1 SEM images of PDMSe microtopographies......................................................................46 3-2 Glass mold used to make PDMSe samples........................................................................46 3-3 AutoCad sketches of proposed topographies.....................................................................47 3-4 Measurements taken to calculate nuclear form factor.......................................................47 3-5 Layout of channel topographies.........................................................................................48 3-6 Change in wettability induced by the 20 m spaced ridges (grey squares) and Sharklet AF (black triangles) topographies compared to smooth PDMSe....................48 3-7 Comparison of contact angles predicted by the model to contact angles measured on the surfaces.........................................................................................................................49 3-8 SEM images of 2 m diameter pillars in PDMSe..............................................................49 3-9 Ulva settlement on smooth (SM) and textured PDMSe.....................................................50 3-10 Ulva settled on smooth and textured PDMSe....................................................................50 3-11 PVEC alignment on smooth (SM) and textured PDMSe...................................................51 3-12 Endothelial cells grown on smooth and textured PDMSe.................................................51 4-1 Chemical Reactions between gelatin and glutaraldehyde..................................................68 4-2 Crosslinking mechanism of genipin...................................................................................69 4-3 Mold design for creating smooth PDMSe wells for casting gelatin..................................70 4-4 Process for preparing topographically patterned PDMSe wells........................................70 4-5 Process for crosslinking gelatin films with glutaraldehyde...............................................71 4-6 Representative stress-strain curves for GEN and GTA crosslinked gelatin......................72 4-7 Initial Youngs modulus versus crosslinker concentration for GEN and GTA crosslinked gelatin.............................................................................................................73 4-8 Elongation at break versus crosslinker concentration for GEN and GTA crosslinked gelatin.................................................................................................................................73 9

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4-9 Ultimate tensile strength versus crosslinker concentration for GEN and GTA crosslinked gelatin.............................................................................................................74 4-10 Stress-strain curves of GEN and GTA crosslinked gelatin................................................74 4-11 Effect of strain rate on the initial modulus of GEN crosslinked gelatin............................75 4-12 Effects of post-processing on the initial modulus of GEN crosslinked gelatin.................75 4-13 Swelling of GEN and GTA crosslinked gelatin for 7 days in water..................................76 4-14 Swelling at 20 h of GEN and GTA crosslinked gelatin films............................................77 4-15 Representative plot used to calculate diffusion coefficients..............................................77 4-16 Representative plot used to calculate the time exponent for diffusion kinetics.................78 4-17 Mass loss of GEN and GTA crosslinked gelatin samples after swelling for 7 days in water...................................................................................................................................78 4-18 Profilometry images of channel topographies replicated in different materials................79 5-1 Example of convention used for naming topographies....................................................100 5-2 Processing of DAPI images to measure cell density and nuclear orientation..................100 5-3 Processing of images to measure cell area, elongation and orientation...........................101 5-4 Processing of Alexa fluor 488 images to measure alignment of focal adhesions............102 5-5 SEM images of PDMSe replicates of silicon wafers patterned by different processing methods............................................................................................................................102 5-6 WLIP images of topographies formed by the DRIE process...........................................103 5-7 WLIP images of PDMSe topographies formed by the photoresist process.....................103 5-8 WLIP images of gelatin channels....................................................................................104 5-9 PVECs grown on PDMSe topographies in the preliminary assay...................................105 5-10 Density of PVECs on PDMSe topographies in the preliminary assay............................106 5-11 Fluorescent images of PVECs grown on PDMSe and polystyrene syrfaces...................107 5-12 Density of PVECs on topographies in the fluorescent assay...........................................108 5-13 Mean cell area for PVECs on topographies in the fluorescent assay..............................108 10

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5-14 PVEC elongation on topographies in the fluorescent assay............................................109 5-15 PVEC orientation on topographies in the fluorescent assay............................................109 5-16 Orientation of PVEC nuclei on topographies in the fluorescent assay............................110 5-17 PVEC focal adhesion orientation on topographies in the fluorescent assay....................110 5-18 Histograms of alignment indices for focal adhesions on topographies in the fluorescent assay..............................................................................................................111 5-19 Light microscope images of PVECs grown on topographies for Assay 3.......................112 5-20 PVEC density on topographiess in Assay 3.....................................................................113 5-21 PVEC coverage on topographies in Assay 3...................................................................113 5-22 PVEC area on topographies in Assay 3...........................................................................114 5-23 Light microscope images of PVECs grown on topographies for assay 4........................115 5-24 PVEC density on topographies in Assay 4......................................................................116 5-25 PVEC confluence on topographies in Assay 4................................................................116 5-26 PVEC spreading on topographies in Assay 4..................................................................117 5-27 PVEC elongation on topographies in Assay 4.................................................................117 5-28 PVEC orientation on topographies in Assay 4................................................................118 6-1 Original design of flow chamber.....................................................................................132 6-2 Modified design of flow chamber....................................................................................132 6-3 Layout of samples for the final shear study.....................................................................133 6-4 PVECs on topographies before exposure to flow in the preliminary shear study...........134 6-5 Density of PVECs on topographies before and after flow in the preliminary shear study.................................................................................................................................135 6-6 PVECs grown on topographies in the final shear study..................................................136 6-7 Density of PVECs on PDMSe topographies before and after flow in the final shear study.................................................................................................................................137 6-8 Elongation of PVECs on topographies before flow in the final shear study...................137 11

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6-9 Orientation of PVECs on topographies before flow in the final shear study...................138 6-10 Retention of PVECs based on topography in the final shear study.................................138 6-11 Average area for PVECs on topographies before and after flow in the final shear study.................................................................................................................................139 B Diagram of flow between parallel plates.........................................................................159 12

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LIST OF ABBREVIATIONS ANOVA Analysis of Variance AFM Atomic force microscope ATS Allyltrimethoxysilane BHK Baby hamster kidney CH Channel DAPI 4',6-Diamidino-2-phenylindole DMF Dimethylformamide DRIE Deep reactive ion etching ECM Extra Cellular Matrix FG Fibrinogen FN Fibronectin FOV Field of View GEN Genipin GTA Glutaraldehyde HBSS Hanks balanced salt solution HMDS Hexamethyldisilazane Hyal Hyaluronic acid HyalS Sulfonated hyaluronic acid MDCK Madine Darby canine kidney MeI Methyliodide MEQ Molar equivalent NFF Nuclear form factor PBS Phosphate buffered saline 13

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PDMS Polydimethylsiloxane PDMSe Polydimethylsiloxane elastomer PR Photoresist PS Polystyrene PVEC Porcine vascular endothelial cell RFGD Radio frequency glow discharge RGD Arginine-glycine-aspartate SEM Scanning electron microscopy SK Sharklet AF SM Smooth SMC Smooth muscle cell TCP Tissue culture polystyrene TRITC Tetramethylrhodamine isothiocyanate UV Ultraviolet WLIP White light interference profilometry CP Microcontact printing 14

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENGINEERED MICROTOPOGRAPHIES TO INDUCE IN VITRO ENDOTHELIAL CELL MORPHOLOGIES STABLE TO SHEAR By Michelle Lee Carman August 2007 Chair: Anthony Brennan Major: Biomedical Engineering Large diameter (>6 mm) synthetic grafts have an established record of clinical success, but consistently occlude at smaller diameters. Attempts to improve patency by seeding grafts with endothelial cells have failed due to removal of cells under high shear. Endothelial cells are known to elongate with flow, requiring cells to break focal adhesions and form new ones. A means of inducing alignment of adhesions before exposure to flow could improve retention. This is the first known work to investigate the influence of microscale topographies on inducing cellular alignment to improve retention. Initial studies examined the efficacy of existing wetting models. A series of engineered topographies were generated in polydimethylsiloxane elastomer (PDMSe) and contact angles of four solvents were measured. Results correlated strongly with classical models (y=0.99x) with a coefficient of determination of 0.89. Data were compared with settlement of algae spores and porcine vascular endothelial cells. Packing density of algae spores and alignment of endothelial cells followed similar trends, suggesting wettability of topographies may be a strong factor in determining biological responses. Based on insights from the wettability studies, topographies were designed to induce cytoskeletal alignment of endothelial cells. Gelatin was selected as a potential base material and 15

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glutaraldehyde and genipin were investigated as crosslinking agents. Mechanical properties of gelatin films with varying crosslinker concentrations were determined. Genipin stabilized gelatin more efficiently, exhibiting relatively high modulus and tensile strength while minimizing swelling during hydration. Porcine vascular endothelial cells were cultured on a series of microscale topographies in genipin-crosslinked gelatin and fibronectin-adsorbed PDMSe. Cells did not grow on gelatin, most likely due to cytotoxicity of unreacted genipin. Cells grew to confluence on topographies formed in fibronectin-treated PDMSe. Focal adhesions and overall cell shape aligned with the underlying topography. The topographies led to significantly smaller mean cell areas, more closely approaching that of cells in vivo. Microscale topographies enhanced cell spreading but not retention after 2 minutes of 2 Pa of flow-induced shear stress. After flow, cells on smooth controls decreased spreading by 60% and tended to form isolated aggregates. Cells on microtopographies maintained spreading, suggesting better viability. 16

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CHAPTER 1 INTRODUCTION Cardiovascular disease is the leading cause of death in the United States. In 2003 685,089 U.S. deaths were attributed to the disease which correlates to one death every 45 seconds [1]. Although synthetic large diameter grafts have an established record of clinical success, they consistently fail if used at vessel diameters of 6 mm or less. Currently, the preferred treatment of partially occluded vessels is minimally invasive angioplasty and stenting. However, for severe cases angioplasty and stenting are not an option and bypass grafts are necessary. The current standards for coronary bypass grafts are autologous mammary artery followed by saphenous vein despite drawbacks including donor site morbidity and limited supply. As early as the 1970s, researchers sought to improve the antithrombogenic nature and hence the patency of artificial vascular grafts though the incorporation of endothelial cells onto the inner lumen of the graft surface. They have shown that endothelial cells grow to confluence on a wide variety of substrates, but are removed easily when exposed to shear stresses equivalent to those present in natural human arteries. Attempts to improve endothelial cell adhesion have included surface modifications of the graft materials. Substrates have been treated though adsorption of adhesion proteins (e.g., albumin, extracellular matrix, gelatin and fibronectin), carbon deposition, photo discharge, and plasma discharge. Protein adsorptions were aimed at providing selective sites for cell adhesions. The remaining techniques were focused on increasing the density of non-specific reactive surface groups or altering surface wettability to influence the adsorption of proteins. Although these methods have been successful at increasing cell density and improving adhesion, there is still a need to develop a surface capable of supporting endothelial cells under high shear stress. 17

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Endothelial cells in vivo elongate in the direction of blood flow in regions of high shear stress. It is logical to assume that endothelial cells would tend to alter their morphology in this way when exposed to shear in vitro as well. In fact, researchers report that cells maintain attachment and undergo elongation if subjected initially to low shear rates followed by increasing rates up to physiological shear rates [2, 3]. It seems likely that the problem with current attempts to seed vascular grafts with endothelial cells might lie in the fact that cells are seeded in static conditions. It is hypothesized here that these cells are removed when exposed to flow because they must break focal adhesions to adapt their morphology. This implies that a method of increasing cellular alignment under static conditions could improve endothelial cell retention after flow-induced shear stress is applied. Cells alter their shape based upon the underlying surface morphology, a phenomenon referred to as contact guidance [4]. This has been widely studied over the past several decades with a diverse selection of cell types, surface materials, and topographical features. The design and formation of microscale features has been made possible through advances in fabrication techniques by the microelectronics industry. Simple geometries such as channels and pillars are often used, although more complex geometries have been investigated as well. In particular, channels topographies have been shown to result in cell elongation parallel to the long axis of the channels. For endothelial cells and fibroblasts, cells adhere almost exclusively to the valleys of channels with widths of 5 m or greater. Consequently, gaps in cell coverage exist wherever the channels are separated by ridges. The work presented here builds upon these studies in order to increase cytoskeletal alignment of endothelial cells without a disruption in cell confluence. Initially, results of colleagues studying both endothelial cells and marine fouling by algae are examined. Surfaces 18

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used in their studies are evaluated for the influence of topography on surface wettability. A correlation between the topographical influences on wettability and biological settlement is then made. Based upon this work as well as the diverse literature on contact guidance, topographies are designed with the goal of supporting shear-stable endothelial layers. Topographical dimensions are selected based on their ability to influence the formation of focal adhesions rather than their ability to influence the shape of the cells as a whole. Focal adhesions are typically between 2 and 3 m in diameter, and so topographical features are developed with lateral dimensions of ~2 m. The height of topographical features is minimized in order to prevent disruptions in cell confluence. Polydimethylsiloxane elastomer (PDMSe) surface modified with adsorbed fibronectin has been widely used by colleagues in the study of contact guidance of mammalian cells. PDMSe is examined here as well as a hydrogel material system. Gelatin is derived from collagen which is a natural component of vascular walls. Crosslinked gelatin is investigated for its mechanical stability, ability to replicate microscale topographical features and potential to support cell growth. Glutaraldehyde and genipin were the crosslinking reagents investigated. Glutaraldehyde is commonly used as a fixative for protein systems but is known to be cytotoxic in its unbound state [5]. Genipin is a natural plant extract and suppliers claim a lower level of toxicity. The primary objective of this work is to determine if microscale topographies induce alignment of endothelial cells and whether this morphological change results in improved resistance to removal by flow induced shear stress. This is the first known attempt to evaluate the influence of engineered topographies to endothelial cell retention during exposure to flow. To achieve this goal, the influence of microscale topographies on biologic settlement was 19

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correlated with changes in surface wettability. Then gelatin was investigated as a potential substrate material and mechanical properties and diffusion kinetics were improved through the optimization of crosslinking. Microscale topographies were next created in the crosslinked gelatin as well as fibronectin-treated polydimethylsiloxane elastomer. The surfaces were evaluated for the ability to induce orientation of endothelial cells without disrupting confluence. Finally, adhesion of the endothelial cells to the topographically modified materials was evaluated using a parallel plate flow chamber. Specific aim 1. Wettability is correlated with biological settlement on engineered topographies. Contact angles of water, methyliodide, isopropanol, and dimethylformamide are measured using the sessile drop technique on smooth and micropatterned PDMSe. Measured angles are correlated with values predicted by classical wetting theories. The wettability results are correlated with previously published results of algae settlement and nuclear alignment of endothelial cells. One-way analysis of variance (ANOVA) with Tukeys multiple comparisons test are ( = 0.5) used to compare groups. Specific aim 2. Measure the mechanical stability of gelatin films crosslinked with glutaraldehyde and genipin. Gelatin films (10% w/v in water) are crosslinked by genipin (1.4, 2.9, and 4.3% MEQ) and glutaraldehyde (2.9, 4.3 and 5.9% MEQ). Samples are evaluated for the ability to increase modulus, elongation at break and ultimate tensile strength while reducing the percent swelling with water. One way ANOVA with Tukeys multiple comparisons test ( = 0.5) are used to determine significant differences in mechanical properties as a result of varying crosslinking reagent and concentration. Specific aim 3. Demonstrate that cells will align on topographies with 2 m lateral dimensions while maintaining confluence equal to that of smooth PDMSe. Percentage of 20

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covered surface area, cell density, and alignment of focal adhesions are determined. At least 90% surface coverage must be achieved to be considered confluent. Alignment of focal adhesions is indicated when the average offset angle to topography approaches zero. One way ANOVA followed by pair-wise t-tests ( = 0.5) were used to determine significant differences between surface types. Specific aim 4. Demonstrate that cell alignment induced by micropatterns increases cell retention when subjected to flow generating 2 Pa of shear stress on the sample substrate. Percentage of covered surface area and cell area before and after exposure to flow are determined. Endothelial cell retention is calculated by comparing cell densities before and after flow. One way ANOVA followed by pair-wise t-tests ( = 0.5) are used to determine if the topographies increase cellular retention. 21

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CHAPTER 2 BACKGROUND Introduction This work proposes the use of microscale topographies to improve endothelial cell retention to biomaterials with the long-term goal of the development of a successful small diameter vascular graft. In order to fully understand the rationale for this research, normal vessel anatomy and the role of the endothelium must first be considered alongside other attempts to improve endothelial cell adhesion to graft materials. These are discussed below as well as the extensive collection of research on cellular responses to microscale topography and efforts to model wettability of textured surfaces. Vessel Anatomy and the Endothelial Layer Three distinct layers make up the walls of blood vessels. The innermost layer is referred to as the tunica intima (or tunica interna) and includes the endothelial lining with an underlying layer of connective tissue. In arteries, an internal elastic membrane exists in the outer region of this innermost layer. Beneath the tunica intima lays the tunica media. Sheets of smooth muscle cells are supported by a framework of loose connective tissues which bind the tunica media to the outermost layer, the tunica adventitia (or tunica externa). The tunica media of arteries is thicker compared to veins and contains an external elastic membrane. Collagen (dominant in veins) and elastin (dominant in arteries) fibers provide support. The endothelium is a made up of a confluent layer of simple squamous cells which regulate cardiovascular physiology. It provides a continuous, selectively permeable barrier between the arterial wall and circulating blood. Additionally, the endothelium controls platelet activation, adhesion, and aggregation and smooth muscle cell (SMC) proliferation and migration. 22

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If allowed to occur uncontrolled, aggregation and proliferation of platelets and SMCs would lead to vessel occlusion. Endothelial Seeding of Graft Surface As early as the 1970s, researchers sought to improve the antithrombogenic nature and hence the patency of artificial vascular grafts though the incorporation of endothelial cells onto the inner lumen of the graft surface [2, 3, 6-33]. They have shown that endothelial cells grow to confluence on a wide variety of substrates, but are removed easily when exposed to shear stresses equivalent to those present in natural human arteries. Attempts to improve endothelial cell adhesion have included surface modifications of the graft materials. Substrates have been coated with adhesion proteins (e.g. albumin, extracellular matrix, gelatin and fibronectin), which have led to improved short term (<3 hours) cellular retention during exposures to flow-induced shear stress [15, 18, 28-30, 32, 33]. Longer exposures, however, show a rapid decrease in cellular attachment on many of these surfaces which has been attributed to desorption of adhesion proteins from the substrate [29, 32, 33]. Additionally, exposed adhesion proteins have been found to promote platelet adhesion and activation [32, 34]. Consequently, gaps resulting from insufficient initial cell seeding or removal due to shear promote the formation of fibrin and fibrous encapsulation of the graft which leads to occlusion. Other attempts to improve endothelial adhesion have included carbon deposition, photo discharge, and plasma discharge treatments. These treatments were aimed at either increasing the density of reactive surface groups or influencing the adsorption of proteins. Success of these techniques has been limited due to their unspecific interaction with cells and poor control over protein orientation [33]. A more promising treatment has involved the use of a peptide sequence found in fibronectin. Covalent binding of synthetic versions of the arginine-glycine-aspartate 23

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(RGD) sequence has been shown to overcome late term removal of cells and resist platelet adhesion and activation [13, 15, 32]. Shear-Induced Changes in Endothelial Cells Endothelial cells located in vascular regions of relatively high shear stress (e.g., arteries larger than 0.5 mm in diameter) tend to be elongated in the direction of flow. In these cells, actin filament bundles which terminate at focal adhesions are aligned parallel to flow in an apparent attempt to compensate for the stress [35]. Endothelial cells grown in static culture do not exhibit these same qualities. Instead, the cells tend to be polygonal in morphology with only a small number of stress fibers confined to the cell periphery [36]. These fibers are assumed to be responsible for maintaining cell spreading and preventing contraction. When cultured cells are exposed to flow-induced shear stresses, cytoskeletal and sometimes morphological changes are induced. These changes are accompanied by a stiffening of the cell which is related to an increase in stress fiber density [37]. These responses have been found to be dependent on both the magnitude of the shear stress and the duration of the exposure. At 0.2 Pa of wall shear stress, an increase in the density of actin filament bundles occurs between 2 and 3 hours of exposure, but cellular alignment does not occur and the stress fibers do not exhibit preferential orientation in the direction of flow [36]. After 7 hours of exposure to 1 Pa of wall shear stress, cells elongate and stress fibers align with the flow direction accompanied by the coalescence of focal contacts so that they are fewer in number but greater in size [38]. For a cell grown in static culture to align itself in this manner, it must break many of the focal adhesions it created with the surface and form new ones [38, 39]. It is hypothesized here that during this transitional period cells are removed from a surface. If this is the case, then a method that would cause the cells to align prior to implantation would be advantageous. 24

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In the 1990s Ballermann et al. of Johns Hopkins University significantly improved in vivo endothelial cell retention by preconditioning cell-seeded grafts with in vitro shear stress [2, 3, 40]. They found that exposing cell seeded grafts to 0.1 Pa wall shear stress for 3 days and then 2.5 Pa for a subsequent 3 day period significantly improved cell retention and reduced neointimal thickness on aortic interposition grafts in rats. This supports the hypothesis that alignment of the actin cytoskeleton prior to implantation is advantageous. Micropatterning of Cells It has long been known that cells respond to the shape of the substrate on which they grow. The earliest known report of this was made by Harrison in 1914 in which he observed that fibroblasts found in embryonic nervous tissue from frogs take on a polygonal shape when cultured against smooth glass, but become drawn-out when grown on spider silk [41]. The term contact guidance was later used to describe the phenomenon when Weiss performed a similar experiment with nerve cells grown on glass fibers [4]. In the past several decades, literature on cellular responses to topography has expanded rapidly. Researchers have investigated numerous combinations of cell types and topographical geometries and dimensions. Appendix A provides a table summarizing the pertinent literature. The degree of contact guidance varies with topographical dimensions and geometry as well as the cell type studied. As an example, Clark et al. examined chicken embryonic cerebral neurons and two epithelial cell types: baby hamster kidney (BHK) and Madin Darby canine kidney (MDCK) [42]. All three cell types were grown on grooved polymethylmethacrylate (Perspex) with channel widths and spaces ranging from 4 to 24 m and depths ranging from 0.2 to 1.9 m. In all cases, alignment increased with decreased spacing and increased depth. Depth had a greater influence than spacing for the dimensions studied and the MDCK cells were more sensitive to topography than BHK cells. 25

