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The Control of Normal and Tumor Cell Behavior using Novel Materials

Permanent Link: http://ufdc.ufl.edu/UFE0041427/00001

Material Information

Title: The Control of Normal and Tumor Cell Behavior using Novel Materials
Physical Description: 1 online resource (122 p.)
Language: english
Creator: Lee, Ji
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The Control of Normal and Tumor Cell Behavior Using Novel Materials Cell interactions with nanostructured biomaterials are of broad interest because of applications in controlling tissue response to biomedical implants. In particular, mammalian cell adhesion to and proliferation on implanted devices is desirable for preventing the blockage of cardiovascular stents and tumor stents. Current methods to prevent stent blockage include coating with slow-release chemicals, but these have a finite lifetime requiring stent removal which causes clinical complications. The potential of densely coated nanorod monolayers for controlling normal mammalian cell adhesion involved in cardiovascular stent blockage was first explored. Densely packed nanorods were fabricated with a solution-based technique. NIH 3T3 fibroblasts and vascular endothelial cells were unable to adhere on dense nanorods. Cells could not assemble focal adhesions, and were poorly spread. Cell survival in adherent cells was reduced by more than 100-fold on nanorods. This reduction was not due to changes in protein adsorption nor due to toxicity of unknown dissolving material into the solution. The adhesion of tumor epithelial cells on nanorods was investigated. The morphology of tumor epithelial cells cultured on nanorods was rounded compared to flat surfaces and was associated with decreased cellular stiffness and non-muscle myosin II phosphorylation. Tumor cell number was decreased by nearly 50%, although proliferation and survival in adherent cells was unaltered. Single tumor cell motility was significantly increased on nanorods compared to flat surfaces coupled with a decrease in cell adhesion. Collectively, the results appear to support a model in which nanorods interfere with integrin clustering at the nano-scale, preventing cell adhesion and spreading and resulting in decreased cell survival. Nanostructured surfaces may be a promising approach to decrease cell adhesion. The role of nuclear-cytoskeletal connections in mediating cell mechanosensitivity was investigated. Endothelial cell adhesion and motility was found to be significantly perturbed in the absence of nesprin-1, a nuclear-cytoskeletal linker. Cells lacking nesprin-1 were observed to lose the ability to sense substrate rigidity. The results suggest a new role for nesprin-1 in mediating cell mechanosensing. A new method for studying wound healing under realistic conditions in vitro was developed. The method involves creating defined patterns of damaged, necrotic cells with PDMS stamping. This novel assay permitted the quantification of wound healing rates in the presence of cell debris. Experimental results with this assay suggest that cell migration in the presence of cell debris is a two step process requiring migration and myosin dependent phagocytosis.
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 Ji Lee.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Lele, Tanmay.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041427:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041427/00001

Material Information

Title: The Control of Normal and Tumor Cell Behavior using Novel Materials
Physical Description: 1 online resource (122 p.)
Language: english
Creator: Lee, Ji
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The Control of Normal and Tumor Cell Behavior Using Novel Materials Cell interactions with nanostructured biomaterials are of broad interest because of applications in controlling tissue response to biomedical implants. In particular, mammalian cell adhesion to and proliferation on implanted devices is desirable for preventing the blockage of cardiovascular stents and tumor stents. Current methods to prevent stent blockage include coating with slow-release chemicals, but these have a finite lifetime requiring stent removal which causes clinical complications. The potential of densely coated nanorod monolayers for controlling normal mammalian cell adhesion involved in cardiovascular stent blockage was first explored. Densely packed nanorods were fabricated with a solution-based technique. NIH 3T3 fibroblasts and vascular endothelial cells were unable to adhere on dense nanorods. Cells could not assemble focal adhesions, and were poorly spread. Cell survival in adherent cells was reduced by more than 100-fold on nanorods. This reduction was not due to changes in protein adsorption nor due to toxicity of unknown dissolving material into the solution. The adhesion of tumor epithelial cells on nanorods was investigated. The morphology of tumor epithelial cells cultured on nanorods was rounded compared to flat surfaces and was associated with decreased cellular stiffness and non-muscle myosin II phosphorylation. Tumor cell number was decreased by nearly 50%, although proliferation and survival in adherent cells was unaltered. Single tumor cell motility was significantly increased on nanorods compared to flat surfaces coupled with a decrease in cell adhesion. Collectively, the results appear to support a model in which nanorods interfere with integrin clustering at the nano-scale, preventing cell adhesion and spreading and resulting in decreased cell survival. Nanostructured surfaces may be a promising approach to decrease cell adhesion. The role of nuclear-cytoskeletal connections in mediating cell mechanosensitivity was investigated. Endothelial cell adhesion and motility was found to be significantly perturbed in the absence of nesprin-1, a nuclear-cytoskeletal linker. Cells lacking nesprin-1 were observed to lose the ability to sense substrate rigidity. The results suggest a new role for nesprin-1 in mediating cell mechanosensing. A new method for studying wound healing under realistic conditions in vitro was developed. The method involves creating defined patterns of damaged, necrotic cells with PDMS stamping. This novel assay permitted the quantification of wound healing rates in the presence of cell debris. Experimental results with this assay suggest that cell migration in the presence of cell debris is a two step process requiring migration and myosin dependent phagocytosis.
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 Ji Lee.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Lele, Tanmay.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041427:00001