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Many researchers have investigated the potential use of topography for preventing fibrous encapsulation and subsequent contraction and stiffening of implants [43, 44]. This goal is of particular interest for maintaining the aesthetic appearance and mechanical integrity of breast implants. Campbell and von Recum examined the influence of pore size of Versapor filter materials (acrylic copolymers on nylon supports) implanted subcutaneously in canines on fibrous tissue growth [43]. Nonadherent, contracting capsules were formed on materials with pore sizes less than 0.5 m. Thin, tightly adhered capsules were formed on implants with pore sizes ranging from 1.4 to 1.9 m and inflammatory tissue infiltrated pores sizes greater than 3.3 m. Schmidt and von Recum later performed a similar experiment in which 2, 5 and 8 m wide/spaced grooves were patterned into silicone elastomer and implanted into rabbits [44]. In general it was found that the 2 and 5 m grooves (depths ranging from 0.4 to 0.6 m) resulted in fewer attached cells and thinner fibrous capsules than the smooth control and 8 m grooves. Additionally the surfaces were tested in vitro with murine peritoneal macrophages and cellular alignment was observed. In a more recent study, van Kooten and von Recum have shown that in addition to altering cellular morphology, topography can increase the density of focal adhesions [45]. Fibroblasts grown on 2 m wide/spaced grooves were shown to have a greater density of focal adhesions than the same cells grown on 8 m wide/spaced grooves and smooth fibronectin-coated silicone substrates. A 0.5 m groove depth was used for the study. Walboomers et al. have shown that topography can be tailored to increase cell density relative to a smooth substrate [46]. Fibroblasts were grown on both smooth and microgrooved (1-10 m wide and 0.5-5.4 m deep) polystyrene. It was shown that for samples of equal projected planar areas, the number of adhered fibroblasts was increased on all topographies 26

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relative to the smooth control. When the numbers were corrected to account for the surface area added by roughness, however, the 1.5 m deep grooves reduced cell density as did the 1 and 2 m wide grooves. Fibroblasts are the most common cell type used in studies with microscale topographies. Literature specific to endothelial cell responses to topography is considerably more limited. Matsuda and Sugawara bovine aortic endothelial cell attachment on 20 to 130 m wide channels and found that alignment increased with decreased channel width [47]. Mrksich et al. grew bovine capillary endothelial cells on polyurethane coated with gold and patterned with alkane thiols and adsorbed fibronectin [48]. They examined V-shaped channels that were 25 and 50 m wide and had equal spacing. They showed that endothelial cells adhere to ridges or valleys, depending on which was patterned with the alkane thiol and therefore adsorbed fibronectin. Palmaz et al. examined migration of human aortic endothelial cells on nitinol containing 1, 3, 15 and 22 m channels [49]. Cells aligned with all channels and migration was greater on larger channels. Uttayarat et al. studied smaller channel topographies in fibronectin treated polydimethylsiloxane elastomer (PDMSe). Channel widths ranged from 2.7 to 5.5 m and depths ranged from 0.2 to 4.6 m [50]. Cell proliferation was similar on all substrates and cell elongation and alignment increased with channel depth. Focal adhesions formed in channels for all topographies except the 4.6 m deep channels. Barbucci and Magnani investigated the influence of the combination of topography and chemical patterning on cell behavior [31, 51]. They observed that endothelial cells increasingly align themselves on ridges as the topographical spacing is reduced from 100 to 10 m. Similar results were found by Wilkerson et al during the study of endothelial cell growth on ridges ranging in spacing from 20 to 5 m [52, 53]. Feinberg et al has also examined endothelial cell growth on topography and chemical patterns [53, 54]. 27

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They found that cells are disrupted by topographies with profile heights greater than 1 m and that focal adhesions form almost exclusively on fibronectin (FN) regions of FN-treated PDMSe. Furthermore, the area of individual focal adhesions does not vary with surface treatment and is approximately 2 m 2 Topography and Wettability Although topography is clearly a significant factor in determining cell confluence and shape, the mechanism for this response has not been elucidated. The change in wettability of a surface that results from surface roughness, i.e., topography, is likely a contributing factor. Wettability is often characterized in terms of the three phase contact angle which relies on the relative interfacial tensions according to Youngs equation given below [55]: cosLVSLSG (2-1) Youngs equation assumes the surface is chemically and topographically homogeneous and does not take into account the dynamic nature of wetting. Many groups have demonstrated the bidirectional nature of surface wetting and therefore, one must consider dewetting as well [56-58]. It has been hypothesized that the hydrophobicity and hence the force required to affect spreading is a function of the hysteresis between advancing and receding angles. Numerous groups have studied the wetting characteristics of rough surfaces. The earliest report to correlates wetting with topography was made by Wenzel, who assumed the contours of the topography are fully wetted [59]. The apparent contact angle on the textured surface (cos ) was related to the Youngs contact angle on a smooth surface of the same material as follows: cos*cosr (2-2) Wenzel defined the roughness ratio (r) as the actual surface area divided by the area of the surface when projected onto a two dimensional plane. 28

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A more detailed approach by Cassie and Baxter proposes an alternative to the Wenzel equation [60]. They evaluated the wetting by water of waxy surfaces which where not just rough, but also porous. Under this condition, water did not follow the contours of the topography and instead rested upon a composite structure of wax and air. The ratios of the areas of liquid beneath the drop in contact with solid and air relative to the planar surface area were termed f 1 and f 2 respectively. The resulting contact angle ( D ) for the porous surface was then thermodynamically determined to be the following: 21coscosffD (2-3) Marmur[57] and Qur et al.[61] have independently examined the thermodynamics of the wetting regimes to determine when air entrapment will be energetically favored. Qur et al. defined the variable s as the fraction of liquid beneath the drop in contact with solid. It is equivalent to the f 1 term in Cassie and Baxters relationship. Air entrapment would be favored for liquids of sufficiently high surface tensions (>cos -1 [(f 1 -1)/(r-f 1 )]) and would be metastable for liquids satisfying the condition of 90<
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Application to this Work Based on the background work presented here, studies were developed to examine the potential of microscale topographies to induce endothelial cell morphologies that increase resistance to shear removal. In the following chapters, studies are presented that examine the influence of microscale topographies on cellular attachment. In the first study, wettability is correlated with the biological response of algae spores (Ulva) and porcine endothelial cells. Later, genipin and glutaraldehyde crosslinked gelatin is investigated for its ability to replicate and maintain fidelity of microscale features. Then the gelatin topographies are evaluated alongside fibronectin-treated PDMSe topographies for potential to support endothelial cell growth. Cell density, confluence and orientation are measured in relation to the microscale features. Finally, endothelial cell retention to microscale topographies is measured after exposure to flow-induced shear stress in a parallel plate flow chamber. 30

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CHAPTER 3 INFLUENCE OF TOPOGRAPHY ON WETTABILITY AND BIOADHESION Notice of Previous Publication The contents of this chapter were originally published in Biofouling 2006 by Taylor and Francis [62]. It is reprinted here in accordance with the copyright agreement. Introduction Reports on cellular responses to topographical cues on both nanometer and micrometer scales have increased in the past few decades [63-66]. Appropriately scaled nanotopographies have been shown to prevent cell attachment by prohibiting formation of focal contacts [66, 67]. Alternatively, cells can respond to microscale features by altering their shape such as elongating along grooves [45, 63]. In the area of marine fouling, topography has been shown to alter settlement of algae [68, 69], barnacles [70] and bacteria [71]. The change in wettability of a surface that results from surface roughness (i.e., topography) is likely a contributing factor to these responses. Wettability is often characterized in terms of the three phase contact angle which relies on the relative interfacial tensions according to Youngs equation [55]: cosLVSLSG (3-1) Youngs equation assumes that the surface is both chemically and topographically homogeneous and does not take into account the dynamic nature of wetting. Many groups have demonstrated the bidirectional nature of surface wetting and therefore, one must consider dewetting as well [56, 58, 72]. Numerous groups have studied the wetting characteristics of topographically rough surfaces. The earliest report that correlates wetting with topography was made by Wenzel [59], who assumed the contours of the topography become fully wet and the change in contact angle is 31

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due to an increase in surface area that topography provides. Wenzel defined a roughness ratio (r) as the actual surface area divided by the area of the surface when projected onto a two dimensional plane to account for the change in wetting in terms of contact angle as follows: coscos*r (A more detaile 3-2) d approach by Cassie and Baxter [60] proposed an alternative to the Wenzel equatd f1 ion. They evaluated the wetting by water of waxy surfaces which where not just rough, but also porous. Under this condition, water did not follow the contours of the topography and instead rested upon a composite structure of wax and air. The ratios of the areas of liquid beneath the drop in contact with solid and air relative to the planar surface area were termeand f 2 respectively. The resulting contact angle ( D ) for the porous surface was then thermodynamically determined to be the following: 21coscosffD (3-3) More recently, Qurboth Wnd for et al. [61, 73-75] demonstrated that for a given surface, regimes of enzel and Cassie-Baxter behavior exist across a range of liquid surface tensions. They defined the variable s as the fraction of liquid beneath the drop in contact with solid. It is equivalent to the f 1 term in Cassie and Baxters relationship. Air entrapment, fully wetted, awicking occurs for liquids of sufficiently high, moderate, or low surface tensions respectively. In the case of wicking, the liquid is drawn into the topography at the advancing edge so that the drop rests on a composite surface of liquid and solid. The corresponding relationships and criteria for each case are given below. Air entrapment: *f )1(cos1cos1 1111cosfrf (3-4) Fully wetted: for coscos*r 1111111cos1cosfrffrf (3-5) 32

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Wicking: )1cos (cos1 1*f for 1111cosfrf (3-6) icated that the air entrapment state would be mfollowing condition: Qur et al. also indetastable for the 111 1f cos90fr. (3-7) In the present study, a series of enginpolydimethylsiloxane elastomer (PDMSe). The engineered patterns include a biomimetic inspir Changes as been develd a eered microtopographies was created in a ed design that is based upon the configuration of placoids of fast moving sharks.in wettability were measured and compared against the values predicted by the Wenzel andCassie-Baxter relationships. Some of these surfaces were then selected to test the hypothesis thatwettability influences the contact-sensing processes used by living cells. For this purpose weselected two well-characterized but contrasting model systems to represent both marine and biomedical fouling, viz. the motile spores of the marine alga Ulva (syn. Enteromorpha), and porcine vascular endothelial cells (PVECs) which form the inner lining of arteries. The green algal genus Ulva (formerly Enteromorpha) is the most common macroalga contributing to soft fouling of man-made surfaces throughout the world [76] and h oped extensively as a model system for experimental studies [77-80]. Fouling is initiateby the settlement and subsequent adhesion of motile spores, a process which is influenced byvariety of surface-associated cues. We have previously used engineered microtopographies in PDMSe to identify surface features that promote settlement [68, 69] Our hypothesis in the present study was that topographic patterns that mimic a natural antifouling surface, viz. the placoid structure of shark skin, may provide a surface with low settlement properties. 33

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Endothelial cells are widely used as models in which to study the influence of substratumorphology on adhesion and contact-mediated growth of animal cells [31, 51, 52, 81-8 m 4]. We have dy A platinum catalyzed PDMSens Silastic T-2) was chosen for due to its high transparency and reproducibility. The PDMSe is filled with micron and sub-m and ribs (Fig. 3-1). Channels, pits were 5 m wide and spaced 5, 10, and 20 m apart. The rib designs are a reducd, chosen to use porcine vascular endothelial cells (PVECs) for our study because of the localavailability of a well characterized cell line [85]. The pig has been shown to be an ideal preclinical model for vascular research, as in vitro tests have concluded that the coagulation and fibrinolytic systems of swine closely resemble that of humans [86, 87]. In the present stuPVECs were used to investigate the affect of feature spacing (5 to 20 m) on cellular orientation.This response was then correlated with the influence of topography on wettability. Materials and Methods Material (Dow Corning Corporatio this study icron silica particles. In the unmodified state, the polymer is known to promote minimal bioadhesion because of its combination of low surface energy and low modulus [69]. The PDMSe was prepared by mixing ten parts by weight of resin with one part by weight curing agent. The PDMSe was typically cured at ~ 22C for 24 hours. Pattern Designs The features studied included channels, ridges, pillars, pits ridges, pillars, and tion of the scales of fast moving sharks. We refer to this biologically inspired pattern as the Sharklet AF because it is an antifouling topography that was inspired by, but does not reproduce, the skin of the shark. The ribs are 2 m wide, spaced 2 m apart, and have lengths ranging from 4 m. Both 1.5 and 5 m high channel and pillar features were investigatewhereas the ribs of the Sharklet AF were 4 m high. 34

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Silicon Wafer Processing Patterns were etched into silicon wafers using stan dard photolithography techniques as afers were then critically cleaned using a piranha etch (50:1 H2SO4 and H sferred to PDMSe in either negative (channels, pits and Sharklet AF) or 3-1). Negatives were replicated directly from the etched wafere glass slides. Se was cured in a glass mold (Fig. 3-2) as described previously [68]. Smooith ee described previously [88]. W 2 O 2 ) at 120C for 10 min followed by subsequent rinsing in acetone and ethanol prior toeach replication with PDMSe. Hexamethyldisilazane was used to methylate the surfaces in order to prevent adhesion. Pattern Transfer and Die Production Patterns are tran positive (pillars and ridges) form (Table so that the PDMSe topography is inverted compared to the silicon wafer. For example, pillars in the wafer would transfer as pits into the PDMSe. Positives were generated by first solution casting polystyrene (0.15 g/mL in chloroform) against the wafer followed by curing thPDMSe against the polystyrene. Epoxy dies (Epon 828 with Jeffamine D230, 9.7:2.73 by weight) were then made from both positively and negatively patterned PDMSe. Sample Production Samples included PDMSe films that were either free standing or adhered to In both cases, the PDM th samples were cast directly off the glass, while patterned samples were produced by casting against epoxy or silicon dies. For glass-backed samples, slides were first pretreated w0.5% allyltrimethoxysilane (ATS) in a 95% ethanol/water solution to improve adhesion. Thrreplicates of each pattern type were produced. Fidelity of the surface features was verified with the aid of light microscopy and SEM. 35

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Contact Angle Measurements Wettability was evaluated on free -standing PDMSe films containing 5 m high channels, pits, and Sharklet AF and 1.5 m high ridges by the Sessile drop method with 2 L drops. This method looks at advancing contact angles measured in the first few seconds of contact. Video capture goniometry was used and angles were measured with ImageTool software. Liquids included in the study were nanopure water (17 M*cm resistivity), methylene iodide, and dimethylformamide. Surfaces were rinsed with ethanol and dried at 80C prior to testing with each liquid. One drop was placed on three replicates of each pattern and two angle measurements, one from the left and one from the right, were taken per drop. In this manner, six measurements were taken per pattern. Drops were viewed down the lengths of channels and ribs. Comparison with Model Wettability data were compared against values predicted by Qurs combined model of Wenzel and Cassie-Baxter relations. Predicted contacted angles were calculated from the model using the roughness ratios and solid surface fractions of each topography. For both relations, the contact angles on rough surfaces are related to the contact angle on the smooth surface. In order to account for this, data were normalized by dividing the contact angles for each liquid on textured surfaces by the angle the same liquid makes on smooth PDMSe. Normalized values predicted by the models were then plotted against normalized values measured on the surfaces. Linear regression was performed to test the validity of the models. Predicted Wetting on Novel Topographies Once the model was determined to give a good approximation of the wetting across engineered topographies, it was used to predict the effectiveness of proposed topographies. 36

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Topographies considered include d circular pillars, square pillars, star-shaped pillars, ring-shaped pillar in t ated for sly ]. The 5 m Sharklet AF topography was evaluated using the same protocol except for o the ultrahydrophobic nature of the Sharklet AF topography, samp fluorescence microscope and ]. Thirty images were taken of each of the 3 replic flat of s, a combination of triangular and circular pillars, a gradient array of circular pillars, and hexagonal pillars (Fig. 3-3 and Table 3-2). Both 1 and 3 m features heights were evaluated. The Sharklet AF topography was also considered at these depths to determine effectivenessaltering wettability. Ulva Zoospore Assay PDMSe samples containing 5 m wide ridges and pillars spaced 5, 10 and 20 m apart a1.5 and 5 m heights in addition to the 5 m deep Sharklet AF topography were evalusettlement of Ulva spores. Three replicates of each type were tested and all samples were backed by glass slides. Settlement data for the pillars and ridges have been published previou[68, 69 one slight deviation. Due t les for this study were shipped to the bioassay site in nanopure water to ensure air was displaced from the features and the samples were fully wetted during the assay. Ulva zoospores were obtained from fertile plants of Ulva linza collected from Wembury Beach, UK (50 N; 4 W) and prepared for experiments as previously described [79]. Briefly, 10 mL of spore suspension (adjusted to 2x10 6 mL -1 ) is added to each sample and incubation is carried out for 60 min in the dark followed by fixation with 2% glutaraldehyde inartificial seawater (Instant Ocean). Settled spore counts were taken using a 10X objective witha Zeiss Kontron 3000 image analysis system attached to a Zeiss epi video camera as described by Callow et al. [68 ate samples to quantify the number of attached spores. For smooth samples and bothand textured areas of patterned samples, images were taken at 1 mm intervals along the axis 37

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the slide. Spore settlement data are reported as the mean number of adhered spores (x = 90) with 95% confidence limits. Porcine Vascular Endothelial Cell (PVEC) Assay Free-standing PDMSe samples containing 5, 10, and 20 m spaced, 5 m wide ridges at both 1.5 and 5 m heig hts were evaluated. In order to promote cell attachment, surfaces were (FN) using the method of Ostuni and Whitesides [89]. This protein adsorgma) uring hich would affectpplied coated with fibronectin ption makes the surface hydrophilic and improves cellular adhesion. Topography coupled with fibronectin should induce cellular alignment within channels to maximize contact area while minimizing tension on the cell membrane. Briefly, lyophilized bovine plasma FN (Siwas dissolved in 2 mL of 0.22 m filtered water at 37C for 45 minutes and then diluted to 50 g/mL in Hanks Balanced Salt Solution (HBSS). Sterilized samples were placed in individual wells of a 24-well plate, and FN was added in 0.5 mL aliquots to each sample. Dexposure to vacuum (100 kPa) to remove trapped air, samples were left to incubate for 1 h atroom temperature. Air must be removed to prevent denaturizing of the protein w adhesion [90]. The FN solution was aspirated out and then the samples were washed 3 times with HBSS. PVECs obtained from the main pulmonary artery of 6 to 7-month-old pigs were suby Dr. Edward Blocks lab between passages 2 and 5 [91]. Cells were seeded at a density of 2x10 5 cells per sample in 1 mL of serum-free media. Serum-free media was selected because the adhesion protein was already adsorbed on sample surfaces. The cells were incubated at 37C and 5% CO 2 for 48 to 72 hours. Samples were fixed with cold 10% n-buffered formalin for 20 minutes. The cell bodies were then stained for 20 minutes in 1% crystal violet solution. Hematoxylin (Richard Allan 38

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Scientific) was used to stain cell nuclei so that nuclear elongation could be used to quantify contact guidance. Cells w ere stained in hematoxylin for 2 minutes g a Nikon Optiphot microThis asurements methylene iodide and dimethyl formamide were reduced by as muchs. Cells were imaged on the surface at 200X magnification usin scope and Matrox image capturing system. Multiple images were taken at each feature width that included at least 5 nuclei. Images were analyzed to measure the nuclear form factor (NFF) of each nucleus. The NFF is the log of the ratio of the cell length to width (Fig. 3-4). measurement was adapted from a procedure introduced by Dunn and Heath which requires the measurement of the length and width of nucleus at its widest point [92]. A 5x5 grid was superimposed on the images, and 5 nuclei were chosen per image, each from a separate square of the grid. Using this method, at least 20 nuclei per topography type were quantified. Statistical Methods Results are reported using mean values and 95% confidence intervals. One-way analysis of variance (ANOVA) and multiple comparison tests (Tukey, 95% confidence interval) were used to compare groups. Results Contact Angle Me As expected, topography increased water contact angles and decreased both methylene iodide and dimethyl formamide contact angles (Table 3-3). The most effective topography ataltering wettability was the Sharklet AF, whereas the 20 m spaced ridges and pits behaved the most like smooth PDMSe. The water contact angle on PDMSe was increased by as much as 20%, while the contact angles of as 35% and 33%, respectively. Droplets on the pits and Sharklet AF maintained a circular contact area, whereas droplets on the channels and ridges elongated along the feature 39

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Because of their cylindrical, rather than spherical, geometry the contact angles on the channeand ridges must be treated as estimates. ls ifficulty daries, the feature height was the only difference sensed by the spreading dropsn ge topographies. Increased feature spacing and decreased pattern depth resulted in diminished contact angle changes the Receding contact angles were not evaluated by the captive air method because of din bubble placement. For example, the channel patterns were only 0.33 cm wide (Fig. 3-5) andthe topographies became invisible when wetted. Consequently, it was impossible to be certain the air bubble remained in the desired region. In addition, the Wilhelmy plate technique was also deemed unsuitable because of the inability to prepare a proper sample (same topography onall sides). The effect of topography height was examined by comparing the 5 m spaced, 5 m deep channels against the 5 m spaced and 1.5 m deep ridges. Because droplets were placed away from the pattern boun The water contact angle was significantly higher on the 5 m deep channel compared to the 1.5 m deep ridge. The effect of feature spacing was examined by looking at trends withithe pits, channels, and rid Comparison with Model The sessile drop contact angle data were compared against the model to determine its viability for use in prescreening ideas for new patterns. The 1.5 m high ridges have relativelylow roughness factors (1.1 to 1.3), and solid fractions (0.2 to 0.5). On these surfaces water droplets appear to follow the situation proposed by Wenzel (Fig. 3-6) rather than the metastable state described by Qur. The Sharklet AF topography, on the other hand, has a high roughness factor (5.0) and moderate solid fraction (0.47), and all three test liquids followed situations described by Cassie (air pockets or wicking). 40

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Qur proposed that air entrapment would be favored for liquids of sufficiently high surface tensions (>cos -1 [(f 1 )/(rf 1 )]) a nd would be metastable for liquids satisfying the condis. ettability on Novel Topographies that the 3 m tall mixed star pattern will be the n increase in water contact angle of approximately 31 relative to smoo held tion of 90<
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Ulva fect pearent occurred almost entirely in val er orientation was most pth. At the 1.5 m depth only the 5 m wide ridges and smooth surfaces showed a significant difference. One of the difficulties in the analysis is the fact that the nuclei are not round, but rather elongated in Zoospore Assay The ridge topographies enhanced spore settlement (Fig. 3-9). The most significant efwas observed on the 5 m spaced, 5 m high ridges, which increased settlement by 150% relative to the smooth surface. This dimension is roughly equivalent to the diameter of the shaped swimming spore at its widest point and the diameter of the settled spore (Fig. 3-10). As spacing increased, the density of settled spores approached that of smooth PDMSe. Settlement density decreased on the shorter 1.5 m high ridges compared to the 5 m high ridges, but still remained at least as high as the density on smooth PDMSe. Settlem ley regions for all ridge topographies. The Sharklet AF topography, which has feature dimensions smaller than the spore body, significantly reduced settlement density by ~86% relative to smooth PDMSe (Fig. 3-9).Spores avoided the 2 m wide channels and were largely confined to defects and slightly widspaces (~3 m) located between adjacent Sharklet AF diamonds (Fig. 3-10). PVEC Assay Cell growth on smooth PDMSe was random with respect to orientation. Consequently, NFFs were not significantly different from zero (Fig. 3-11). Cells attached to ridge patterned substrata became aligned with the topographies. The PVECs settled almost entirely in the valleys formed by adjacent ridges similar to the Ulva spores (Fig. 3-12). Cell strongly directed by the 5 m deep, 5 m spaced ridges. NFFs varied directly with feature height and inversely with feature spacing for 5 m deep features (Fig. 3-11). The NFFs are significantly different for all widths at the 5 m de 42

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random directions on th e smooth FN treated PDMSe samples. Consequently, the mean nuclear form e-Baxter equations for the topographies investigated, but ido c principles as nsider a cell settling on a textured surface. If the cell is too large to rest between or on er res. This would increag unfavorable energy barriers. This would be very useful, particularly in limiting marine fouling. factors for smooth areas are near zero, but with a large standard deviation. Discussion Researchers have independently shown that topography alters wettability [59, 60] and surface energy influences bioadhesion [93-97]. This study investigated the interrelationship of all three factors (topography, wettability and adhesion) simultaneously using two different cell types viz. motile algal spore which choose where to settle and adhere and cultured animal cells(PVECs) which are known to adapt to underlying substrate morphology. The wetting response was well described by the Wenzel and Cassi eally one would also like to predict the effect topography has on bioadhesion. It is interesting to note that both biological models responded to the channel topographies by trying tfill the valleys either through settlement packing (Ulva) or by elongation (PVECs). This suggests that their responses are governed by the same underlying thermodynami wettability. Co top of the features, it must bridge, align, or conform to their shape. Bridging is similar tothe air pocket state and alignment is similar to the wicking states described by the Cassie-Baxtrelation. Alternatively, conforming resembles Wenzel behavior. Consider a surface that an organism will settle on but for which it has a relatively low affinity (e.g., PDMSe for Ulva spores). If the topography of this surface is engineered to expand the Cassie-Baxter regime, then the organism may be induced to bridge the featu se tension along the unsupported regions of the organisms membrane. Additionally, bridging would reduce the area of contact between the organism and surface, which would decrease the adhesion strength. Thus, bridging reduces the potential for settlement by creatin 43

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Using this hypothesis, the Sharklet AF topography was engineered to enhance hydrophobicity with dimensions smaller than the Ulva spore so that bridging wou ld be necessary for settlement to occur. It is important to nharklet AF design is biomimetically inspirated 99]. on n ote that the S ed rather than a true biomimic. Although the basic pattern of the placoids has been maintained, the dimensions have been reduced and the tips of the ribs have been flattened. As designed, spores avoided the topography which resulted in an 86% reduction in settlement density. This result provides the first demonstration that engineered microtopographies can inhibit the settlement of spores of marine algae. In addition to preventing settlement, topographies can also be engineered to promote it. Consider an organism settling on a surface which it prefers (e.g., PVECs on fibronectin-coPDMSe). If the topography of this surface is engineered to expand the Cassie regime, then the organism may be induced to align with topographies. This was evident in the PVEC studypresented here. For the topographies studied, the Cassie-Baxter regime increased with increasedspacing and increased depth. Similarly, PVECs showed increased alignment with increased spacing and depth as demonstrated with NFFs. This result is consistent with research by von Recum et al. demonstrating rat dermal fibroblasts become increasingly oriented on 0.5 m deep microgrooves as the width is reduced from 10 to 2 m [44, 45, 98, Microbubbles on surfaces are reported to denature surface adsorbed proteins, which increases cell adhesion [90]. Fibronectin pretreatment of PDMSe was used in the PVEC assay to convert the surface to a hydrophilic surface needed for initial cell attachment. To minimize any potential artifacts in the PVEC assay that could be caused by differential fibronectin adsorptithrough the presence of microbubbles, all PDMSe surfaces were degassed during fibronectiadsorption to eliminate surface-adsorbed air bubbles from topographies. It can therefore be 44

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concl Thus, d t rtant to note that bioadhesion is complex and does n r the uded that it is unlikely that the results on PVEC alignment can be ascribed to artifacts caused by microbubbles. In the case of Ulva spore settlement, it can also be reported that hydrophilic modification of the topographies (which would eliminate microbubble formation) did not alter the inhibition of zoospore settlement by the Sharklet AF topography [100].it is concluded that Ulva zoospores were contact sensing the topography and were not influenceby the presence of microbubbles [101]. These results suggest that wettability models can be useful in predicting cellular contacguidance for engineered topographies. It is impo ot only rely on surface energetics but is also species-specific [102]. The material modulusand surface elasticity of the cell membrane are also important to consider. The process is furthecompounded by the variety of adhesive proteins that an organism may secrete. Additionally, theuse of wettability models is limited by their assumption that the droplet is much larger than topographical features. This allows for line tension effects to be neglected. Investigation of wetting of these topographies by picoliter-sized drops may provide greater insight. Althoughfurther investigation is needed, these relationships may eventually be used to develop models capable of predicting the contact guidance of cells and microorganisms. Such a model would beof value in the biomedical device and marine coating industries. 45

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5 m spaced pits. B) 5 m w Figure 3-1. SEM images of PDMSe microtopographies. A) 5 m diameter, 5 m diameter, ide, 20 m spaced channels. C) 5 m wide and 20 m spaced ridges. D) Sharklet AF topography. Pillars, pits, channels, and Sharklet Figure 3-2. Glass mold used to make PDMSe samples. The epoxy and silicon dies were used AF are all 5 m deep, while ridges are 1.5 m high. Scale bars represent 20 m. only for patterned samples. Epoxy or PDMSe Silicon Die Metal Spacer Glass Plates 1x3 Glass Slide (optional) 46

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Figure 3-3. AutoCad sketches of proposed topographies. A) 2 m diameter, d pillars. triangles and 2 m pillars. C) 4 m wide, 2 m spaced stars. D) 2 m spaced square pillars. E) Rings with 2 m inner diameter and 6 m outer diameter, spaced 2 m apart. F) 4 and 2 m wide stars. G) 2 m diameter pillars spaced 1, 2 and 4 m apart in a gradient array (repeat unit designated by triawith 12 m long sides and spaced 2 m apart. I) 2 m wide, 2 m spaced channels. Scale bars represent 20 m. 2m space wide, 1 m B) ngle). H) hexagons Figure 3-4. Measurements taken to calculate nuclear form factor where L is the length of the nucleus parallel to the ridges and W is the width of the nucleus orthogonal to the ridges. 47

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. Layout of channel topographies. Each channel width (5, 10 and 20 m) is contained within a 1 cm by 0.33 cm area. Figure 3-5 -1.0-0.50.00.5 1.0 -1.0-0.50.00.51.0cos cos Figure 3-6. Change in wettability induced by the 20 m spaced ridges (grey squares) and Sharklet AF (black triangles) topographies compared to smooth PDMSe. Both measured data and model predictions are given. Dashed lines are used to indicate the metastable air pocket state proposed by Qur. 48

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y = 0.99xR2 = 0.890.50.70.91.11.30.50.70.91.11. 3 Normalized Measured AngleTheta* / ThetaNormalized Predicted Angl e Theta* / Theta Figure 3-7. Comparison of contact angles predicted by the model to contact angles measured on the surfaces. Data were normalized withespect to contact angles on smooth r PDMSe. Linear regression indicates a near 1:1 relationship (slope-0.99) with high correlation (R 2 = 0.89) to the data. Figure 3-8. SEM images of 2 m diameter pillars in PDMSe that are A) 5 m high and 4 m spaced and B) 3 m high and 2 m spaced. Increased height causes pillars to bend. Scale bars represent 15 m. 49

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pgp 0400800120016002000SM5R10R20RSMShark5R10R20R 5 m deep1.5 m deep Settlement Density (spores/mm2) Figure 3-9. Ulva settlement on smooth (SM) and textured PDMSe. Topographies studied included the Sharklet AF (Shark) in addition to 5 m wide ridges that were 5, 10,and 20 m spaced (5R, 10R, and 20R) and either 1.5 or 5 m high. The Sharklet AF topography was evaluated in a separate experiment as indicated by the darker bars. Error bars indicate standard errors of the mean. For all surfaces, counts are based on the mean of 90 counts, 30 from each of 3 replicates. Figure 3-10. Ulva settled on smooth and textured PDMSe. A) Smooth. B) 5 m wide, 5 m spaced, and 5m deep channels. C) 5 m deep Sharklet AF in PDMSe. Images were taken via light microscopy. Scale bars represent 25 m. 50

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-0.100.000.100.200.300.400.50SM5R10R20RSM5R10R20R 5 m deep1.5 m deep Nuclear Form Factor Figure 3-11. PVEC alignment on smooth (SM) and textured PDMSe. Topographies studieincluded 5, 10, and 20 m spaced, 5 m wide ridges (5R, 10R, and 20R) that are both1.5 and 5 m high. Error bars indicate standard errors of the mean. d Endothelial cells grown on smooth and textured PDMSe. A) Smooth. B) 5 m wide, 5 m spaced, and 5 m tall ridges. C) 5 m wide, 5 m spaced, and 1.5 Figure 3-12m tall ridges. Images have been processed to improve contrast. Scale bars represent 50 m. 51

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Table 3-1. Dimensions of Topographies Used in Wettability and Bioadhesion Studies Feature Height (m) Width (m) Spacing (m) Replication Type 1.5 5 5 1.5 5 10 1.5 5 20 5.0 5 5 5.0 5 10 Pillar 5.0 5 20 Positive 5.0 5 5 5.0 5 10 Pit 5.0 5 20 Negative 5.0 5 5 5.0 10 5 Channel 5.0 20 5 Negative 1.5 5 5 1.5 5 10 1.5 5 20 5.0 5 5 Positive 5.0 5 10 Ridge 5.0 5 20 Sharklet AF 4.0 2 2 Negative 52

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Table 3-2. Dimensions of Novel Theoretical Topographies Feature H eigh(m)cingm) thm) t Spa( (s) Wid( (s) 1 2 2 Circular Pilla3 2 2 1 2 2 10 rs Triangle/Circ3 2 2 10 1 2 4 les Star Pillars 3 2 4 1 1 2 Square Pillars3 1 2 1 2 2 Ring Pilla rs 3 2 2 1 2 2 4 Mixed Star P3 2 2 4 1 1 2 3 4 2 illars Gradient P illars 3 1 2 3 4 2 1 2 20 Hexagon Pillars 3 2 20 1 2 2 Channels 3 2 2 1 2 2 Sharklet AF 3 2 2 53

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Table 3-3. Measured Contact Angles on Microtopographies ct Angles () Conta Features Spaci(m)WMeI DMF 1.00 108 4 71 6 55 8 ng f 1 r ater Smooth 1.0 5 0.80 115 2 65 2 50 8 1.8 10 0.91 112 2 69 4 52 4 Pits 0.97 110 6 65 6 56 6 0.50 133 8 51 2 39 6 1.41.1 205 2.0 10 0.67 121 6 62 4 49 8 Channels 20 0.80 116 6 68 48 6 5 0.50 116 8 63 8 46 4 1.7 1.4 1.3 10 0.33 115 8 63 6 46 8 20 0.20 111 6 66 4 52 8 AF 2 0.47 135 3a46 8 35 2 1.2 Ridges 1.1 Sharklet 5.0 aplet wounot settln the surface and had to be captured with video Indicates dro ld e o 54

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CHAPTER 4 CHA RATIN OF GUTARALDEHYDE AND GENIPIN CROSSLINKED GE FILctiCollagen e priprocomn btin and connective tissue. Collagen has beenves fo asterue to its antigenicity extensive further researchuld bedete imthne system. Alternatively, gelatin has relatively low antigenicity. Gelatin is for d collagen, through hphysicand calade protein to destroy the triple-helix structure. wound dressings, adsorbent surgical pads, and vascular graft sealants. Gelatin has the advantage of being a natural, biodegradable biopolymer. As such, upon implantation no cytotoxicity is evident and over time it can be resorbed and replaced with native collagen. At temperatures above 40C, aqueous gelatin solutions exist in the solvated state allowing them to be cast into a variety of forms. At these elevated temperatures, the polypeptide exists as flexible single coils. Upon cooling, gelation takes place in which the triple helical structure of collagen is partially recovered. Gelation requires the concentration of gelatin in water to be above a certain critical minimum point, typically accepted to be between 0.4 and 1% by weight [103]. A hydrogel results, which allows for the transport of water and nutrients through the bulk. The primary drawback of gelatin lies in poor mechanically stability. In creating a gelatin hydrogel, dry gelatin powder is mixed with water that has typically been heated to above 40C which is the denaturation point for native soluble collagen. Upon allowing the solution to cool, gelation occurs as there is a partial recovery of the collagen triple helix occurs along segments of the polymer chains. At the triple helix regions, three chains are combined to form a type of CERIZA O L LATIN MS Introdu on is th mary tein ponent i one, car lage, ski n i tigated r use a bioma ial, but d wo e need to d rmine the pact on e immu med from denature eating or al hemic degr ation of th In the biomedical sector, gelatin is most commonly used for drug delivery capsules, 55

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crosslink. However, if gelatin is submergus environment long enough it will eventually dissolve completely. Conse must be employed to improve mechanical stability. This is typically accugh chemical crosslinking with bifun as a the ed in an aqueo quently, crosslinking omplished thro ctional aldehydes, diiosocyanates, carbodiimides, epoxy compounds and acyl azide methods [104]. Physical methods such as dehydrothermal treatment and ultraviolet and gamma irradiation have also been used [105-107]. The improvement of mechanical properties depends on the crosslink density and can be modeled using rubber elasticity theory. Briefly,rubber is stretched a retractive force is generated due to the decrease in entropy that occurs as thepolymer chains become stretched. The basic equation that relates the retractive stress () to extension ratio () is given below: 21McIn this equation, represents density and M RT. (4-1) s. ently ymer, but is known to exhibit localized cytotoxic effects as it is released durin ed c denotes the molecular weight between crosslinkAlthough the stress-strain relationship is not linear, it is clear that increasing the crosslink density (which decreases M c ) results in higher modulus values. Glutaraldehyde (GTA) is the most common crosslinker for gelatin systems and it primarily reacts with lysine and hydroxylysine amino acid residues. Although it reacts heterogeneously with gelatin (Fig. 4-1), it only requires one GTA molecule to form a crosslink. GTA efficistabilizes the biopol g material degradation [5]. Genipin (GEN) provides an alternative to dialdehyde crosslinking. GEN is a natural crosslinker that is obtained from an extract (geniposide) of gardenia fruits. Two GEN moleculescombine to form a single crosslink between primary amino groups (Fig. 4-2). The mechanism begins with a ring-opening condensation reaction with a primary amine [108] and is complet 56

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with a dimerization reaction which may involve free radicals [109]. GEN has been shown tonearly as efficient as GTA at stabilizing collagen-based biomaterials, but with a much lowerassociated cytotoxicity [110]. In comparison to GTA, GEN fixation of cell-free xenogenivascular grafts results in the formation of a more consistent endothelial layer in vivo [111]Attempts to generate microscale topographies in gelatin films have been limited. Yaal produced gelatin ridges adhered to a glass substrate by modification of conventional photolithography techniques [112]. Briefly, gelatin was first spin coated onto glass and then photoresist was coated on top. UV photolithography was then used to generate the desired pattern in the photoresist and then the three-layer structure was immersed in GTA to crosslink the exposed regions of gelatin. The be c ng et gelatin microtopgraphy was revealed after rinsing in acetone to strip away photoresist and immfidelity of the resulting patterns depended on both the line width (at least 5 m) of the gelatin chieved for dimensions greater than 10 mo wide) microscale features. ersion in hot water to remove uncrosslinked gelatin. The and spacing between features (at least 10 m). Yu et al. produced parallel grooves (5-500 m wide) in a chitosan-collagen-gelatin composite via photolithography and replication from a silicone intermediate [112]. Good reproducibility was a In the present study, gelatin films were crosslinked with varying concentrations of GTA and GEN. These films were evaluated for their potential at producing stable microtextured cell culture substrates. Mechanical properties (tensile and swelling) were determined in addition tthe ability to produce fine (~2 m Materials and Methods PDMSe Mold Preparation Smooth polydimethylsiloxane elastomer (PDMSe) wells were produced using Dow Corning Corporations Silastic T-2. The resin and curing agent were mixed in a 10:1 ratio, 57

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degassed and poured over two 3 inch by 2 inch microscope slides that were adhered to one another. Large top and bottom glass plates separated by 5 mm spacers were used to ensure a surface on the back of the wells (Figure 4-3). The PDMSe was allowed to cure for 24 h at rtemperature and then removed from the mold. Excess silicone was cut away, leaving about a flat oom 1cm bess are ith give ~0.5 mm thick PDMSe film. Cured films were then removed from the wafers and the placed on a clean HMDS-treated glass plate with 3 mm spacers at theoximately 120 g of PDMSE was ver the film and slides and a second clean, HMDS treated glass plate efore order around the well Topographically patterned PDMSe molds were prepared in a two-step curing proc(Fig. 4-4). First, PDMSe (10:1 ratio of base resin to curing agent) was cast directly from topographically modified silicon wafers. The wafers were prepared using standard photolithography techniques (processing performed by James Schumacher). Wafers were prepared using deep reactive ion etching (DRIE) as previously described in Chapter 3. The target dimensions of the topography were 2 m wide channels separated by 2 m wide ridges that 1 m tall. For the wafers, the channels are etched into the surface and are therefore consideredto be negative features. When PDMSe is cast against the wafer, channels replicate as ridges protruding out from the surface and so the topography is inverted. In an initial cure step, PDMSe was then cast against clean wafers within a glass mold wspacers to e desired pattern portion cut out leaving ~2 mm thick border of smooth PDMSe around thedges. Two 2 in x 3 in glass microscope slides were adhered to one another and then treated with hexamethyldisilazane via vapor deposition. The textured film was then suctioned pattern-side down to the center of the microscope slides. This was then corners of the plate. Appr mixed, degassed and poured o was laid over top. The PDMSe was allowed to cure overnight at room temperature b 58

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being removed from the mold. Excess silicone was then cut away, leaving ~1 cm border of smooth PDMSe around the well. Gelatin Film preparation Gelatin derived from bovine calf skin was supplied as a dry powder (Sigma). Uncrosslinked gelatin films w ere prepared by first dissolving the powder in 50C nanopure water at a 1m o level of the r ents viously measured by Cuevas [94]. d 50.8 mm by 76.2 mm by 2 mm PDMSe well. A clean glass slide was then across the well in 0% (wt/v). For each film, 8 mL of the heated gelatin solution was poured into a 50.8 mby 76.2 mm by 2 mm PDMSe well. A clean glass slide was then across the well in order tthe solution. A desiccator lid was placed above the well in order to slow evaporationwater. The film was allowed to dry for 24 hours at room temperature. GTA and GEN were used to stabilize smooth gelatin films. Four GTA concentrations were investigated: 2.2, 3.2, 4.3 and 9.1 wt/wt% which correspond to 2.9, 4.3, 5.9 and 13% molaequivalents (% MEQ), respectively. GEN crosslinking was carried out at 3 different crosslinkerconcentrations: 4.8, 9.1 and 13 wt/wt% (1.4, 2.9 and 4.3% molar equivalents). Molar equivalare based on a molecular weight of 1.2x10 5 g/mol for gelatin as pre The average molecular weight of repeat units was assumed to be 65 g/mol. GTA crosslinking was performed on partially dried gelatin films. Five hours after initialcasting of uncrosslinked gelatin, 4 mL of an appropriate GTA solution was pipetted on top of the film (Figure 4-5). The desiccator lid was then placed above the well and the GTA was alloweto react overnight at room temperature. GEN crosslinking was carried out by bulk mixing. GEN was first dissolved in nanopure water and heated to 50C. Gelatin was then slowly added while mixing to a final concentration of 10% (wt/v). The solution was then placed in a 50C oven for 5 minutes to ensure the gelatincompletely dissolved. For each film, 8 mL of the heated gelatin-GEN solution was poured into a 59

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order to level the solution. A desiccator lid was placed above the well in order to slow evaporation of the water. The film was allowed to react and dry for 24 hours at room the smooth films, a topographically patterned GEN crosslinked gelatiannels d. Postcin (2 in/min). Additionally GEN-crosslinked samples witho temperature. In addition to n film (2.9 % MEQ) was also prepared. The topography included 2 m wide chformed between 2 m spaced ridges that were 1 m tall. Soxhlet Extraction of Gelatin For some studies, gelatin films were Soxhlet extracted in nanopure water for 72 hours to remove residual unreacted GEN. Samples were then immersed in fresh nanopure water and allowed to equilibrate for 24 hours before mechanical tests were performe uring of Gelatin In order to evaluate the thermal stability of GEN crosslinked gelatin exposed to moderate heat, two films of each GEN concentration were immersed in water and placed in a 50C oven for 3 hours. Samples were then immersed in fresh nanopure water and allowed to equilibrate for 24 hours before mechanical tests were performed. Mechanical Testing Samples for tensile testing were cut using an ASTM D1822-68 type L dog bone die (1 in gauge length and 3.1 mm cross-sectional width). Three specimens were cut from each gelatin film. Samples were tested using an Instron 4301 with Series IX software. All samples were tested at a crosshead speed of 50.8 mm/m ut post-processing (Soxhlet extraction or postcuring) were tested at a second, slower crosshead speed of 5 mm/min to determine the effect of strain rate. Pneumatic grips were used with the pressure set to 24 psi. 60

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Swelling Study Swelling studies were carried out in order to further characterize the abilities of GGEN to stabilize gelatin films. A 12 mm diameter circular punch was used to cut out samplefrom smooth, hydrated films. Four samples of each type were punched. Samples were placedthe oven at 50C for 3 hours to dry and the initial weight o TA and s in f each sample disc was measured. al wells of 12-well culture plates. Then 3 mL of nanopure water was aple was then determine the hydrated weight. Percent swelling was then calculated by comp Samples were placed in individu dded to each well. At ten time points (5, 10, 15, 20, 30, 60, 120, 240, 1200 and 10000 min) the water uptake in the gelatin samples was measured. For each time point, samples were removed from water and their surfaces blotted dry with a task wipe. Each sam immediately weighed to aring the hydrated (W t ) and initial dry weights (W o ) according to the following equation: %100*% ootWWW Swelling (4-2) s were dried for 3 hours at 50C and the ples throughout the study was then calcut e that n 0.5X, 1X and 2X magnifications. High resolution was needed for the present study and so the 50X external objective and 2X FOV were used for a combined magnification of 100X. After the swelling study was complete, sample final dry weights (W f ) taken. The mass change of the sam lated by substituting W f for W o in Equation 4-2. Evaluation of Microscale Gelatin Features The fidelity of the topographically patterned gelatin film was evaluated using white lighinterference profilometry. A Wyko model NT1000 profilometer coupled with Vision 32 software was used for all measurements. The microscope has 2 lenses: the exterior objectivwas available in three magnifications namely 5X, 20X and 50X and the internal field of view (FOV) lens which was available i 61