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1 T HE CONTROL OF NORMAL AND TUMOR CELL BEHAVIOR USING NOVEL MATERIALS By JIYEON LEE 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 2010

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2 2010 Jiyeon Lee

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3 To my dear husband, Choon, and my baby

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4 ACKNOWLEDGMENTS I acknowledge the support and guidance of my doctoral advisor, Dr. Tanmay Lele. He was helpful throughout my time at the University of Florida, encouraging me to study challenging research topics with the right path. I would like to thank my other doctoral committee members for their time and patience spent with me. I appreciate the material science knowledge Dr. Fan Ren has given me. In collaboration with his lab, I could study nanostructure for designing novel biomaterials. In addition, I must thank Dr. Peng Jiang and Dr. Stephen Pearton for their willingness to participate in my doctoral review process. I thank Dr. Yiider Tseng for the opportunity to serve as a teaching assistant and his helpful discussion of my research. I thank Dr. Richard Dickinson and Dr. Kirk Ziegler for their insightful viewpoints in my research. I appreciate Dr. Jason Weaver s help for allowing me to learn his lab work. I greatly appreciate a valuable discussion with Dr. Shamik Sen and Dr. Sanjay Kumar in the Department of Bioengineering at University of California, Berkeley. Dr. Anand Gupte in the Department of Me dicine at University of Florida, gave me an opportunity to study tumor cells which I am extremely grateful. Also, I must thank many colleagues working with me in Dr. Lele s lab. Dr. Hengyi xiao taught me many important lab procedures during my first days in the lab. TJ Chancellor, Bob Russell, and Jun Wu were always helpful to work together in the lab. Nicole Clarke was my co worker in the lab and she was very helpful for me to perform the experiment. David Lov ett was supportive during my last days in the lab. Additionally, I appreciate a great deal of support from Dr. Ren s group colleagues, Byung Hwan Chu and Dr. Byoung Sam Kang. I thank my family for their support and understanding. My parents always encourage me with their constant trust. My sisters and brothers in law have always

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5 helped me. I must thank my father in law and mother in law for their support to my husband and me during our stay in USA. I really appreciate patient understanding of my dear husband, Choon Jae Ryu. He has always inspired me and been helpful. With his broad knowledge of computer knowledge and mechanical engineering, he is also my good discussion partner. I thank him for his constant love and support. Lastly, I thank my precious baby