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Profilometr y was performed on both dry (ambient conditions for 24 h) and rehydrated (imm o ong Mechanical Testing of Gelatin Films Tensile testing of GEN and GTA crosslinked gelatin films indicated that the mechanical prope. Interestingly, both chemistries were equally efficire led with a value of 280 14 kPa. A direct comparison with ersed in nanopure water for 24 hours) gelatin films. The surface of the rehydrated film wasblown dry with nitrogen gas immediately preceding testing. The Vision 32 software was used tmeasure the channel dimensions (width, spacing and height). Six measurements per dimensions were taken at random across the sample. Statistical Analysis Results are reported as mean values with 95% confidence intervals. For each study, one way analysis of variance ( = 0.05) was used to determine if any significant differences amthe treatment means existed. As appropriate, Tukeys multiple comparisons test was used todetermine which treatments were significantly different. Results and Discussion rties are influenced by the concentration of crosslinker. As crosslinker concentration is increased, the stress-strain curves become steeper (Fig. 4-6), indicating stiffness is increased. For both GEN and GTA, the initial Youngs modulus of the gelatin increased significantly with increased crosslinker concentrations (Fig. 4-7) ent at stabilizing the gelatin in terms of modulus. No significant differences existed between GEN and GTA at concentrations of 2.9 and 4.3% MEQ. Mean moduli values we~120 and ~155 kPa, respectively, for these two concentrations. This contradicts results published by Cuevas which state GEN is significantly more efficient at crosslinking gelatin [113]. The reason for the difference is not immediately clear, but might be attributed to differences in the length of storage of GEN. Increasing the GTA concentration to 5.9% MEQto a significant increase in modulus 62

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GEN uld s were observed for the highest and lowest h GEN and GTA. Elongation values across all treatments ranged from 42 9 increase the ultimate tensile strength compared to GEN, particularly at a concentration of 2.9 % MEQ. However, high variability in s. ith by N s could not be made at this concentration. A 5.9% MEQ GEN crosslinked gelatin film conot be produced due to GENs low solubility in water (slightly less than 0.02 g/mL). Elongation at break for gelatin was also affected by crosslinker concentration. An increase in crosslinker amount tended to decrease the elongation at break (Fig. 4-8). Significant differences for the mean elongation value concentrations of bot % to 82 16%. As with modulus, elongation did not differ significantly among the GENand GTA samples at either 2.9 or 4.3 % MEQ. Ultimate tensile strength (also known as break stress) did not vary significantly with crosslinker amount or chemistry (Fig. 4-9). GTA tended to the data prevented definitive conclusion Mechanical testing of GEN crosslinked gelatin was carried out at both 5 mm/min and 50.8 mm/min (2 in/min) strain rates in order to determine how the time scale of perturbations affects the mechanical stability of the gelatin. No obvious differences were observed between the characteristic stress-strain curves for the two strain rates at any of the crosslinker concentrations (Fig. 4-10). Similarly, the initial Youngs modulus did not vary significantly wstrain rate either (Fig. 4-11). The thermal stability of GEN crosslinked gelatin was evaluated by processing samples two methods. The 50C postcure for 3 hours did not significantly affect the gelatin at GEconcentrations of 2.9 and 4.4% MEQ (Fig. 4-12). However, at 1.4% MEQ the initial Youngmodulus was decreased significantly by the heat, lowering the modulus from 98 19 to 67 3 kPa. Soxhlet extraction significantly reduced the initial modulus for all GEN 63

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concentrations. In particular, the 1.4% MEQ GEN crosslinked gelatin samples were so degraded that the films fell apart under their own weight and dog bone specimens could not prep ared. At 2.9 an ironments. Dried samples of GEN and GTA crossllling nker resulted in a decrease in swn s the d from 520 10% to 370 20% across a range of 2.9 to 5.9% MEQ ks 2nd d 4.3% MEQ of GEN, Soxhlet extraction of the samples resulted in ~50% reduction of the initial Youngs modulus. Swelling Studies of Gelatin Films The goal of this chapter was to choose an optimum crosslinker for later cell studies withtopographically modified gelatin. With this in mind, it was necessary to limit the swelling of gelatin over time when immersed in aqueous env inked gelatin were immersed in nanopure water for up to 7 days and their percent swewas plotted with time (Fig. 4-13). The GEN crosslinked samples tended to level off more quickly and at a lower swelling percentage than the GTA samples. For both GEN and GTA, increasing the concentration of crossli elling. By 1200 min (20 h), the only treatments that did not significantly differ in the degree of swelling were 1.4% MEQ GEN and 5.9% MEQ GTA (Fig. 4-14). With the exceptioof these two, all of the GTA samples swelled to a greater extent than all of the GEN samples. The swelling of GEN crosslinked samples decreased from 360 20% to 100 10% acrosslinker concentration increased from 1.4 to 4.3% MEQ. Similarly, the swelling of GTA crosslinked samples decrease of the crosslinker. These results suggest that GEN samples would be better than GTA samples at maintaining the fidelity of microscale topographies during culture The transport of water through each film was evaluated assuming Fickean diffusion. Thediffusion coefficient (D) was determined by the following equation which is based on Ficlaw [114, 115]: 64

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2/124lDtWWtIn this equation, W (4-3) fusion coefficient and l is the average thickness of the gel sion coefficient for each sample was calculated from the slope efficient of gelatin did not signifon t and W represent the mass of water in the hydrogel at time t and infinite time respectively. D is the dif during the swelling study. The diffu of a linear regression to W t /W versus the square root of time (Fig. 4-15). It is important to note that the weight of the GTA crosslinked gelatin samples leveled off between 480 and 1200 minutes (4 and 20 h) but underwent a sharp weight increase between 1200 and 10000 minutes (roughly 1 and 7 days). This suggests that the network of the material underwent degradation during this time period and so the infinite weight time point was selected to be1200 minutes to exclude the effects of degradation. The diffusion co icantly vary with type or concentration of crosslinker used (Table 4-1). The average diffusion coefficient was 13 2 m 2 /s. To further evaluate the diffusion kinetics, a generalized rate equation was used to determine whether Fickean diffusion was rate determining mode of mass transport. The equatiused was as follows [114, 116]: ntktW. (4-4The term k is a characteristic constant of the gel and n is a characteristic exponent whicdepends on the primary mode of transport of the penetrant. Fickean kinetics de W) h fines n = 0.5. Value time (Fig. 4-16). The values for n varied significantly with crosslinker type but not concentration (Table 4-1). The value of n was 0.39 0.07 for GEN samples and 0.50 0.06 s of n between 0.5 and 1.0 indicate that the desorption process is non-Fickean. The characteristic exponent, n was calculated from the slope of the linear regression of log (W t /W ) versus the log of 65

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for GTA samples. This suggests the rate of diffusion of water through GTA crosslinked gelatin is governed by Fickean diffusion, whereas other processes are significant for GEN crosslinked gelatiipin ed exhibited ly EvaluFeature widths did not vary significantly from the silicon wafewidths of 1.6 to 1.7 m (Table 4-1). Feature depths were not as accurately replicated. Comp r. n. The value of n might have been decreased because of a desorption of unreacted genIn order to determine if GEN and GTA crosslinked gelatin degrades or leaches crosslinker into the water, the weights of dried samples before and after the 7 day swelling study were compared (Fig. 4-17). The mass loss of GTA crosslinked samples was 7.4 0.9% and it did notvary significantly with crosslinker concentration. In contrast, the mass loss of GEN crosslinksamples increased with increasing crosslinker concentration. The 4.3% GEN samplesa 20.2 2.3% mass loss, which was significantly greater than that of 1.4% and 2.9% GEN samples which lost 11.9 5.4% and 14.7 1.2% respectively. Some of the mass loss is likedue to the leaching of unreacted genipin from the gelatin into water. The total mass loss is similar to the amount of GEN in the original film, which suggests that at least some of the change in mass is due to a loss of gelatin ation of Microscale Gelatin Features GEN (2.9% MEQ) crosslinked gelatin was able to replicate the 2 m wide microchannels as shown in the profilometry images (Fig. 4-18). r master, with both substrates having channel widths of 2.3 to 2.4 m and ridge ared to the silicon wafer, channel depth decreased from 0.90 0.02 m to 0.77 0.02 mfor dehydrated gelatin. The features in the rehydrated gelatin appeared even shallower with a depth of 0.55 0.01 m. However, the true rehydrated feature height may have been largeBecause the profilometry measurements were taken in air, water from the bulk of the gelatin may have diffused to the surface and partially filled the channels. The replication of the feature depths could possibly be improved if the PDMSE mold were hydrophilically modified prior to 66

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casting the gelatin. This could be accomplished either through plasma treatment or acid immersion and would allow the gelatin to fully wet the contours of the PDMSe. Conclusion Both GEN and GTA stabilized the gelatin films. For equal crosslinker concentrations (% MEQ), GTA provided a slight advantage at improving tensile properties (similar modululonger elongation at break), but GEN was significantly better at stabilizing gelatin against swelling in water. Although GEN is known to be less cytotoxic than GTA, some concern remains about whether cells will grow on the GEN crosslinked gelatin surfaces. Swelling studiesindicate that unreacted genipin will likely be leached from the gelatin samples. Soxhlet extraction in water was not found to be an acceptable method of removing residual unreacted GEN from gelatin because the mechanical properties of the films deteriorated too much. Assuch, the 2.9% MEQ GEN was chosen as the best crosslinker concentration to proceed withcell culture work because it should provide s but into the best trade-off of enhancing tensile properties and ount of unreacted GEN. Topographically modif resistance to swelling while minimizing the am ied gelatin films crosslinked with 2.9% MEQ GEN were found to replicate 2 m wide features adequately, although the PDMSe mold would need to be hydrophilically modified in order for feature depth to be replicated accurately. 67

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Figure 4-1. Chemical Reactions between gelatin an d glutaraldehyde [117]. 68

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Figure 4-2. Crosslinking mechanism of genipin [108, 109, 118]. 69

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Metal SpacerPDMSe2x3Glass SlideGlass PlatesABC Metal SpacerPDMSe2x3Glass SlideGlass Plates Metal SpacerPDMSe2x3Glass SlideGlass PlatesABC Figure 4-3. Mold design for creating smooth PDMSe wells for casting gelatin. A) Pour PDMSe over 2 in by 3 in glass slides and allow ot cure in the glass mold shown. B) Remove PDMSe and slide from the mold. C) Peel away slide an cut out well. Patterned Silicon Wafer SpacerPDMSeGlass Plates SpacerPDMSe2x3Glass SlideGlass PlatesABCD Patterned Silicon Wafer SpacerPDMSeGlass Plates SpacerPDMSeGlass Plates SpacerPDMSe2x3Glass SlideGlass Plates SpacerPDMSe2x3Glass SlideGlass PlatesABCD Figure 4-4. Process for preparing topographically patterned PDMSe wells. A) Cure PDMSe against patterned wafer. B) Suction patterned PDMSe to glass slides. C) Pour fresh PDMSe on top and allow to cure. D) Remove PDMSe from mold and cut out the well. 70

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71 Dry 5hrs Add GTA React Overnight Remove from Mold Uncrosslinked Gelatin in PDMSe Mold Figure 4-5. Process for crosslinking gelatin films with glutaraldehyde.

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0.000.050.100.150.200.250.00.20.40.60.81.0Strain (mm/mm)Stress (MPa) 2.9% MEQ 4.4% MEQ 5.8% MEQ 0.000.050.100.150.200.2 5 0.00.20.60.81.0in (mm/mm)Stress (MPa) 0.4Stra 1.4 % MEQ 2.9% MEQ 4.3% MEQ AB Figure 4-6. Representative stress-strain curves for GEN and GTA crosslinked gelatin. A) GEN crosslinked gelatin. B) GTA crosslinked gelatin. Tensile testing was carried out at a crosshead speed of 50.8 mm/min (2 in/min). 72

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01002003001.42.94.35.9Crosslinker Concentration (%MEQ)Modulus (kPa) GEN GTA**** ** *** ** ** Figure 4-7. Initial Youngs modulus versus crosslinker concentration for GEN and GTA crosslinked gelatin. Tensile testing was performed at a crosshead speed of 50.8 mm/min (2 in/min). Asterisks denote groups with means that are not statistically different (Tukeys Test, = 0.05). () 0204060801001201.42.94.35.9Crosslinker Concentration (%MEQ)Elongation at Break (%) GEN GTA* ** *** *** Figure 4-8. Elongation at break versus crosslinker concentration for GEN and GTA crosslinked gelatin. Tensile testing was performed at a crosshead speed of 50.8 mm/min (2 in/min). Asterisks denote groups with means that are not statistically different (Tukeys Test, = 0.05). 73

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0501001502002503003501.42.94.35.9Crosslinker Concentration (%MEQ)Ultimate Tensile Strength (kPa) GEN GTA Ultimate tensile strength versus crosslinker concentration for GEN and GTA crosslinked gelatin. Tensile testing was performed at a crosshead speed of 50.8 mm/min (2 in/min). Significan Figure 4-9.t differences did not exist among any of the means (Tukeys Test, = 0.05). 0.000.050.100.150.200.25 0.00.20.40.60.81.0Strain (mm/mm) Stress (MPa) 1.4%, 2 in/min 1.4%, 5 mm/min 2.9%, 2 in/min 2.9%, 5 mm/min 4.3%, 2 in/min 4.3%, 5 mm/min Figure 4-10. Stress-strain curves of GEN and GTA crosslinked gelatin. Testing was performed at 2 in/min (solid lines) and 5 mm/min (dashed lines). 74

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0501001502001.42.94.3Genipin Concentration (%MEQ)Modulus (kPa) 5 mm/min 50.8 mm/min** *********** igure 4-11. Effect of strain rate on the initial modulus of GEN crosslinked gelatin. Asterisks 0.05). F denote groups with means that are not statistically different (Tukeys Test, = 0501001502001.42.94.3Genipin Concentration (%MEQ)Modulus (kPa) Fresh 50C Post cure Extracted*** *** *** *********** Figure 4-12. Effects of post-processing on the initial modulus of GEN crosslinked gelatin. Samples were tested without post-processing (Fresh), after heating for 3 h in a 50C oven while immersed in water (50C Postcure), or after being Soxhlet extracted in water for 72 h (Extracted). Mechanical testing was performed at a crosshead speed of 50.8 mm/min (2 in/min). Asterisks denote groups with means that are not statistically different (Tukeys Test, = 0.05). 75

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02004006008001000110100100010000 Degree of Swelling (wt%) GTA-2.9% GTA-4.3% GTA-5.9% 0110100100010000 Time (min) 2004006008001000Time (min)Degree of Swelling (wt%) GEN-1.4% GEN-2.9% GEN-4.3%A B Figure 4-1s represent standard errors. 3. Swelling of GEN and GTA crosslinked gelatin for 7 days in water. A) GEN crosslinked gelatin. B) GTA crosslinked gelatin. Samples were initially dried overnight in a 50C oven. Error bar 76

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() 01002003004005006001.42.94.35.9Crosslinker Concentration (% MEQ)Swelling (%) GEN GTA Figure 4-14. Swelling at 20 h of GEN and GTA crosslinked gelatin films. Swelling was carried out in nanopure water for 20 h. All sample means are significantly different from one another (Tukeys Test, = 0.05). y = 0.14xR2 = 0.98y = 0.13xR2 = 0.99y = 0.15xR2 = 1.00y = 0.14xR2 = 1.000.000.200.400.600.80012345Time1/2 (min)Wt / W Sample 1 Sample 2 Sample 3 Sample 4 Figure 4-15. Representative plot used to calculate diffusion coefficients. Fractional water uptake (Wt/W) is plotted against the square root of time. The diffusion coeffiis calculated from the slope of this plot according to Equation 4-3. cient 77

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y = 0.44x 0.73R2 = 0.98y = 0.53x 0.90R2 = 0.97y = 0.46x 0.79R2 = 0.97y = 0.46x 0.79R2 = 0.99-0.6-0.5-0.4-0.3-0.2-0.10.00.00.51.01.5Log Time (min)Log Wt/W Sample 1 Sample 2 Sample 3 Sample 4 Figure 4-16. Representative plot used to calculate the time constant for diffusion kinetics. The log of fractional water uptake (Wt/W) is plotted against the log of time. The time exponent (n) for the welling kinetics is ca lculated from the slope of this plot according to Equation 4-4 -25.0-20.0-15.0-10.0-5.00.01.42.94.35.9Crosslinker Concentration (% MEQ)Change in Loss (%) GEN GTA Mass loss of GEN and GTA crosslinked gelatin samples after swelling for 7 days inwater. Samples were dried for 3 h at 50C. Figure 4-17 78

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Figure 4-18. Profilometry images of channel topographies replicated in different materials. A) Silicon wafer. B) PDMSe mold. C) Dry gelatin. D) Rehydrated gelatin. Gelatin samples were crosslinked with 2.9% MEQ genipin. Images are of 48x76 m2 sample areas. Table 4-1. Diffusion Parameters Determined for GEN and GTA Crosslinked Gelatin Crosslinker Crosslinker Concentration (% MEQ) Diffusion Coefficient, D (m2/s) Time Exponent, n 1.4 13 2 0.46 0.08 2.9 13 1 0.39 0.05 GEN 4.3 13 2 0.36 0.03 2.9 15 2 0.54 0.06 4.3 12 2 0.49 0.07 TA G 5.9 14 2 0.47 0.04 79

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Table 4-2. Feature Dimensions of Topographically Modified Substrates Measured by WLIP Material Ridge Width (m) Channel Width (m) Feature Depth (m) Silicone Wafer 2.3 0.1 1.7 0.1 0.90 0.02 PDMSe 1.6 0.1 2.4 0.1 0.90 0.01 Dehydrated Gelatin 2.7 0.1 1.3 0.1 0.77 0.02 Rehydrated Gelatin 2.4 0.1 1.6 0.1 0.55 0.01 80

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CHAPTER 5 ENDOTHE LIAL TH ON TOPOGRAPHICNED SUBSTRIntrods have sprove thbogenic nce the on of endothelial cells onto the inner lumen of the graft surface [2, 3, 6-33]. They have shown that endothelial cells grow to confluence on a wide variety of substrates, but are removed when exposed to shear stresses equivalent to those present in natural human arteries. Attempts to improve endothelial cell adhesion have included surface modifications of the graft materials by coating with adhesion proteins such as albumin, extra cellular matrix, gelatin and fibronectin [15, 18, 28-30, 32, 33] as well as non-specific treatments such as carbon deposition and plasma discharge [33]. These methods have found some success at improving short term adhesion, but cellular retention is not maintained for longer exposures (>3 h). A more newly developed treatment has involved the use of a peptide sequence found in fibronectin. Covalent binding of synthetic versions of the arginine-glycine-aspartate (RGD) sequence has been shown to overcome late term removal of cells and to resist platelet adhesion and activation [13, 15, 32]. Another approach to improving endothelial resistance to shear looks at cell morphology in natural arteries. Endothelial cells located in vascular regions of relatively high shear stress tend to be elongated in the direction of flow and have actin filament bundles which terminate at focal adhesions that are aligned parallel to flow [35]. Alternatively, endothelial cells grown in static tend to be polygonal in morphology with only a small number of stress fibers confined to the cell periphery [36]. When statically cultured cells are exposed to shear, cytoskeletal and sometimes morphological changes CELL GROW ALLY PATTER ATES uction Researcher ought to im e antithom nature and he patency of artificial vascular grafts though the incorporati 81

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are induced. These changes are accomiffening of the cell which is related to an increase in stress fiber density [upon the magnitude and duration of shear exposure, cells elongate and strewith the flow direction accompanied by thee e ich ations ves m. Similar results were ging in ns panied by a st 37]. Depending ss fibers align coalescence of focal contacts so that they are fewer in number but greater in siz[36, 38]. In order for a cell grown in static culture to align itself in this manner, it must break many of the focal adhesions it created with the surface and form new ones. It islikely that during this transitional period cells are removed from a surface. If this is thcase, then a method which would cause the cells to align prior to implantation would be advantageous. It has long been known that cells respond to the shape of the substrate on whthey grow. [41]. In the past several decades, literature on cellular responses to topography has expanded rapidly. Researchers have investigated numerous combinof cell types and topographical geometries and dimensions as listed In Appendix AConfining the discussion to only the area of endothelial cells, Barbucci and Magnani investigated the influence of the combination of topography and chemical patterning oncell behavior [31, 51]. They observed that endothelial cells increasingly align themselto ridges as the topographical spacing is reduced from 100 to 10 found by Wilkerson during the study of endothelial cell growth on ridges ranspacing from 20 to 5 m [52]. Additionally, Feinberg has shown that cells confluence is disrupted by topographies will profile heights greater than 1 m and that focal adhesioform almost exclusively on fibronectin (FN) regions of FN-patterned polydimethylsiloxane elastomer (PDMSe) [54]. Additionally, he showed that the area of 82

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individual focal adhesions does not vary with surface treatment and is approximately 2 m 2 This work proposes that microscale topographies can be used to orient the cytoskeletal components of endothelial cells. In the following studies, microscale channel and topographies were generated in PDMSe and gelatin. The height of the topographical features was maintained at ~1 m so as not to disrupt endothelial cell spreading. Because this height was significantly shorter than for Sharklet AF (3 m) which was introduced in Chapter 3, the new name of Sharklet CE was developed to indicate it is cel l enhancing. The topographies of primary interest had lateral dimensions of ~3 othelial f ial is cast against For convenience, the following naming scheme was developed by the Brennan group: [depth]_[topography type]_[width]x[spacing] m so that focal adhesions could be supported. Additionally, the height of the primary topographical features was maintained at ~1 m so as not to disrupt endcell spreading. Porcine endothelial cells were cultured on the topographies and cell density, confluence, density and spreading were examined. Additionally the ability otopographies to align focal adhesions and nuclei was investigated Materials and Methods Engineered Topographies Silicon wafer masters were prepared using standard photolithography techniques(processing performed by James Schumacher). Both channel and Sharklet CE patterns were included with feature heights of ~1 m for most topographies. Negatives of these topographies were generated in the silicon wafers, so that positives (channels are defined by ridges protruding out of the surface) would be formed once a materthem. For example, a channel in the wafer becomes a ridge in the replicating material. 83

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All feature dimensions are given in micrometers. The depth was identified as if thefeatures w ere etched below the surrounding planar surface as in the case of the silicon wafers in the CH re e of : deep reactive ion etching (DRIE) and photoresist rocess, first the photoresist t long enough to fully penetrate the photoresist. In areas of the mt master and + if the features were raised above the surrounding material ainitial replicating material (Fig. 5-1). The topography type was classified as either for channels or SK for Sharklet CE. Although, the target lateral dimensions we2x2 for most of the topographies in the following studies, the true dimensions for somthe topographies were closer to 3x1 (see Results section). For the purpose of naming topographies, the actual dimensions were used. As an example, +1_CH_1x3 refers to positive channel features that are 1 m tall, 1 m wide and 3 m spaced. The names and descriptions of all topographies used are given in Table 5-1. Two-dimensional representations were first created using AutoCAD and then electronically transferred in chrome onto quartz optical photomasks using e-bream lithography. Clean silicon wafers were then coated with positive photoresist via spin coating. Two techniques for generating topography were then used (PR) exposure. In the PR p layer was exposed to UV ligh ask where no chrome is present, the UV light is transmitted and chemically alters the photoresist to make it more soluble. Then wafers were exposed to a developer solution to remove all regions exposed to UV. In this manner, the pattern was reproduced in the photoresist. In the DRIE process, wafers were then exposed to reactive ion etching so that the features are transferred into the underlying silicon. Etched wafers were cleaned via a piranha etch (50:1 of H 2 SO 4 andH 2 O 2 ) at 120C for 10 minutes followed by subsequen 84

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rinsing in acetone and ethanol prior to each replication with PDMSe. Clean wafers wertreated with hexamethyldisilazane (HMDS) to prevent adhesion by generating unreactivmethyl groups on the surface. The HMDS was applied by vapor deposition under 28 inHg (95 kPa) vacuum. PDMSe Mold Preparation Smooth and patterned PDMSe wells were formed using Dow Corning Corporations Silastic T-2 as previously described in Chapter 4. Preparation of Gelatin Films Gelatin derived from bovine calf skin was supplied as a dry powder (Sigma). Genipin crosslinked (10 w/w%) gelatin films were prepared as previously described in e e Chapr The to an generate ~1 mm thick films. ter 4. Briefly, the appropriate amount of genipin was dissolved in nanopure wateand heated to 50C. Then gelatin was added to create a 10 wt/v% aqueous solution.mixture was then cast into the PDMSe mold (smooth or with topography) and allowedreact for 24 h at room temperature. In order to minimize the amount of residual unreacted GEN in the films, samples were immersed in nanopure water for 3 days at room temperature. The nanopure water was exchanged every 24 h. Prior to cell seeding, all samples were sterilized in 70% ethanol and rinsed 3 timesin phosphate buffered saline (PBS). They were then equilibrated in PBS at 37C inincubator for 4 h. Preparation of PDMSe Films PDMSe films were cast directly against silicon wafers. The base resin and curing agent were mixed (10:1 by weight), degassed and poured over top of the silicon wafer.Curing was carried out at room temperature for 24 h in a glass mold with spacers to 85

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Characterization of Topographically Modified PDMSe and Gelatin Films Dimensions of PDMSe and gelatin topographies were analyzed using scanning electron microscopy (SEM) and white light interference profilometry (WLIP). Fibronectin Adsorption to Samples ma fibronectin (Biomedical Technologies, Inc.) was dissolribed by the Samples were first minutes. Samples were rinsed 3 times with PBS and placelace d es 2 and 3. Cells were timycotic solution. They were incubated at 37C and 5% CO2. ere passaged every 3-4 days when they were ~90% conflun of Lyophilized bovine plas ved (1 mg/mL) in 0.22 m filtered water at 37C for 45 minutes as descmanufacturer. The solution was then diluted to 50 g/mL in PBS. sterilized in 70% ethanol for 30 d into the bottoms of the wells of culture plates. Gelatin samples were held in pwith PDMSe washers placed above the samples. Enough fibronectin solution was addeto just cover the surfaces (same volume for each sample) and allowed to react for 30 minutes (preliminary assay) or 1 h (all other studies) at room temperature. Samples were rinsed 3 times with PBS prior to cell seeding. Cell Culture, Imaging and Processing Porcine vascular endothelial cells (PVECs) were supplied by Dr. Edward Block(Veterans Administration Hospital, Gainesville, FL) between passag previously harvested from the pulmonary artery of 6 to 9 month old pigs [85, 91]. Cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serumand 1% antibiotic-an Unless used immediately, cells w ent. Cells were not typically used beyond passage 5. Prior to seeding cells on test surfaces, they were detached from the culture flasks by incubation with 1 mL of 0.05% trypsin for 10 minutes. Then 2 mL of media was added to stop the enzymatic reactio 86

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the trypsin. Cell concentration was measured using a hemocytometer and 1% c rystal violet was carried out for 4 days using 12-well culture platesere f ined d (less than 4 h). Preliminary cultures scope at 400X magnification. PDMSe and gelati s stain. Then the solution was diluted to achieve the desired seeding density. Preliminary assay The preliminary cell culture assay and two replicates of each sample described in Table 5-2. Cells (passage 6) wseeded at a density of 3.5 x 10 4 cells/mL with 1 mL being applied to each sample. Due to a lack of availability, cells could not be obtained between passages 2 and 5 at the time othe preliminary study. For the samples indicated, fibronectin treatment was carried outfor 45 minutes. On day 4, cells were washed twice with PBS and stained with 0.1% Mayers hematoxylin (Sigma) for 8 minutes. They were then washed twice with water and stawith Eosin Y (Sigma) for approximately 30s. Cells were then washed twice with 95% ethanol. Cells were covered in PBS until image were imaged on a Zeiss Axioplan 2 micro n discs were removed from the well plates prior to imaging. A needle was used tominimize bending of the sample substrates. When necessary, the backside of the sample(the side without cells) was rinsed with isopropanol to remove residual H&E stains. Celldensities (cells/mm 2 ) were determined manually for each image. Immunofluorescent assay The immunofluorescence assay was carried out until confluence was reached oncontrols (4 days) using 6-well culture plates. Cells (passage 5) were seeded at a density of 1.0 x 10 4 cells/mL and 2.5 mL per well. Two replicates of each sample described in Table 5-3 were included. All samples were fibronectin treated for 1 h. 87

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On day 4, cells were rinsed twice with PBS and then fixed in 4% formaldehyde f5 minutes. Then permeability of the cell membranes was increased using 0.3% Trito or n XPBS) for 5 minutes. In order to stain for focal contact adhesions, cells were in the cell nuclei. Cells were then rinsed 5 times with PBS and covered in PBh d sity, cell area and dhesions Cell density and orientation of nuclei were calcu 2). ImageJ 100 (prepared with treated with mouse anti-vinculin primary antibody (1:400 in PBS) for 1 h at 37C. Cells were then rinsed 5 times in PBS and treated with goat anti-mouse conjugated to Alexa Fluor 488 (Molecular Probes, 1:400 in PBS)) for 1.5 h at 37C. After 5 rinses with PBS, cells were treated with 5 m phalloidin-TRITC for 12 h at 37C in order to stain the actin cytoskeleton. Before placement in the incubator, 4 L DAPI per mL PBSwas added to sta S until being imaged. Fluorescent imaging was carried out using a Zeiss Axioplan 2 microscope with epifluorescence and digital capture system. Each sample was imaged at 2 locations eacfor 200X and 400X magnifications. At each location, 4 separate images were acquired.The first used white light and captured the topographical pattern. The remaining 3 useUV light though appropriate filters to capture the nuclei (DAPI), actin (TRITC) and focaladhesions (Alexa-Fluor 488) separately. Images obtained for the fluorescent cell culture assay were processed using ImageJ software to create composite images as well as determine cell den orientation of focal contact a lated from images of DAPI at 200X magnification. The image was first rotated sothat the underlying topography (based on corresponding optical image) was oriented vertically. Then the image was converted to an 8-bit black and white image and ellipseswere fitted to the cell nuclei using the Analyze Particles feature (Fig 588

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genere unction. Hermans parameter is typically used to describe the degredom orientation. from e ated a result file which includes a count of the ellipses, lengths of major and minor axes, and angle formed between major axis and horizontal reference line. Cell density was calculated by dividing the number of ellipses by the area of the field of view (0.095 mm 2 ). Elongation of nuclei was found by dividing the length of the major axis by the length of the minor axis. Orientation angles were found by subtracting the output anglby 90, the angle formed between the topography and horizontal reference line. In thismanner, orientation angles ranged from -90 to +90. A negative sign in the orientationangle indicates that the nuclei are offset in the counterclockwise direction as opposed to the clockwise direction. The nuclear orientation within a given image was determined using Hermans orientation f e of orientation of fibers within a composite. It is calculated from the following equation: 1cos22f. (5-1) In Equation 5-1, is the trigonometric average. Hermans parameter (f) ranges from -1 (perpendicular orientation to topography) to +1 (parallel orientation to topography). A value of zero indicates ran Cell area, elongation and orientation were determined from composite images of actin (Phalloidin-TRITC) and nuclei (DAPI) at 400X magnification (Fig 5-3A). Individual cells were traced in green (Fig 5-3B) and then these lines were separatedthe image using the RGB split feature. The green image was converted to black and white and the interior of the cells filled with black (Fig 5-3C). The Analyze Particlesfeature was used and ellipses were fitted to the cells. ImageJ generated result files containing the area of each cell (based on number of pixels and not an elliptical fit) th 89

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lengths of the major and minor axes, and the angles formed between the major axes and ahorizontal reference line. Elongation and orientation of cells were calculated using the same methods as for nuclei. Focal contact adhesions were analyzed from images of vinculin at 400X magnification (Fig. 5-4A). Images were converted to black and white (Fig. 5-4B) and then the Analyze Particles feature was used and ellipses were fitted to each focal adhesion (Fig. 5-4C). Due to inherent noise in the vinculin imaging, it was necessary to only include particles of the appropriate size (20 to 80 pixels) and shape (major axis at least 25% longer than minor axis). Hermans orientation of the focal adhesions is calculated using the same methods as for nuclei. Cell culture assay 3 The third cell culture assay was carried out for 4 days using 6-well culture plates. Cells (passage 5) were seeed in 4% formaldehyde for 5 minear. age area (i.e. confluence). The brightness and contrast were first adjusted and then the image ded at a density of 1.0 x 104 cells/mL and 2.5 mL per well. Two replicates of each sample listed in Table 5-3 were included. All samples were fibronectin treated for 1 h. On day 4, cells were rinsed twice with PBS and then fix utes. Cells were then rinsed thee times with PBS before being stained with 1% crystal violet for 2 minutes and rinsed with distilled water until water remained clCells were immediately imaged using a Zeiss Axioplan 2 microscope at 50X and 200X. Five images per sample were taken at each magnification. Cultures were imaged on a Zeiss Axioplan 2 microscope at 200X magnification. Amanual count of cells in each image was made and densities (cells/mm 2 ) for each imreported. Images were then processed using ImageJ software to determine cell coverage 90

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was converted to an 8-bit black and white image. Under Set Measurements only area and area fraction were selected and then the Analyze Particles tool was used to findthe area fraction (ratio of blac k pixels to total pixels) for all cell groups. The average area per cehe area of Table treated for 1 h. ere rinsed twice with PBS and then fixed in 4% formaldehyde for 5 min0X. e taken at each magnification. es tool was used to find the area f ll for each image was then calculated by multiplying the area fraction by tthe field of view (0.38 mm 2 ) and then dividing by the number of cells. Cell culture assay 4 The fourth cell culture assay was carried out until confluence was achieved on controls (6 days) using 6-well culture plates. Cells (passage 5) were seeded at a densityof 1.0 x 10 4 cells/mL and 2.5 mL per well. Two replicates of each sample listed in5-4 were included. All samples were fibronectin On day 6, cells w utes. Cells were then rinsed thee times with PBS before being stained with 1% crystal violet for 2 minutes and rinsed with distilled water until water remained clear. Cells were immediately imaged using a Zeiss Axioplan 2 microscope at 200X and 40Thee images per sample wer Cultures were imaged on a Zeiss Axioplan 2 microscope at 200X and 400X magnifications. Cell confluence was determined from the 200X images using ImageJ software. The brightness and contrast were first adjusted and then the image was converted to an 8-bit black and white image. Under Set Measurements only area andarea fraction were selected and then the Analyze Particl raction (ratio of black pixels to total pixels) for all cell groups. Cell density, area, elongation and orientation were determined from 400X images. A manual count of cells in each image was made and densities (cells/mm 2 ) for each image reported. Coverage area was determined as mentioned above and the average area 91

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per cell for each image was then calculated by multiplying the area fraction by the area of the field of view (0.092 mm 2 ) and then dividing by the number of cells. In order to determine cell elongation and orientation, 12 cells from each image were traced and filled in black using ImageJ software. Cells were selected randomly with the only criteria being that their full outline could be observed. ImageJ software was the n used to fit olored cells. Elongation was measured as the length of the major axis tne way analysis of variannd Gelatin ore ype, but there was not a ellipses to the black-c o minor axis of the cell. Orientations were measured according to Hermans orientation parameter as outlined in the immunofluorescence assay above. Statistical Methods All results are reported as mean values standard errors. O ce (ANOVA, = 0.05) was performed for each dataset to determine if any statistical differences exist among the groups. Pair-wise t-tests ( = 0.05) were performed as appropriate to determine which groups were statistically different. Results Characterization of Topographically Modified PDMSe a PDMSe samples replicated both the PR and DRIE wafers with high fidelity. Differences in fidelity between PR and DRIE replicates were evaluated using SEM images (Fig. 5-5). The DRIE process led to thinner ridges (wider channels) that mclosely matched the target dimensions. WLIP was used to determine the dimensions of the features (Figs. 5-6 though 5-8). The profilometry data verified that the lateral dimensions of the topographies varied based on the wafer processing t significant difference in feature height (Table 5-5). 92

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Preliminary Cell Culture Assay As expected, PVECs did not grow on unmodified PDMSe substrates, but grew tconfluence on fibronectin-adsorbed PDMSe (Fig. 5-9). Additionally, PVECS grew on all topographically modified PDMSe surfaces. Cells appeared slightly more elongated othe +1_SK_2x2 and +3_SK_2x2 topographies compared to smooth and channel-modPDMSe. Cell densities did not vary significantly among smooth PDMSe and topographically modified PDMSe surfaces pretreated with fibronectin (Fig. 5-10). Cells were not observed on gelatin surfaces. This may be due at least o n ified in part to the samples. Because light could not be transmitted though the sample, the Zere PVECs grew to confluence on smoig. 5-11). ll surfaces tested (Fig. ned ed to all other surfaces. Additionally, cells on the +1_CH_1x3 (PR) pattern were significantly smaller (~1500 m2) than those on the PS controls. Cells on all surfaces tended to be elongated by ~80% opacity of the gelatin eiss Axioplan 2 microscope could not be used and instead samples were viewed using a Nikon scope lit though the objective. Even in this configuration, no cells wobserved. Immunofluorescent Cell Culture Assay oth PS and all PDMSe substrates (F Cell densities were similar, ranging between 450 and 650 cells/mm 2 for a 5-12). The +1_SK_3x1 (PR) patterned PDMSe yielded a significantly higher density of cells compared to all other surfaces except the +1_CH_2x2 (DRIE) patterPDMSe. No significant differences existed among the remaining groups. The average surface area of each cell showed greater variability than cell density (Fig. 5-13). Cells tended to be largest on the smooth PS and PDMSe surfaces withaverage areas of ~2000 and ~1800 m 2 respectively. The +1_SK_3x1 (PR) pattern resulted in significantly smaller cells (~1300 m 2 ) compar 93

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(Fig 5-14). No significant differen ces in elongation of cells were detected between smoo rfaces, as came partiath orientation on these two surfaces were significantly different from smooth R) topography. hy no signifent s on ereas e th and topographically modified samples, although the orientation of the elongationappeared more random on the smooth surfaces. The topographies were found to enhance alignment of cells, nuclei, and focal adhesions parallel to topographical features (Figs. 5-15, 5-16 and 5-17). Orientation of these elements were essentially random on smooth polystyrene and PDMSe suthe Hermans orientation parameters did not significantly differ from zero. Nuclei be lly aligned parallel to the topographical features as indicated by an increase in Hermans parameter. Although Hermans parameter for the +1_CH_1x3 (PR) topography was significantly different from zero, it was not significantly different from f for smooPDMSe. The Sharklet CE topographies generated the highest degree of orientation (f~0.3) and PS, smooth PDMSe and the +1_CH_1x3 (P Alignment of cells followed the same trend as the orientation of nuclei. All four topographies significantly enhanced orientation relative to smooth PS and PDMSe. The greatest degree of cell orientation was observed on the +1_SK_2x2 (DRIE) topograpwhich yielded a Hermans orientation of ~0.45. Despite a trend for the Sharklet CE topographies to increase orientation of cells relative to channel topographies icant differences were observed. Analysis of focal contact adhesions indicated that all surfaces promote alignmwith the long axis of the underlying topography. Histograms of focal adhesion anglePS and smooth PDMSe surfaces indicated an even distribution across all angles, whhistograms for topographically modified PDMSe surfaces indicated a peak at or near th 94

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topography angle (Fig. 5-18). Orientation was significantly enhanced on all four topographies relative to smooth PS and PDMSe surfaces. The greatest degree of orientation was observed on the two Sharklet CE topographies. Hermans orientation parames ed than the smooth PDMSe contro3 DRIE) eters for these two were ~0.44. Although orientations on channels topographiwere not significantly different from that of the Sharklet CE topographies, Hermans parameter tended to be lower at a value of ~0.31 for each. This is consistent with the trends observed for cell bodies and nuclei. Cell Culture Assay 3 Cells did not grow to confluence during the four culture days of assay 3 (Fig. 5-19) in which fibronectin treatment was increased to 1 hour from 30 minutes in the preliminary assay. Cell densities were highest on the two Sharklet CE surfaces and lowest on the two controls (PS and smooth PDMSe). All of the topographically modifiPDMSe surfaces induced significantly greater cell densities l (Fig. 5-20). Additionally, all topographies with the exception of the +1_CH_1x(PR) pattern yielded a greater density of cells than on the PS control. The +1_CH_2x2 (DRIE) pattern provided the greatest density of cells, significantly greater than on all other surfaces except the +1_SK_3x1 (PR) pattern. Cells were significantly more denseon the +1_SK_3x1 (PR) pattern than the remaining surfaces except +1_CH_2x2 (pattern. Although the densities of cells on the Sharklet CE topographies were greatest, their degree of confluence did not vary significantly from the PS and smooth PDMSe controls (Fig. 5-21). Additionally, these did not vary significantly with the confluence on 95

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the +1_CH_2x2 (DRIE) surface. The +1_CH_1x3 (PR) pattern, however, resultedsignificantly less surface coverage than all other surface types. Cells tended to spread less (smaller area) on the topographically modified surfacesthan on the smooth PS and PDMSe controls (Fig. 5-22). The +1_CH_1x3 (PR) pattern did not vary significantly from the PS control, however. Cell areas did not vary significantly among the four different patterns, although the two Sharklet CE topographies had the lowest mean call area in values. nt long mong the two Sharklet CE topographies and these led to 55 and 22% increathe +1_CH_2x2 (DRIE) topography (Fig. 5-25). Confluence was not statistically different on this surface compared to smooth polystyrene, smooth PDMSe, +1_CH_2x2 Cell Culture Assay 4 Cells proliferated and were nearly confluent on all surfaces (Fig. 5-23). Cells on the smooth polystyrene and PDMSe controls varied in shape and had no apparerange orientation. Most cells were more elongated than typical endothelial cells, and their appearance may be evidence of a mixed cell population. It is possible that either fibroblast or smooth muscle cells contaminated the primary culture. In contrast to the cells on the smooth controls, cells appeared to orient with the longaxis of features for all topographies studied. The topographies increased cell density relative to the smooth polystyrene and PDMSe controls (Fig. 5-24). This affect was greatest on the -3_CH_5x5 (DRIE) surface and became less apparent as the spacing of the channels increased to 10 and 20 m. There was no significant difference in cell density a sed in cell density compared to the smooth polystyrene and PDMSe controls respectively. Quantitative analysis of the images indicated that the cells were most confluent on 96

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(DRIE) and -3_CH_5x20 (DRIE). Between 95 and 97% of these surfaces were coverewith cells. Increasing the depth of the Sharklet CE topograph d y led to a small but signif e at observed in vivo (Fig. 5-26). The 2) were observed on tissue culture polystyrene and the smallest cells tudied. The affect of top to cing ere not s orientation parameter ranged from 0.94 to 0.98 for cells on all topog onectin icant reduction in confluence as the surface coverage dropped from 97 to 93%. The-3_CH_5x5 (DRIE) topography resulted in the most disrupted confluence with only 89%of the surface are covered. As was observed in the earlier assays, the topographies tended to reduce thaverage area occupied per cell to a value closer to th largest cells (~1000 m (~500 m 2 ) were grown on the -3_CH_5x20 (DRIE) topography. Increasing the spacing between 5 m wide channels to 20 m led the cell area to approach that for smooth PDMSe (~800 m 2 ). The areas of cells on +1_CH_2x2 (DRIE), +1_SK_2x2 (DRIE), and +3_SK_2x2 (DRIE) were similar at ~600 m 2 Cells elongated and oriented with features for all topographies s ography was more significant in this assay than any of the previous. The possibility of a mixed cell population might account for the difference among the assays. Cell elongation was similar on the +1_CH_2x2 (DRIE), +1_SK_2x2 (DRIE), +3_SK_2x2(DRIE) and -3_CH_5x5 (DRIE) topographies which had mean values ranging from 3.64.0 (Fig. 5-27). For the 5 m wide channels, elongation decreased with increased spabetween channels. Cell elongation on smooth polystyrene and PDMSe controls wsignificantly different and were 1.7 and 1.8, respectively. Herman raphies with the +1_CH_2x2 (DRIE) topography resulting in the largest mean value(Fig. 5-28). It is unclear what caused the dramatic results. An older supply of fibr 97

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was used in this study, but the increased alignment may have more to do with the biological variability in the cell source. Hermans orientation parameter for the smoocontrols was not significantly different from zero, indicating random alignment of cells. Discussion Endothelial cells are n th ot able to thive on the GEN crosslinked gelatin samples. This ibe esponse. The only excepry Cell spreading, nuclei and focal s most likely due to cytotoxic effects of residual unreacted GEN. This might overcome with a more robust wash process, possibly involving longer leach times and immersion in PBS at 37C to more closely simulate the cell culture conditions. When combined with fibronectin treated PDMSe, all four topographies with 2 mlateral dimensions and 1 m heights support endothelial cell growth. The subtle differences in topography generated by the photoresist and deep reactive ion etch processes did not result in significant differences in cellular r tion was a slight increase in cell density and decrease in average cell area observed for the +1_SK_3x1 (PR) topography relative to its DRIE counterpart during the immunofluorescence assay. This difference was not observed in either the preliminaassay or assay 3, suggesting it may have been an outlying occurrence. The microscale topographies tend to increase endothelial cell density relative to smooth FN-treated PDMSe, although the differences were not always significant for all assays. Additionally, cells on these topographies tend to spread across a smaller area and more closely approach the size observed in porcine arteries. adhesions are found to orient with the underlying topographies. In comparison to the channel topography of the same dimensions, the Sharklet CE topography tends to increase endothelial cell density and orientation of cytoskeletal components. 98

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An increase in height of the Sharklet CE topography from 1 to 3 m resulslight (~3%) but significant decrease in the confluence of endothelial cells. Cell densityand orientation are not significantly affected by the change in height. Cellular ts in a elongation and density decreased signiacing increased from 5 to 20 m on 3 m d. E) ) tion had n the hydra_CH_2x2 (DRIE) and +1_SK_2x2 (DRIE) topographies are effective at orienting cytoskeletal ficantly as sp eep channels, This is consistent with results observed by Wilkerson et al [52, 62]Although cellular orientation on this topography is similar to that on +1_CH_2x2 (DRIand +1_SK_2x2 (DRIE) topographies, cell confluence was decreased significantly (~6%indicating it is not a good candidate for cell seeding. It is important to note that all of the PDMSe topographies were treated with fibronectin prior to cell culture. It is unclear what if any affect the protein adsorpon the shape of the topography. It is conceivable that the protein may have filled itopography to some degree so that the cells were presented with a somewhat smoother surface than what is indicated by the reported feature dimensions. If this work is carried forward in the future, the fibronectin adsorption to the topographies should be characterized. This might be accomplished with the use of immunofluorescently labeledfibronectin. A confocal microscope could be used to analyze the dimensions of the ted, labeled protein layer. Alternatively, the samples could be freeze dried after fluorescent labeling to lock in the structure. Then a microtome could be used to section the samples so that the Zeiss microscope with epifluorescence could be used to determine the thickness of the fibronectin layer in relation to the underlying topography. Conclusion Fibronectin treated PDMSe is a better substrate for culturing endothelial cells than fibronectin treated genipin crosslinked gelatin. In the PDMSe substrate, the +1 99

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comp onents while maintaining cellular confluence. These topographies will be evaluatedfor their ability to improve endothelial cell retention to shear in Chapter 6. AB features are raised above the plane of the surrounding material and B) Figure 5-1. Example of convention used for naming topographies. A) Positive (+) negative (-) features are formed below the plane of the surrounding material. Figur e 5-2. Processing of DAPI images to measure cell density and nuclear orientation. A) Image of nuclei on +1_SK_2x2 (DRIE) at 200X. B) Conversion to black and white. C) Ellipses fitted to nuclei and counted. D) Magnification of ellipses to demonstrate measurement of orientation angle and elongation. Blue arrows indicate topography direction. Scale bars represent 50 m. 100

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Figure 5-3. Processing of images to measure cell area, elongation and orientation. A) Portion of overlaid mage of nuclei (DAPI) and actin (phalloidin-TRITC for +1_CH_2x2 (DRIE) at 400X. B) Cells outlined in green. C) Cells filled with black for area calculation. D) Ellipses fitted to cells for measurement of orientation angle and elongation. Blue arrows indicate topography direction. Scale bars represent 25 m. 101

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images to measure alignment of focal adhesions. A) Image of focal adhesion (400X). B) Image converted to black Figure 5-5. SEM images of PDMSe replicates of silicon wafers patterned by different processing methods. A) Photoresist. B) Deep reactive ion etching. Target dimensions of the channel topographies were 2 m wide, 2 m spaced and 1 m depth. Scale bars represent 10 m. Figure 5-4. Processing of Alexa Fuor 488 and white. C) Image of ellipses fitted to adhesions and measured for alignment. Scale bars represent 25 m. 102

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Figure 5-6.) PDMSe replicate of (C). Images are of 48x76 m2 sample areas. WLIP images of topographies formed by the DRIE process (100X). A)-1_CH_2x2 silicon wafer. B) +1_CH_2x2 PDMSe replicate of (A). C1_SK_2x2 silicon wafer. D) +1_SK_2x2 F igure 5-7. WLIP images of PDMSe topographies formed by the photoresist process (100X). A) +1_CH_1x3 PR. B) +1_SK_3x1 PR. Images were taken at 100X magnification and are of 48x76 m sample areas. 103

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Figure 5-8. WLIP images of gelatin channels (100X). Gelatin topographies were replicated from the -1CH3x1 DRIE silicon wafer via a PDMSe intermediate. A) Dehydrated gelatin. B) Hydrated gelatin. Images are of 48x76 m2 sample areas. 104

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105 Figure 5-9. PVECs grown on PDMSe topographies in the preliminary assay. A) Smooth without fibronectin. B) Smooth. C) +1_CH_1x3 (PR). D) +1_CH_2x2 (DRIE). E) +1_SK_3x1 (PR). F) +1_SK_2x2 (DRIE). Fibronectin was adsorbed to all surfaces unless otherwise indicated. Topography directions are indicated by arrows. Images were captured at 400X. Scale bars represent 25 m.

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Figure 5-10. Density of PVECs on PDMSe topographies in the preliminary assay. All samples were pretreated with fibronectin (FN) unless stated otherwise. Data was obtained though manual count of images at 400X magnification. Groups of statistically indistinct means are indicated by tie bars (t-test, = 0.05). 050100150200250300350400+1 SK2x2SM+1 CH1x3+1 CH2x2+1 SK3x1SM -no FNDensity (cells/mm2) 106

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107 Figure 5-11. Fluorescent images of PVECs grown on A) TCP, B) smooth PDMSe and C-F) topographically modified PDMSe. All PDMSe samples were pretreated with adsorbed fibronectin. The light microscope views at the left of images C though F show the underlying topography to the fluorescently labeled PVECs to the right of the images. Images were captured at 400X magnification and scale bars represent 25 m.

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108 0200400600800+1 CH1x3PS+1 SK2x2SM+1 CH2x2+1 SK3x1Cell Density (#/mm2) Figure 5-12. Density of PVECs on topographies in the fluorescent assay. Cells were grown on fibronectin-adsorbed PDMSe surfaces and PS. Data was obtained though processing of fluorescent DAPI images at 200X magnification. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). Figure 5-13. Mean cell area for PVECs on topographies in the fluorescent assay. Cells were grown on fibronectin-adsorbed PDMSe surfaces and TCP. Values were generated though the processing of overlaid images of cell nuclei (DAPI) and actin (phalloidin-TRITC). Tie bars connect groups with means that are not statistically different (t-test, = 0.05). 0500 1000 150020002500PS+1 CH2x2+1 SK2x2SM+1 CH1x3+1 SK3x1Cell Area (m2)

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1.01.41.82.2PSSM+1 CH1x3+1 CH2x2+1 SK3x1+1 SK2x2Major Axis / Minor Axi s on topographies in the fluorescent assay. Cells were grown on Figure 5-14. PVEC elongation fibronectin-adsorbed PDMSe surfaces and TCP. Tie bar connects group with means that are not statistically different ( = 0.05). -0.4-0.20.00.20.40.60.8PSSM+1 CH1x3+1 CH2x2+1 SK3x1+1 SK2x2Hermans Orientation Paramete r in the fluorescent assay. Cells were grown on Figure 5-15. PVEC orientation on topographies fibronectin-adsorbed PDMSe surfaces and PS. Smooth PS and PDMSe do not significantly alter orientation away from random (Parameter=0). All topographies significantly increase orientation. Tie bars connect groups with means that are not statistically different (t-test, = 0.05) 109

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-0.3-0.20.00.20.30.5PSSM+1 CH1x3+1 CH2x2+1 SK3x1+1 SK2x2Hermans Orientation Parameter 6. Orientation of PVEC nuclei on topographies in the fluorescent assay. Cells were grown on fibronectin-adsorbed PDMSe surfa Figure 5-1ces and PS. Smooth PS and PDMSe do not significantly alter orientation away from random (Orientation Parameter=0). All topographies significantly increase orientation relative to smooth PS. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). -0.20.00.20.40.60.8PSSM+1 CH1x3+1 CH2x2+1 SK3x1+1 SK2x2Hermans Orientation Parameter PVEC focal adhesion orientation on topographies in the fluorescent assay. Cewere grown on fibronectin-adsorbed PDMSe surfaces as well as PS. Error bars indicate standard errors. Orientation on smooth PS and PDMSe surfaces are notsignificantly different from random (param Figure 5-17lls eter=0). All topographies significantly increase orientation. Tie bars connect groups with means that are not statistically different (t-test, = 0.05) 110

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3%6%9%12%15%18% 0%Alignment Index Frequency 0%Alignment Index Figure 5-18. Histograms of alignment indices for focal adhesions on A) TCP, B) smooth PDMSe, C) +1_CH_1x3 (PR) PDMSe, D) +1_CH_2x2 (DRIE) PDMSe, E) +1_SK_3x1 (PR) PDMSe, and F) +1_SK_2x2 (DRIE) PDMSe. An alignment index of zero indicated the adhesion is perfectly aligned with the topography, while indices of -1 and 1 indicate adhesions are off-angle from topography by 90 clockwise and 90 counter-clockwise respectively. 3%6%9%12%15%18%Frequency 0%3%6%9%12%15%18%Alignment IndexFrequency 0%3%6%9%12%Alignment IndexFrequency 15%18% 0%3%6%9%12%15%18%Alignment IndexFrequency 0%3%6%9%12%15%18%Alignment IndexFrequency -1 0 1-1 -1 0 1-1 0 1-1 0 1A B D E C 0 1-1 0 1F 111

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Figure 5-19. Light microscope images of PVECs grown on topographies for Assay 3. A) PS, B) smooth PDMSe, C) +1_CH_1x3 (PR) PDMSe, D) +1_CH_2x2 (DRIE) PDMSe, E) +1_SK_3x1 (PR) PDMSe, and F) +1_SK_2x2 (DRIE) PDMSe. All surfaces were pretreated with 50 g/mL fibronectin for 1 h. Topographies are oriented from left to right as indicated by arrows. Scale bars represent 50 m. 112

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0100200300400500SMPS+1 CH1x3+1 CH2x2+1 SK3x1+1 SK2x2Density (cells/mm2) Figure 5-20. PVEC density on topographies in Assay 3. All surfaces were pretreated with 50 g/mL fibronectin for 1 h. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). 020406080PSSM+1 SK2x2+1 SK3x1+1 CH2x2+1 CH1x3Percentage of Area Covered (%) 1. PVEC coverage on topographies in Assay 3. All surfaces were pretreated with 50 g/mL fibronectin for 1 h. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). Figure 5-2 113

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114 Figure 5-2250 nect groups with means that are not statistically different (t-test, = 0.05). PVEC area on topographies in Assay 3. All surfaces were pretreated with g/mL fibronectin for 1 h. Tie bars con 020040060080010001200SMPS+1 CH1x3+1 CH2x2+1 SK3x1+1 SK2x2Area per cell (m2)

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Figure 5-23. Light microscope images of PVECs grown on topographies for assay 4. A) TCP. B) smooth PDMSe. C) +1_SK_2x2 PDMSe. D) +3_SK_2x2 PDMSe. E) +1_CH_2x2 PDMSe. F) -3_CH_5x5 PDMSe. G) -3_CH_5x10 PDMSe. H) -3_CH_5x20 PDMSe. All topographies were replicated from deep-reactive-ion-etched wafers. All surfaces were pretreated with 50 g/mL fibronectin for 1 h. Topographies are oriented vertically in all images. Scale bars represent 100 m. 115

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05001000150020002500PSSM-3 CH5x20-3 CH5x10+1 SK2x2+3 SK2x2+1 CH2x2-3 CH5x5Density (cells/mm2) Figure 5-24. PVEC density on topographies in Assay 4. All surfaces were pretreated with 50 g/mL fibronectin for 1 h. Tie bar connect group with means that are not statistically different (t-test, = 0.05). 8084889296100 +1 SK2x2SMPS+1 CH2x2-3 CH5x20+3 SK2x2-3 CH5x10-3 CH5x5 Percentage of Area Covered (%) 5. PVEC confluence on topographies in Assay 4. All surfaces were pretreated w50 g/mL fibronectin for 1 h. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). Figure 5-2ith 116

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igure 5-26. PVEC spreading on topographies in Assay 4. All surfaces were pretreated with 020040060080010001200PSSM-3 CH5x20-3 CH5x10+1 SK2x2+1 CH2x2+3 SK2x2-3 CH5x5Area per cell (m2) F 50 g/mL fibronectin for 1 h. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). 012345PSSM-3 CH5x10-3 CH5x20+3 SK2x2-3 CH5x5+1 CH2x2+1 SK2x2Major Axis / Minor Axis Figure 5-27. PVEC elongation on topographies in Assay 4. All surfaces were pretreated with 50 g/mL fibronectin for 1 h. Tie bars connect groups with means that are not statistically different (t-test, = 0.05). 117

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-0.50-0.250.000.250.500.751.00SMPS-3 CH5x10-3 CH5x20+1 CH2x2-3 CH5x5+3 SK2x2+1 SK2x2Hermans Orientation Parameter 8. PVEC orientation on topographies in Assay 4. All surfaces were pretreated w50 g/mL fibronectin for 1 h. Tie bars Figure 5-2ith connect groups with means that are not statistically different (t-test, = 0.05). Table 5-1. Names of Topographies Topography Description Name 1 m wide channels formed between 3 m wide ridges that protrude out of the surface by 1 m +1_CH_1x3 3 m wide and 1 m spaced channels formed below the surface to a depth of 1 m -1_CH_3x1 2 m wide channels formed between 2 m wide ridges that protrude out of the surface by 1 m +1_CH_2x2 3 m wide and 1 m spaced ribs arranged in the Sharklet AF pattern. Ribs protrude out of the surface by 1 m +1_SK_3x1 2 m wide and 2 m spaced ribs arranged in the Sharklet AF pattern. Ribs protrude out of the surface by 1 m +1_SK_2x2 2 m wide and 2 m spaced ribs arranged in the Sharklet AF pattern. Ribs protrude out of the surface by 3 m +3_SK_2x2 5 m wide and 5 m spaced channels formed below the surface to a depth of 3 m -3_CH_5x5 m wide and 10 m spaced channels formed below the -3_CH_5x5 5 m widesurface to a 5 surface to a depth of 3 m and 20 m spaced channels formed below the depth of 3 m -3_CH_5x5 118

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Table 5-2. Samples for Preliminary Assay Materials Topography Fibronectin (Y/N) # of Replicates Smooth Y 2 Smooth N 2 +1_CH_2x2 (DRIE) Y 2 +1_CH_3x1 (PR) Y 2 +1_SK_2x2 (DRIE) Y 2 PDMSe +1_SK_3x1 (PR) Y 2 Smooth Y 2 Smooth N 2 +1_CH_2x2 (DRIE) Y 2 Genipin Crosslinked Gelatin +1_CH_2x2 (DRIE) N 2 Table 5-3. Samples for Immunofluorescence Assay and Assay 3 Material Topography Fibronectin (Y/N) # of Replicates T CP Smooth Y 2 Smooth Y 2 +1_CH_2x2 (DRIE) Y 2 +1_CH_3x1 (PR) Y 2 E) Y 2 PDMSe ) Y 2 +1_SK_2x2 (DRIK_3x1 (PR +1_S Table 5-4. Samples for Assay 4 Material Topography Fibronectin (Y/N) # of Replicates PS Sm ooth Y 2 Smooth Y 2 +1_CH_2x2 (DRIE) Y 2 2 2 2 +1_SK_2x2 (DRIE) Y PDMSe +3_SK_2x2 (DRIE) Y 3_CH_5x5 (DRIE) -3_CH_5x10 (DRIE) Y -3_CH_5x20 (DRIE) 119

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Table 5-5. Feature Dimensions of Topograp hies Determined by WLIP aphy NaDepth (m) Material Topogr me Width (m) Spacing (m) +1_C H_3x1 (PR) .8 0.11.2 00.66 0.02 2 .1 +1_CH _2x2+1_SK_3x (DRIE) .3 0.11.7 00.86 0.03 1 (PR) .5 0.11.5 00.70 0.02 (DRIE) .8 0.12.2 00.89 0.02 Hydrated Gelatin (DRIE) .4 0.11.6 00.55 0.01 2 .1 2 .1 PDMSe +1_SK_2x 2-1_CH_2x2 1 .1 2 .1 Dehydrated Gelatin 1.3 0.1 0.77 0.02 -1_CH_2x2 (DRIE) 2.7 0.1 120

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CHAPTER 6 INFLUENCE OF TOPOGRAPHY ON SHEAR STABILITY OF ENDOTHELIAL CELLS uctione overall goal of thstigantiascaletopographies to improve ende to fed shs. Tance in tucces diamular own in Chapter 5, microscale topographies can be used to orient endothelial cells along with eir nuclei and focal adhesions. It is proposed that this will stabilize the cells against removal by shear stresses applied parallel to the orientation direction. Parallel plate flow chambers often have been used to evaluate cellular adhesion [71] as well as flow induced changes to cell structure [119, 120] and physiological functions [121]. These chambers generate constant shear stresses across the test substrates as defined by the following equation: Introd Th is research was to inve te the pote l for micro othelial cell resistanc low induc ear stresse his would be a significant adv he development of a s sful small eter vasc graft. As sh th whQw26 (6-1) In this relationship w refers to the shear stress at the wall (ie. sample substrate), is the viscosity of the fluid medium, Q is the volumetric flow rate, and h and w define the chamber height and width respectively. A derivation of Equation 6-1 can be found in Appendix B. In this study, porcine vascular endothelial cells were cultured on microscale topographies. Both channels and the Sharklet CE topography were produced in fibronectin-treated polydimethylsiloxane (PDMSe) elastomer. For both microtopographies, the protruding features (ridges and ribs) were 3 m wide, 1 m spaced and 1 m tall. Porcine vascular endothelial cells (PVECs) were cultured on these substrates and then exposed to a physiological shear stress (2 Pa). Cellular retention and morphological changes were then evaluated. 121

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Materials and Methods Design of Parallel Plate Flow Chamber The top and bottom plates of the chamber were generously supplied by Dr. Roger Tran-Son-Tays group. They are made of Lexan polycarbonate and the upper plate contains inlet and outlet ports (Fig. 6-1). The lower plate has an indentation and cut-out designed to fit a 1 inch by 3 inch glass slide. Preliminary tests of the flow chamber with a plain glass slide proved that it has a tendency to leak. Consequently, the chamber design was modified to create a better seal (Fig. 6-2). A glass slide was placed in the indentation to create a level surface and then the height and width of the chamber were defined by the use of specially prepared PDMSe gaskets. The gaskets were prepared so that the chamber dimensions (height x width) were 0.4x17.0 mm for the preliminary study and 0.5x12.5 mm for the final study. A Harvard compact infusion pump (Model 975) was used to provide continuous and steady flow. The pump held two 60 mL syrinProdtched ared PDMSe, wafers were rinsed with ethanol and then treated with hexamethyldizilazane (HMDS) via vapor phase ges and these were connected though a Y-shaped adapter. The second highest flow setting was used to deliver fluid at 60 mL/min. This arrangement resulted in shear stress of 2 Pa on the test substrate for both studies based on an approximate viscosity of PBS and media (~1 cP = 0.001 Pa*s), uction of PDMSe Topographies Topographically modified PDMSe films were prepared by direct casting against esilicone wafers. Two topographies were included: 2 m channels separated by 2 m wide ridges that are 1 m tall (+1_CH_2x2) and the Sharklet CE pattern with 2 m wide and 2 m spaced ribs that are 1 m tall (+1_SK_2x2). The topographically modified wafers were prepby James Schumacher using a 19 second DRIE cycle. Prior to replication with 122

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deposition for approximately 5 min.er sheets were taped to one side of er were placed on top of one of the plates and 1 mm thick f base e m. The +1_CH_2x2 topography was 2 in long and the +1_SK_2x2 topography was 1 in lond poured over top. A second polyester sheet covered glass plate was spacers. The PDMSe was allowed to cure for 24 h at room During this time, polyest two clean glass plates. HMDS treated waf spacers were placed at the corners of the plate. Silastic T2 was mixed (10:1 ratio oresin to curing agent), degassed and poured over the wafers. The top place was lowered onto thPDMSe until it rested on the spacers. The PDMSe was allowed to cure for 24 h at room temperature before being removed from the mold. Preliminary Shear Study Sample gasket preparation A razor blade was used to cut 16 mm wide samples from each topographically modified PDMSe fil g. The samples were cut to leave a 3 mm border at the ends of each length. In order to prepare each sample gasket, transparency films were cut to create thee 17 mm by 77 mm strips. Three strips were taped together using double sided tape (a total of two tape layers) and then the laminate was taped to a polyester-sheet-covered glass plate. Next a topographically modified PDMSe sample was suctioned pattern-side-down to the center of the transparency laminate. Spacers (2 mm thick) were placed in the corners of the plate. PDMSe was mixed, degassed an then lowered on top until it rested on the temperature and then the sample gasket was cut from the film. Samples were sterilized by soaking in 70% ethanol for 1 h. Then the depression of thesample gasket was treated with fibronectin (Biomedical Technologies, Inc.). It was supplied as a1 mg/mL solution of 0.03 M Tris Cl buffer (pH 7.8) in 30% glycerol. It should have been diluted to 50 g/mL in phosphate buffered saline (PBS), but it was mistakenly diluted using nanopure water. The indentation of each sample gasket was filled with 0.5 mL of the fibronectin solution 123

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and it was allowed to adsorb for 1 h at room temperature. Samples were then rinsed 2 times each with PBS and covered with PBS until cell seeding. Cell culture Porcine vascular endothelial cells were generously supplied by Dr. Block (Veterans Administration Hospital, Gainesville, FL). PDMSe sample gaskets were placed in the bo150 mm diameter Petri dishes. Four samples of each ttom of type (smooth, +1_CH_2x2 and PVECs (passage 3) were diluted to 100,000 cells/mL in 10% FBS hen the indentations of each sample gasket were filled with the cell sO2 and cells were llel rmaldehyde for 10 minutes. They +1_SK_2x2) were included supplemented RPMI media. T uspension (~0.5 mL per sample) to give a seeding density of 4,000 cells/cm 2 Cells were allowed to settle for 4 h and then more media was added so that the final level was approximately 2mm above the gasket. Samples were incubated at 37C and 5% C allowed to grow for 7 days. Shear treatment Two samples of each type were exposed to a wall shear stress of 2 Pa using the paraplate flow chamber. Phosphate buffered saline (PBS, 1X)) was used as the fluid medium and flow was applied at 60 mL/min for 2 minutes which represents the longest time frame possible for the 2 Pa wall shear stress on the substrates based on the syringe pump used. Staining and imaging Samples (both static and shear treated) were first fixed in 4% fo were subsequently rinsed 3 times with PBS and then stained with 1% crystal violet for 5minutes. Slides were gently rinsed in water until water remained clear. Imaging was performed using a Zeiss Axioplan 2 microscope at 400X magnifications. Eight images per sample were taken at each magnification for a total of 16 images per surface treatment and shear combination 124

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Images were taken from the central 1 cm wide by 2.5 cm long area of the shear samples to ensure fully developed flow had been applied. Cell density was measured by processing of the images software. Final change in PDMSe was 76.2 mm (3 in) long section was cut from the gasket center to dimensions. Note that all although the gasket was 2 mm thick, it compressed to 1.6r e PDMSe n the culture media. This was problematic because the endothelial cells settleks degassed and poured over top. A second glass plate was pressed above the mold until it rested using ImageJ Shear Study Gasket preparation Samples were prepared separately from the gasket for the final shear study. Thewas made so that smaller volumes of media would be needed during cell culture and so that all samples could be seeded with the same suspension of cells. Spacers (2 mm thick) were placedthe corners of a clean glass plate. PDMSe (50 g) was mixed, degassed and poured over top. A second clean glass plate was then lowered on top until it rested on the spacers. The allowed to cure for 24 h at room temperature and then the outline of the gasket was cut from thefilm. A 12.7 mm (0.5 in) wide and define the chamber mm thick when clamped into the flow chamber. As such, the gasket provided a chambeheight of 0.5 mm when combined with a 1.1 mm thick sample. PDMSe culture well preparation During a second preliminary study (not presented here) it was found that th samples tended to float i d on the Petri dish instead of the samples. To prevent this, a PDMSe culture well was produced which provides better adhesion to the samples. To produce the culture well, two staccontaining five 2 inch by 3 inch glass slides each were taped together using double-sided tape. The two stacks were taped side-by-side to a clean glass plate to create a 4 inch by 3 inch moldSpacers (6 mm thick) were placed around the mold and then PDMSe (160 g) was mixed, 125

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on the spacers. The PDMSe was allowed to cure for 24 h at room temperature and then it was removed from the mold. Excess PDMSe was cut away, leaving ~5 mm thick border around the 4inch by 3 inch well. The well was then placed in a 15 mm diam eter Petri dish to provide support. final shear study were 12.7 mm (0.5 in) wide, 76.2 mm (3 in) long and 1.1 mm t After that time, they were rinsed of 50 g/mL fibronectin was pipetted over the samples in the win the Sample preparation Samples for the hick. Each sample contained regions of smooth, +1_CH_2x2 and +1_SK_2x2 (Fig. 6-3). A razor blade was used to cut 25.4 mm wide and 12.7 mm long sections from each topographically modified PDMSe film. For each sample, one section of each of the topographies was suctioned patterned-side down to a clean glass plate with a half inch (12.7 mm) long gap between them. A fresh batch of uncured PDMSe (30 g per samples) was then mixed, degassed and poured over top. Spacers (1.1 mm thick) were used and a second glass plate was then pressed over top. The PDMSe was allowed to cure for 24 h at room temperature and then the 1 inch by 3 inch samples were cut from the film. Four samples were suctioned to the bottom of the PDMSE culture well and then the well and samples were immersed in 70% ethanol for 1 h to sterilize. thee times with PBS and then 20 mL ell. The fibronectin solution was allowed to adsorb for 1 h and then the samples were rinsed thee times with PBS just prior to cell seeding. Cell culture Porcine vascular endothelial cells (passage 2) were generously supplied by Dr. Block (Veterans Administration Hospital, Gainesville, FL). PVEC density was determined using a hemocytometer and then the suspension was diluted to 30,000 cells/mL in 10% FBS supplemented RPMI 1640 media. Twenty-five milliliters were placed above the samples 126

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PDMSE culture well to give a seeding density of 10,000 cells/cm 2 Samples were incubated at 37C and 5% CO 2 and cells were allowed to grow for 3 days. Shear treatment A razor blade wa s used to cut each sample in half down its long axis. One half was immed ing a and then stained with 1% crystal violet for 5 min. sing ImageJ software as described in Statisant rd errors diately fixed and stained as described below and the other half was exposed to 2 Pa of wall shear stress using the parallel plate flow chamber. In this manner, four samples were shear testeand four were not. The samples were suctioned into the indentation of the gasket usminimal amount of vacuum grease. Serum-free RPMI 1640 media was used as the fluid medium and shear was applied for 2 minutes which represents the longest time frame possible for the 2Pa wall shear stress based on the syringe pump used. Staining and imaging Samples (both static and shear treated) were first fixed in 4% formaldehyde for 10 minutesThey were subsequently rinsed 3 times with PBS Slides were gently rinsed in water until water remained clear. Imaging was performed using a Zeiss Axioplan 2 microscope at 400X magnifications. Twelve images were taken per surface treatment and shear combination. Images were taken from the central 1 cm wide and 38mm long area to ensure fully developed flow had been applied. Cell density, elongation andorientation were measured by processing the images u Chapter 5. tical Methods Two way analysis of variance (ANOVA, = 0.05) was used to determine if any significdifferences exist among the topography and shear treatments. Mean values 2 standaare reported. 127

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Results Preliminary Shear Study Endothelial c ells did not grow well for the preliminary shear study. The cells grew sporaly rupted s to be drawn. In general, the density of ture did not vary among the smooth and topographically modified PDM2x2 l. of cell culture, it was discovered that nearly all of the cells settled and proliferated against a far edge of the well. Only this sample was used in the analysis of rese nearly confluent on the smooth and topographically modified sections of the remaining sample prior to exposure to flow (Fig. 6-6). Cell growth was much more consistent dically across the samples, with areas of good cell growth surrounded by regions neardevoid of healthy cells (Fig. 6-4). The poor proliferation was attributed to the error in fibronectin treatment mentioned in the description of methods. The protein was dissolved in water rather than PBS which would have disrupted the conformation of the protein and disits ability to adsorb evenly to the PDMSe surfaces. Because of the wide discrepancies in cell proliferation within a sample, the variability in cell density was too great for any definitive conclusion cells grown in static cul Se samples (Fig. 6-5). The mean cell density on all samples was ~300 cells/mm 2 After exposure to 2 Pa of shear stress on the test substrate, greater differences in mean cell density were observed among the surfaces. Although statistical differences do not exist, the +1_SK_topography tended to improve cellular retention whereas the +1_CH_2x2 topography tended todecrease cellular retention relative to smooth PDMSe. Final Shear Study The floor of the PDMSe sample well for the final shear study must not have been leveAfter 3 days on one sample located ults. Cell density before exposure to flow on the other 3 samples was far too low (<1 cell/mm 2 ) to evaluate cell retention. Cells wer 128

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than observed in the preliminary study. Cell density did not significantly vary with surface cell density before flow among all surfaces was 520 60 cells/mm2 (Fig. lts DMSe to ). andom orientation (Fig. 6-9). The +!_SKs ll d that PVECs on both the +1_SK_2x2 ographies appeared to maintain better spreading than cells on smooth fibron ced Alternatively, PVEC spreading on both topographies topography and the average 6-7). The topographies induced morphological changes in the PVECs consistent with the resuof Chapter 5. Cell elongation increased from 2.3 0.3 on smooth fibronectin-treated P3.7 0.3 and 3.8 0.3 on the +!_SK_2x2 and +1_CH_2x2 topographies respectively (Fig. 6-8Orientation was also increased by the topographies. Hermans parameter for the smooth control was not significantly different from zero, indicating r _2x2 and +1_CH_2x2 topographies induced orientation to a similar degree, with Hermanparameter values of 0.90 0.04 and 0.92 0.01 respectively. Cell density decreased significantly on all topographies after exposure to 2 Pa of washear stress in the flow chamber for 2 minutes (Fig. 6-7). The average settlement density after flow among all surfaces was 220 40 cells/mm 2 and it did not significantly vary with topography. Likewise, cell retention was statistically equivalent on all surfaces with 43 20% of cells retained (Fig. 6-10). Closer inspection of the after flow images reveale and +1_CH_2x2 top ectin-treated PDMSe (Fig. 6-6). The cells on the topographically modified sections werespread relatively evenly across the surface, whereas the cells on the smooth section tended to be isolated to small, dense clusters. Evaluation of mean cell areas before and after flow confirmed these results (Fig. 6-11). Cell spreading on smooth fibronectin-treated PDMSe was redufrom 1,100 100 m 2 to 470 100 m 2 129

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was ~1000 m 2 and did not significantly change after exposure to 2 Pa of shear stress on thesubstrate. test Discussion ecause hat surface chemistry is more imporly opographies enhance the ability of PVECs to maintain cell sprea ad topography to some degree so that the cells were presented with a smoother surface than what is indicated by the reported feature dimensions. In the preliminary shear study, the +1_SK_2x2 topography tended to improve cell retention after exposure to shear whereas the +1_CH_2x2 topography tended to decrease it. Initially these results seemed promising and were consistent with the trend seen with algae spores (Ulva) for similar topographies with 3 m feature heights [122]. Unfortunately, no definitive conclusions regarding cell retention can be drawn from the preliminary study bthe PVECs response was too variable due to inadequate pretreatment of the PDMSe surfaces with fibronectin. The results of this study clearly demonstrate t tant that topography when attempting to support endothelial cell growth. Unfortunately, the trends in endothelial cell retention suggested by the preliminary study could not be confirmed. The final shear study indicates cell retention is not significantimproved by either topography, at least not in terms of cell density. Alternatively, both the +1_SK_2x2 and +1_CH_2x2 t ding during exposure to flow. This is consistent with the hypothesis outlined in Chapter 1which states that cells are removed when exposed to flow because they must break focal adhesions to adapt their morphology. These results suggest that endothelial cell retention might be improved by further optimization of material chemistry and topographical dimensions. The discussion from Chapter 5 on the possible infilling of the topographical recesses with fibronectin needs to be revisited here. It is unclear what if any affect the protein adsorption hon the shape of the topography. It is conceivable that the protein may have filled in the 130

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In the most extreme scenario of infilling, fibronectin may be wicked away from the ridge tops and co mpletely fill the topographical recesses. In such a case, the resulting surface is smooth but still has the potential to direct cth based on patterned chemical and mech than a s faces tall features havinure to in ellular grow anical (due to phase contrast) cues. The fibronectin in the recesses would be thickermonolayer and therefore more susceptible to being partially desorbed by flowing media. Thiwould help explain why so many cells were removed from the topographically modified surdespite the fact that the PVECs were aligned in the flow direction. As such, combining the topographies with a different material or switching to two-dimensional chemical patterns might yield better results. Conclusion This study investigated endothelial cell adhesion to topographies with 1 m g 2 m lateral dimensions. Such topographies incorporated into fibronectin-treated PDMSe help PVECs maintain cell spreading but do not improve cell retention after expos2 Pa of shear stress on the substrate. The adsorbed fibronectin layer should be characterizedorder to gain further insight into the nature of contact guidance observed in this study. Endothelial cell retention may be improved by combining the topographies with a different material or by switching to two-dimensional chemical patterns. 131

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Figure 6-1. Original design of flow chamb Figure 6-2. Modified design of flow chamber. er. 132

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igure 6-3. Layout of samples for the final shear study. White regions depict smooth areas of the sample. F 133

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Figure 6-4. PVECs before exposure to flow in the preliminary shear study. Images were taken at 400X. Thee images (low, middle and high density) are given per surface type to indicate the high variability observed. Scale bars represent 100 m Smooth +1 SK 2x2 +1 CH 2x2 134

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Preliminary S hear Study 0100200300400500SM+1 SK 2x2+1 CH 2x2Cell Density (#/mm2) Before Flow After Flow Figure 6-5. Density of PVECs before and after flow in the preliminary shear study. Smooth and textured PDMSe samples were fibronectin treated. Cells were grown for 7 days and then exposed to 2 Pa wall shear stress for 2 min. There are no significant differences between any of the treatments (ANOVA, = 0.05). 135

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136 Figure 6-6. PVECs grown on topographies in the final shear study. Cells were grown on smooth, +1_SK_2x2 and +1CH_2x2 fibronectin treated PDMSe. Images were taken at 400X before and after exposure to flow. Topographical features are oriented horizontally in the images. Scale bars represent 100 m SM +1 SK 2x2 +1 CH 2x2 Before Flow A fter Flow

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0100200300 40 0500600700800SM+1 SK 2x2+1 CH 2x2Density (cells/mm2) Before Flow After Flow* ** ** ** Figure 6-7. Density of PVECs on topographies before and after flow in the final shear study. Cells were grown on smooth and topographically modified PDMSe for 3 days and then exposed to 2 Pa wall shear stress for 2 min. Asterisks denote statistically indistinct groups (t-test, = 0.05). 012345SM+1 SK 2x2+1 CH 2x2Major Axis / Minor Axis ongation of PVECs on topographies before flow in the final shear study. Cells e grown on smooth and topographically modified PDMSe for 3 days. Tie barote statistically indistinct groups (t-test, = 0.05). Figure 6-8. Elwers den 137

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-0.4-0.20.00.20.40.60.81.0SM+1 SK 2x2+1 CH 2x2Hermans Orientation Parameter Figure 6-9. Orientation of PVECs on topographies before flow in the final shear study. Cells were grown on smooth and topographically modified PDMSe for 3 days. Tie bars denote statistically indistinct groups (t-test, = 0.05). 0102030405060708090100SM+1 SK 2x2+1 CH 2x2Cells Retained (% ) Figure 6-10. Retention of PVECs based on topography in the final shear study. Cells were grown on smooth and topographically modified PDMSe for 3 days and then exposed to 2 Pa wall shear stress for 2 min. Retention does not vary significantly among the surfaces (t-test, = 0.05). 138

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s sterisks denote statistically indistinct groups (t-test, = 0.05). Figure 6-11. Average area for PVECs on topographies before and after flow in the final shear study. Cells were grown on smooth and topographically modified PDMSe for 3 dayand then exposed to 2 Pa wall shear stress for 2 min. A 02004006008001000120014001600SM+1 SK 2x2+1 CH 2x2Area per Cell (m2) Before Flow A fter Flow** ** ** **** 139

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CHAPTER 7 CONCLUSIONS AND FUTURE WORK Conclusions The ability to improve endothelial cell retention to biomaterial surfaces would be a significant advancement in the search for a suitable synthetic small diameter vascular graft. This work is the first known attempt to evaluate the influence of engineered microscale topographies on endothelial cell retention during exposure to flow. The engineered microtopographies were designed to orient endothelial cells in static culture with the goal of increasing their resistance to shear. Engineered topographies significantly influence both wettability and biological adhesion to theories. Itfrom 108 ed topographies. Channel topographies with widths ranging from 5 to 20 m and a depth of 5 m increase alignment of PVECs relative to smooth PDMSe. Endothelial cells settle within the channels and do not migrate over ridges, preventing confluence from being reached. Based on this earlier work with cell adhesion to fibronectin-treated PDMSe microtopographies, it was decided that smaller topographical features would be necessary to achieve confluence. A feature width and spacing of 2 m was selected based on the size of focal adhesions in endothelial cells. Additionally, gelatin was investigated for its ability to be micropatterned and maintain shape. Because gelatin dissolves readily in water under physiological temperatures, two crosslinking systems were investigated for their ability to stabilize the film. Genipin and glutaraldehyde were found to be equally efficient at improving modulus, but genipin is far better at decreasing the swelling of gelatin in water. The results biomaterials. The topographies were developed though insights gained from classical wetting was shown that the water contact angle of hydrophobic PDMSe can be increased 4 to 135 3 though properly scal 140

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indicate that 10% (w/w) genipin in gelatin (2.9% MEQ) is the most efficient choice for stabilizing the films. It was shown that 2 m features were sufficiently replicated in gelatin films of this comafter extending leaching of genipin into water and fibronectin adsorption to the gelatin surface. lls grew to confluence on smooth and micropatterned PDMSe samples with adsorbed fibronectin and the topographies reoccupied per cell more closely approximated that of endothelial cells found in vivo. e. The adsorbed fibronectin layer should be characterized in order to gain further insight into the nature of contact guidance topographies with a different material or by switching to two-dimensional chemical patterns. The ent Future Work Future directions of this project could include further examination of the alignment of cytoskeletal components to microtopographies as well as the examination of the physiological state of the endothelial cells as a function of the topography. In particular, the alignment of actin position. Porcine vascular endothelial cells (PVECs) were cultured on genipin crosslinked gelatin and PDMSe substrates. Unfortunately, the cells would not grow on the gelatin substrates even It is suspected that the toxicity of residual unreacted genipin is too high for the cells to remain viable. Ce sulted in a significant increase in focal adhesion and nuclear alignment. Additionally PVECs on some of the topographies spread so that the average area Endothelial cell adhesion to topographies with 1 m tall features having 2 m lateral dimensions was investigated in shear studies. Such topographies incorporated into fibronectin-treated PDMSe help PVECs maintain cell spreading but do not improve cell retention after exposure to flow generating 2 Pa shear stress on the test substrat observed in this study. Endothelial cell retention may be improved by combining the ability to improved endothelial cell retention would be a great step forward in the developmof viable synthetic small diameter vascular grafts. 141

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filaments with flow has been discussed in the literature and it would be a nice advance ifcould be evaluated on the microtopographies presented here as well. This was one of the goals of the fluorescent cell cu this lture assay in Chapter 5, but the resolution of the images was not high enoug al f the vesse cules to atomic force microscopy (AFM) could be used to probe the presence of E-selectin on endothelial cells cultured on the microtopographies thoug h to achieve this. It would be recommended that a higher aperture objective be used to improve this so that individual actin filament bundles could be distinguished from each other. Even though cells grew to confluence on the textured substrates that does not necessarily indicate that they are performing normal biological functions. Endothelial cells are responsiblefor maintaining the homeostasis of the vasculature. Endothelial cells accomplish this though the release and expression of factors which affect coagulation state, cellular proliferation and leukocyte trafficking. One indicator of endothelial dysfunction is the impairment of endothelinitrous oxide formation which modulates vessel tone [123]. This results in stiffening o l wall which is associated with atherosclerosis. Additionally, it has been shown that endothelial production of nitrous oxide modulates platelet adhesion which is a hallmark of inflammation [124]. Release of nitrous oxide into the culture media by cells grown on the textured substrates should be examined as an indicator of the physiological state of the cells. Another indication of endothelial dysfunction is the induction of adhesive glycoproteins onthe cell surface. During inflammation, activated endothelial cells present selectin moletheir surfaces which bind with lectin molecules on leukocytes in the blood. For instance, E-selectin on endothelial cells (an inducible glycoprotein receptor) binds with Sialyl Lewis X of various leukocytes. As demonstrated by Feinberg, h the use of a tip modified with Sialyl Lewis X [54]. Additionally, leukocytes isolated 142

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from blood could be exposed to endothelial cells cultured on the microtopographies within a parallel plate flow chamber to examine their interaction under controlled shear. It would be interesting to also investigate whether the engineered topographies can be useto sort out robust cells from a population of cultured endothelial cells. Among any given cell population, a distribution of cell viabilities exists. As shown in Chapter 6, the endothelial cells d that re again s ting ort of replican capturing scanning electron microscope (SEM) images of lyophilized samples. main on the engineered topographies maintain spreading better than the cells remaining onthe smooth control. The cells that remain may have survived because of an inherent biological advantage. Dr. Mark Segal (Department of Nephrology, University of Florida) suggests that it would be beneficial to re-culture the remaining cells to confluence and then expose to flowto determine if shear resistance is improved in the second generation. In addition to the proposed studies on actin alignment and endothelial functions, there ialso a need for further research into producing a microtextured hydrogel capable of supporendothelial cell growth. This would be especially important in the development of a tissue engineered vascular graft because the supporting membrane would need to allow the transpwater and nutrients. Genipin crosslinked gelatin, the hydrogel examined here, was capable of ating microscale topographic features with adequate mechanical stability but endothelial cells did not proliferate on it in vitro. This might be able to be accomplished though a better process of leaching unreacted genipin from the gelatin. Genipin is more soluble in ethanol thwater and so an extended (several days) soak in 70% ethanol may be sufficient. Gelatin wouldremain swelled while unreacted genipin should diffuse to the surrounding ethanol. Additionally, extended soaks under in vitro conditions (RPMI 1640 media at 37C) may also work. If work iscontinued with this hydrogel, it would also be beneficial to examine its pore structure by 143

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The ability to improve endothelial resistance to shear removal while maintaining normacellular function would be a significant advance toward engineering an effective l small diameter vascu lar graft. 144

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APPENDIX A Year Author(s) SUMMARY OF LITERATURE ON CELLULAR RESPONSES TO TOPOGRAPHY Table A. Chronological Listing of Literature on Cellular Responses to Topography Material(s) Topography Cell Types(s) Results 19 11 Harrison [125] spider webs cylindrical frog embryonic neuronal cells spindle shape cells long projections align with fibers 1914 Harrison [41] spider webs and clotted plasma cylindrical mesenchymal stem cells from sea urchin embryo frog embryonic neuronal cells chicken embryonic neuronal cells cells aligned to the fibers 1979 Ohara and Black [126] polystyrene epoxy channels 2 and 10 m wide with 5 to 30 m repeat spacing depth not indicated chick heart fibroblasts murine epithelial cells percent aligned cells increased with decreasing spacing cells bridged 2 and 20 m channels 1982 Wilkinson et al. [127] serum-coated glass channels 2 m wide and 2 m deep human neutrophil leukocytes cells were more likely to migrate along channels than across them 1983 Brunette et al. [128] titanium-coated silicon channels (V shaped) 70, 130 and 165 m wide with 80, 140 and 175 m repeat spacing human gingivival cells porcine epithelial cells cells aligned to long axis of channels epithelial cells didnt bend around ridges cells migrated along channels 1986 Brunette [129] titanium coated silicon epoxy replicates and photoresist channels (square and V-shaped) 0.5 to 60 m depth and 30 to 220 m repeat spacing porcine periodontal ligament epithelial cells cells aligned to channels orientation increased with decreased spacing some cells crossed ridges and extended into channels migration directed by channels deep depth enhanced guidance of cells 1986 Dunn and Brown [130] quartz channels 1.65 to 8.96 m wide with 3.0 to 32.0 m repeat spacing and 0.69 m depth chick heart fibroblasts alignment depended greater on ridge width than spacing alignment increased with decreasing ridge width 1986 Brunette [131] titanium coated silicon epoxy replicates and photoresist major channels (square and V-shaped)5 to 120 m depth (width not indicated) minor channels 2 m deep on floor at 54 to major channels human gingivival fibroblasts cells aligned on channels and flat ridges cells oriented preferentially with major channels cells oriented with minor channels if no major channels 145

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Table A. Continued Year Authors Material(s) Topography Cell Types(s) Results 1987 Clark et al. steps 1, 3, 5, 10 and 18 s ic eight sed, cells d more and ss likely to twice as often as other cell types [132] Perspex m height BHK cells as step hincrea chick embryon neural cells chick heart fibroblasts rabbit neutrophils orientewere le cross the step rabbit neutrophils crossed 5 m steps 1987 Hoch et al. [133] polystyrene ridges 0.5 to 100 m Uidges no differentiation on ridges shorter than g wide and 0.5 to 62 m spacing with 0.03 to 5 m heights romyces appendiculatus fungus differentiation maximized on 0.5 m tall r 0.25 m or taller than 1.0 m erm tubes highly oriented on ridgesspaced 0.5 to 6.7 m 1988 Chehroudi et al. [133] ecl rat pl implant model cd ns of d poxy hannels (v shaped) 17 m wide and 10 m deep with 22 m wide ridges porcine periodonta ligament epithelial cells arieta hannels increased epithelial attachment an orientation shorter length epithelial attachment and longer connective tissue attachment to channel se ctioimplant compareto smooth parts channels impeded epithelial down growth 1988 Wood [134] qcdepths mtissue cells highest alignment uartz hannels .98 to 4.01 m wide and spaced with 1.12 to 1.17 m esenchymal cells migrated alongchannels seen on widest repeat spacing 1989 Campbell et al. [43] Versapor filters PDMS coated filters P subcutaneously implanted into dogs res m tissue ore diameters 0.4 to 3.6 m nonadherent, contracting capsules around implants with pore diameters < 0.5 m thin, tightly adhered capsules on implants with pofrom 1.4 to 1 .9pores 3.3 m infiltrated by inflamm atorylittle variation with respect to surface chemistry 146

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Table A. Continued Year Authors Material(s) Topography Cell Types(s) Results 1990 Chehroudi et al. titanium-coated epoxy channels (square and V-shaped)30 m repeat rat parietal implant ls th endothelial cells fi,10 fibroblasts inserted e r to h [135] spacing with 3,10 or 22 m depth; 7 and 39 m repeat spacing with 3 or 20 m depth model endothelial celattached to smooand 3,10 m channels bridged 22 m channels broblasts encapsulated smooth and 3m channels into 22 m channels pithelial down growth greatest on smooth and channels oriented parallel to implant length epithelial down growth least on 10and 22 m channels perpendiculaimplant lengt 1990 Clark et al. [136] Perspex cith equal width and spacing and 0.2 to 1.9 m depths BHK and MDCK ay a hannels 4-24 m repeat w lignment inverselproportional to spacing and directly related to depth lignment influence by depth more than spacing 1991 von Recum [44] silicone deal s in vivo study in rabbits 2res us cm ore d Schmidt and imples 2, 5 and 8 m wide/spaced; 0.38 to 0.46 m high murine peritonmacrophage and 5 m textu yielded less mononuclear cells and thinner fibrocapsules ells on 2 and 5 e m textures werelongated ancontained more pseudopods 1991 Clark et al. [136] Quartz with poly-l-lysine adsorption channels ~130 nm wide and ~130 nm spaced at depths of 100, 210 and 400 nm BHK MDCK chick embryonic neurons Bsins unaffected by the topography HK alignment increased with depth ngle MDCK aligned perfectly on all and elongation increased with depth epithelial islands of MDCK and all embryonic neuro 147

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Table A Continued Year Authors Material(s) Topography Cell Types(s) Results 1993 Clark et al. [137] quartz treated with hydrophobic silane and laminin chemical stripes 2,3,6,12 and 25 m wide with equal spacing and 2 m stripes spaced by 50 m chick embryo neurons murine dorsal root ganglia neurons ed growth cones bridged nching y 15 m alignment decreaswith decreased width narrow non adhesive stripes eurite bran reduced bstripes 1993 Meyle et al. human gingivival co [138] epoxy channels (square) 0.5 m width and sapce with 1 m depth fibroblasts ells aligned with channels and ei ther bridged or conformed tfeatures 1993 Meyle et al. [139] epoxy channels (square) 0.5 m width and space with 1 m depth human gingivival fibroblasts c cellular prostheses did not extend into channel corners ytoskeletal elementsoriented along channels 1993 Oakley and Brunette [140] titanium-coated silicon channels (V-shaped) 15 m wide and spaced with 3 m depth human gingivival fibroblasts m t 20min aat icrotubules orientedat bottom ofchannels actin first observed wall-ridge edges after 40 to 60min most cells had aligned focal contacts by 3hrs 1994 Meyle [141] ated by radio frequency glow discharge sspaced and 1 m deep human gingivival fibroblasts c focal PDMS tre hannels (square) 1 m wide, 4 m ells and contacts aligned with channels 1995 W lated quartz with poly-l-lysine h 0.1 to 1.17 m depths rat optic nerve oligodendrocytes o hippocampal and cerebellar neurons (HCNs) ONOs and ONAs HOetwork Ohen r ebb et al. [142] chrome-pcoated channels 0.13 to 4.01 m wide and 0.13 to 8m spaced wit (ONOs) ptic nerve astrocytes (ONAs) aligned with channels CNs did not align NAs showed extensive n of actin stress fibers while ONOs did not NOs alignment maximized w channel width same as axon diamete 1995 Chesmel and Black [143] polystyrene channels radial arrays of 5 m long channels at 1 intervals;, 0.5 5 rat calavarial cells ein c Es m wide and 0.5 orm deep multi-layer protadsorption onfluence in 4 daysCM in 7 day 1995 Chou et al. [144] titanium-coated silicon treated with radio frequency glow discharge cm repeat spacing depth not indicated human gingivival fibroblasts ctw hannels (V shaped) 3 m wide with 6 to 10 ells aligned and secreted m ore fibronectin on channels ice as much ECM on channels 148

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Table A. Continues Year Authors Material(s) Topography Cell Types(s) Results 1995 den Braber etal. [145] PDMS treated with ultraviolet (UV) ge (RFGD) .5 ion than 10 gn UV an light and radiofrequency glow dischar channels (square) 2, 5and 10 m wide withequal spaces and 0m depth rat dermal fibroblasts 2 and 5 m channels induced strongerorientat m channels rowth lower otreated surface th RFGD treated surface 1995 Meyle et al. [146] silicon dioxid e human fibroblasts gengivival nmonocytes macrophages 100% of fibroblasts n ytes or channels 0.5 m wide and spaced and 1 mdeep keratinocytes eutrophils, and 20% of macrophages aligned o orientation ofkeratinoc meutrophils 1995 Oakley and Brunette [147] titanium coated silicon treated with radio frequency glow discharge channels (V shaped) 15 m wide and spaced and 3 m deep porcine epithelial cells clong wall ells oriented along channels actin filaments and microtubules oriented a and ridges cell alignment less variable within single cells than clusters of cells 1995 Wojciak-Stothard et al. channels 0.5, 5, 10 and .5 s murine P388D1 c nnels caster ls more c-nt [148] quartz 25 m wide with equal spacing and 0and 5.0 m depth macrophages ells spread faster on shallower chaells elongated fon deeper channe cells elongated on wider channels hannels increased F actin during initially attachme 1995 [149] qcs; spacing not indicated Fsation m Wojciak-Stothard et al. uartz hannels 5, 10 and 25 m widths and 0.5, 1, 2 and 5 m depth B HK cells -actin conden at discontinuities in topography condensation typically at right angles to channel edge with 0.6 m periodicity vinculin orientation similar to actin icrotubules formed after 30min 1996 dal. [98] d with radio frequency glow discharge c fibroblasts nel en Braber et silicon treate hannels 2, 5 and 10 m wide/spaced and 0.5 m tall rat dermal proliferation did notvary with tex ture alignment of cells increased with decreasing chanwidth 1996 den Braber et al. [150] Pcnd rafibroblasts cde ent DMS treated with radio frequency glow discharge hannels 1 to 10 m wide and 1 to 10 m spaced with 0.45 a1.00 m depths t dermal ells oriented on ridge 4 m wi channel width and depth did not affect cellular alignm 149

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Table A Continued Year A uthors Material(s) Topography Cell Types(s) Results 1996 Britland et al[151] channels 100, 50, 25, 12 and 5 m repeats with 0.1, 0.5, 1, 3, and ridges and perpendicular to topography B st P and quartz 6 m depths microcontact printing (CP) on smooth, in channels, on HK21 C13 cells aligned strongest to 25 mwide CP and 5 m wide, 6 m deep channels ress fibers and vinculin alignedwith C channels cell alignment enhanced on parallel channels and CP cells aligned to adhesive tracks on channels with matched pitch rength of c stues became more matched when channels became narrower and deeper 1996 Matsuda and Sugawara [47] photoreactive poly(N,N-dimethylacrylamide-co-3-azidostyrene on tissue culture PS channels 130, 80, 60, 40 and 20 m wide spaced 20 m apart bovine aortic endothelial cells c ells avoided photoreactive regions & aligned in channels alignment increased with decreased channel width 1996 Mrksich et al. [48] polyurethane coated with gold and patterned with alkane thiols and adsorbed fibronectin V-sahped channels 25 and 50 m wide with equal spacing; depth not indicated bovine capillary endothelial cells orbed cells only attached to fibronectin regions cells attached to channels or ridges, whichever possessed the alkane thiol and therefore adsfibronectin 1996 Wokciak-Stothard et al. [152] fused silica square ch10 m widths with 30 to 282 nm depths P388D1 macrophages rat peritoneal macrophages c annels 2 and ells activated and spread along channels increased membrane protrusions increased F-actin and vinculin along edges of single steps or channels 1997 Rajnicek et al. [153] quartz coated with poly-l-lysine channels (square) 1, 2 and 4 m wide and 14 embrXenopus spinal cord neurons rat hippocampal ll hippocampal neurons lar nm to 1.1 m deep yonic neurons Xenopus neurites grew parallel to achannels grew perpendicuto shallow, narrow channels and parallel to deep, wide channels 150

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Table A Continued Year Authors Material(s) Topography Cell Types(s) Results 1997 Chehroudi et al. [154] epoxy coated with titanium channels (V-shaped) 35-165 m wide at 30,60 and 120 m depths pits (V-shaped) 35 to rat parietal bone implant model only bm h rient 270 m wide and 30, 60 and 120 m depths mineralizationon topographies one-like foci decreased as channel depth increased ore mineralizationas channel deptincreased bone-like foci oon channels 1997 Rajnicek and McCaig [155] quartz polystyrene both were coated with poly-l-lysine channels (square) 1, 2 and 4 m wide and 14 nm to 1.1 m deep embryonic Xenopus spinal cord neurons rat hippocampal neurons ctaut as e channel, G ein tein ell orientation unaffected by cytochalasin B xol and nocodazole disrupted hippocampal microtubules bdid not affect orientation lignment of neuriteaffected by somcalcium protein, protkinase and protyrosine kinase inhibitors 1998 Chou et al. [156] titanium-coated channels (V shaped) 6 to 10 m repeat spacing with 3 m human gingivival fibroblasts rs c silicon depth cells oriented along channels by 16 hhannels altered the expression and levels of adhered matrix metalloproteinase-2mRNA 1998 den Braber et al. [99] silicone channels (square) 2, 5 and 10 m wide and spaced with 0.5 m depths rat dermal fibroblasts microfilaments and y es fibronectin and vinculin aggregatesaligned with 2 m channels onl vinculin located mainly on ridg vinculin located in channels 1998 van Kooten et al. [157] PDMS channels (square) 2, 5 and 10 m wide with 4, 10 and 20 m spaces and 0.5 m depths human skin fibroblasts s-cs ls topography slowed cell entrance to phase of cell cycle ells proliferated les on 10 m channethan on 2 and 5 m channels 1999 Palmaz et al. [49] nitinol c 22 m al cells c hannels 1, 3, 15 and human aortic endotheli increasing channel sizes increased migration rate ells aligned and elongated with channels 151

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Table A Continued Year Authors Material(s) Topography Cell Types(s) Results 1999 van Kooten and von Recum silicone coated with 5 human skin fibroblasts human umbilical l n size d [45] fibronectin (FN) ridges 2, 5 and 10 mwide and spaced 4, 10and 20 m apart respectively with 0. m heights vein endotheliacells topography influenced initial focal adhesioand density aninitial FN deposition no difference in FN networks by day 6 1999 Walboomers et polystyrene (PS) titanium-coated polystyrene (Ti-PS) id channels 1, 2, 5 and 10 m widths and or Ti-c alignment on PS, sed ed cg al. [158] silicone poly-L-lactic ac(PLLA) -all samples were plasma treated spaces with depths of 0.5 m rat dermal fibroblasts P reproduced better than silicone S and PLLA PS ell proliferation greater on PLLA and silicone surfaces PLLA and silicone surfaces increawith decreas channel width ells aligned on all Ti-PS channels equally reatest alignment on1 m PLLA channels 1999 Walboomersal. [46] et m arrow and polystyrene channels 1-20 m wide and 0.5-5.4 deep rat dermal fibroblasts orientation increasedwith channel depth cells follow contours of shallow and wide channels but bridge ndeep features 2000 Deutsch et al. [159] silicone pillars (rounded) 10 m wide and 10 to 50 m spaced arat myocytes mtached c channels 10 m wide and spaced ll were 5 m deep yocytes at four times as oftento pillars than to smooth PDMSe ells oriented on channels 2000 Pins et al. [160] gelatin collagen-glycosaminoglycan co-precipitate channels 40-200 m wide and 40 to 200 m deep keratinocyte ons cells differentiated toform analogs of basal lamina with invaginati 2002 Petersen et al. [161] agarose gel x5 depths were 18 to 40 m avian chondrocytes s mcined in shells pits (rectangular) 35x5, 35x10, 45x5, 25x5, 25x10, 55x5,15x5, 15x10 and 55m cells isolated in wellore cells isolated per well as dimension increased from 15 to 25 m ells not reta wells >35 m; allower cells retained fewer c 152

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Table A Continued Year Authors Material(s) Topography Cell Types(s) Results 2002 Barbucci et al. sulfonated hyaluronic acid (HyalS) on chemical stripes 10, ricells ripe [31] glass 25, 50 and 100 m wide & spaced HyalS dges in HyalS -step height 300 nm 1 m HGTFN endothelial decreasing stdimensions increases cell locomotion and orientation 2003 Dalby et al. [162] quartz cwide, 2.5 m spaced with 2 m depth imprimary human fibroblasts hannels 12.5 m mortalized cei aligned within channels ells and nucl 2003 Magnani et al. [51] hyaluronic acid (Hyal) and sulfonated s chemical stripes 10, 25, 50 and 100 m;wide and spaced HyalS ridges in HyalS -step height of less than 300 mouse fibroblasts (3T3) human primary fibroblasts bovine aortic endothelial cells human aortic endothelial cells d hyaluronic acid (HyalS) on glas nm to 1 m ecreasing stripe dimensions increased cell locomotion and orientation; 2003 Scheideler et al. [163] ated ) c 0.4 to 2 m deep ridges 1-20 m wide and 0.44 to 2 m tall human foreskin fibroblasts keratinocytes k to ti tanium co silicon (channelsand epoxy replicates (ridges) hannels 1 to 20 m wide and fibroblasts aligned to topography eratinocytes did not align on ridges 2 10 m wide adsorbed fibronectinenhanced cell spreading 2003 Teixeira et al. silicon oxide deposited on silicon channels (width x space)) 70x330, 250x550, 400x800, 0, epths of 0.6 or 0.15 m human corneal lls cth a [164] wafers 650x950, 850x115and 1900x2100 nm with d epithelial ce ells aligned wiridges percent aligned cells independent of lateral dimensions alignment increased with channel depthctin filaments and focal adhesions aligned 2004 Tan and Saltzm an [165] patite on silicon lastMG-63 cell lines c surfaces hydroxya micro 4 m wide and deep ridges spaced 10 m apart and 4 m wide and tall pillars spaced 4 m apart; nano randomly rough human osteoblike Saos-2 and ells aligned parallel to ridges on both micro and micro/nano structured 2004 Wang and Ho [166] chitosanatin m spaced with 10 m depth human microvascular endothelial cells and gel channels 10, 20, 30 and 50 m wide and10 or 50 protein resisistant tri-block copolymer applied to ridges cells restricted to channels by tri-block copolymer cell spreading decreased as channel width decreased 2005 Recknor et al. [167] polystyrene m wide, es 6 m wide, 13 m spaced and 4 m deep for AHPCs astrocytes adult rat hippocampal progenitor cells (AHPCs) gaxially along channels channels 10 20 m spaced and 3 m deeofor astrocytand 1 rat type-1 reater than 85% alignment of astrocytes seen on lamin coated channels AHPCs adhered and extended processes 153

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Table A Continued Year Authors Material(s) Topography Cell Types(s) Results 2005 Uttayarat et al. PDMSe m ells fos nels hies eep celation was similar on all [50] channels 2.7 to 3.9 wide and 3.3 to 5.5 m spaced with depths of 0.2, 0.5, 1.0and 4.6 m Bovine aortic endothelial c cell elongation andalignment increased with channel depth cal adhesion formed in chanfor all topograpexcept 4.6 m dl prolifer substrates 2005 Yang and Ou [112] gand bound to glass cn glass elatin crosslinked with glutaraldehyde ridges at least 5 m wide and at least 10 m spaced with 1.5 m height human l mesenchymastem cells ells selectively adhered to gelatiand away from 2006 Charest et al. [168] polyspaced by 10, 20 or 100 m wide lanes pf osteoblast-like cells walign to r emistry imide channels 8 m wide, 4 m deep, 16 m spaced chemical stripes 10 m wide lanes of FN PEG MC3T3-E1 cells aligned to both topography and chemical patt erns separately hen presented with both, cells topography rathethan ch 2006 Pins et glass treated with tin (FN), fibrinogen (FG) and bovine serum albumin (BSA) c hfibroblasts c al. [169] organosilane and printed with fibronec hemical stripes 600 nm wide and either 10or 40 m spaced and 20 m long uman dermal ells on FN and FGpatterns had greater tendency to spread across adjacent structures than on BSA patterns 2006 Yu et al. [170] chitosan-collagen-gelatin blend c0 h hmesenchymal stem cells ls grated ctopographies smaller hannels 200 and 50m wide and spaced with 80 m depth an d10, 20 and 50 m wide and spaced wit20 m depth uman cells on 200 m channels initially adhered in channeand later mito ridges ells oriented parallel to the 200 m channels dered than cells hinproliferation 2006 Teixeira et al. silicon channespace)) 70x330, n thelial c ch b [171] ls (width x 250x550, 400x800, 650x950, 850x1150, and 1900x2100 nm 600 nm depth for all features primary huma epi cornealcells ell alignment switched from perpendicular toparallel when pitincreased from 400to 4000nm etween 800 and 1600nm pitches both parallel and perpendicular alignment is observed 154

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APPENDIX B CALCULATION OF SHEAR IN PARALL FLOThe following is the calculation of shear stress at the plate for pressure flow through two parallel plates. The important chamber idth (w) of the plats and height (h) of the separation (Fig. B). The calculatior, fd flStart with the equation of continuity assuming constant density as shown EL PLATE W dimensions are the wn assumes lamina ully develope ow with h<
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Substituting this into Equation B-4ing: gives the follow xxgxpyv20. (B-7) 2Because flow direction is perpendicular to gravitational force, gx=0 and Equation B-7 becomes: 22yvxpx (B-8) Because velocity is zero in all directions except x (Equation B-2) and velocity in the x-direction is independent of x (Equation B-3), solution of Navier-Stokes equations for y and z components proves the following: 0zpyp (B-9) Therefore, pressure is only a function of x (or a constant). Equations B-3, B-5 and B-6 indicate that vderiva x is only a function of y (or a constant). In order for Equation B-8 to be true, the tive of P with respect to x must be equal to a constant as shown below: 1Cp x (B-10) 2221Cxpyvx (B-11) Integrating Equation B-11 once gives 3 1 Cyxpyvx. (B-12) Application of the following boundary condition based on symmetry in the y-direction to Equation B-12 156

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00 xv ( yyB-13) gives the equation below: yx pyvx1 (B-14) Integration of Equation B-14 results in the following: 3221Cyxpvx Apply the boundary condition that velocity at the plate (y=0.5h) is zero to Equation B-1to get the equation below: (B-15) 5 22hy. (B-16) 221xpvx Based on symmetry, the maximum velocity occurs at y=0 which gives the following: 2max221hxpx v (B-17) and 2max21hyvvxx. (B-18) Shear stress of a Newtonian fluid is given by the following: yvxyx (B-19) Substitution of Equation B-18 into Equation B-19 and derivation gives the equation below: 2max 8vyx hyx. (B-20) Shear stress at the plate becomes the following: 157

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hvxmax4 hyyx5.0 ugh the parallel plate chamber is the average velocity () multiplied by the cross-sectional area as shown below: (B-21) The flowrate (Q) thro hwvQx (B-22) quation B-18 and dividing by the chamber height as shown in The average velocity can be calculated by integrating E the following equation: max5.0 5.0max21hxxhdyhv 232xhvyv (B-23) Substitution of Equation B-23 into Equation B-22 gives the following: hwvQxmax32 (B-24) Rearranging yields the equation below hwQvx23max (B-25) Substitution into Equation B-21 gives the final equation of shear stress in terms of the volumetric flowrate and cha mber dimensions as shown below: whh25.0 Qyyx6 B-26) ( 158

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Figure B. Diagram of flow between parallel pl ates. 159

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BIOGRAPHICAL SKETCH ichelle Carman, the daughter of Don Mna Fulford and Robert Carman, was born December l school.nd spent the first month of her life under the graduated salutatorian of Forest High School in 1996. From there, she returned to the University e hoosing to remain at UF to study biomedical engineering in graduate school. 8, 1977 at Shands Hospital at the University of Florida where her father was attending denta Michelle was born 10 weeks premature a skillful watch of the NICU. She spent most of her childhood in Ocala, Florida where she of Florida and received her Bachelor of Science degree in chemical engineering in 2000 befor c 174


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