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6 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. 4 page LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 CellSubstrate Adhesion ......................................................................................... 18 Nanostructured Surfaces for Anti fouling Applications ............................................ 19 Organization of this Document ................................................................................ 21 2 NANO SCALE CONTROL OF CELL SUBSTRATE ADHESION ............................ 23 Introduction ............................................................................................................. 23 Control of Cell Adhesion by Intermolecular Spacing ............................................... 23 Protein Adsorption on Nanostructures .................................................................... 24 Alterations in F ocal A dhesions A ssembly by N anoscale T opography ................... 26 Conclusions ............................................................................................................ 27 3 THE CONTROL OF CELL ADHESION AND VIABILITY BY ZINC OXIDE NANORODS ........................................................................................................... 28 Introduction ............................................................................................................. 28 Materials and Methods ............................................................................................ 29 Fabrication of ZnO Nanorods ........................................................................... 29 Prep aration of Substrates for Cell Culture ........................................................ 29 Cell Culture and Adhesion ................................................................................ 30 Immunostaining ................................................................................................ 30 Cell Viability Assay ........................................................................................... 30 Scanning Electron Microscopy (SEM) .............................................................. 31 Time Lapse Imaging ......................................................................................... 31 Results .................................................................................................................... 32 Formation of Uniform ZnO Nanorod Monolayers .............................................. 32 Decreased C ell S preading and F ocal A dhesion F ormation on ZnO N anorods ....................................................................................................... 32 Lack of Lamellipodia and Filopodia Formation on ZnO Nanorods .................... 33 Decreased Initial Cell Spreading on ZnO Nanorods ......................................... 33

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7 Discussion .............................................................................................................. 34 Conclusions ............................................................................................................ 36 4 RANDOMLY ORIENTED, UPRIGHT SIO2 COATED NANORODS FOR REDUCED ADHESION OF MAMMALIAN CELLS ................................................. 43 Introduction ............................................................................................................. 43 Materials and Methods ............................................................................................ 44 Fabrication of Nanorods ................................................................................... 44 Contact Angle Measurements .......................................................................... 45 Cell Culture ....................................................................................................... 45 Immunostaining and Cell Viability Assay .......................................................... 45 Time Lapse Imaging ......................................................................................... 46 Protein Adsorption on Nanorods and Glass ..................................................... 47 Results and Discussion ........................................................................................... 47 Fabrication of SiO2 Coated Nanorods .............................................................. 47 Decreased Cell Adhesion on SiO2 Coated Nanorods ....................................... 48 Protein Adsorption on Nanorods ...................................................................... 49 Sp atial Patterning of Cell Adhesion with Nanorods .......................................... 50 Decreased Cell Survival on Nanorods .............................................................. 50 Conclusions ............................................................................................................ 51 5 MODULATING MALIGNANT EPITHELIAL TUMOR CELL ADHESION, MIGRATION AND MECHANICS WITH NANOROD SURFACES ........................... 63 Introduction ............................................................................................................. 63 Materials and Methods ............................................................................................ 64 Growth of Nanorods ......................................................................................... 64 Cell Culture ....................................................................................................... 65 Cell Viability Assay ........................................................................................... 65 BrdU Staining ................................................................................................... 65 Scanning Electron Microscopy (SEM) .............................................................. 66 Cell Motility Assay ............................................................................................ 66 Cell Stiffness Measurement by Atomic Force Microscopy (AFM) ..................... 67 Western Blotting ............................................................................................... 67 Results and Discussion ........................................................................................... 68 Decrease i n Adhesion of Esophageal Epithelial Cells on Nanorods ................. 68 Viability and Proliferation is Unchanged in Tumor Cells on Nanorods .............. 68 Tumor Cell Cultured on Nanorods have Decreased Contractility ..................... 69 Single Tumor Cell Motility is Increased on Nanorods ....................................... 7 0 Conclusions ............................................................................................................ 71 6 THE ROLE OF NUCLEA R CYTOSKELETAL LINKAGES IN CELL MECHANO SENSING ................................................................................................................ 79 Introduction ............................................................................................................. 79 Materials and Methods ............................................................................................ 80

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8 Cell S ultu re and siRNA K nock D own of N esprin1 ........................................... 80 Fabrication of P olyacrylamide S ubstrates ........................................................ 80 Immunostaining ................................................................................................ 81 Measurement of C el l S preading A rea .............................................................. 81 Cell M otility A ssay ............................................................................................ 82 Results .................................................................................................................... 83 The R igidity D ependence of C ell S preading is A ltered in N esprin1 D eficient C ells .............................................................................................................. 83 Focal A dhesion and S tress F iber A ssembly on PAAms ................................... 83 NKHUVECs have A ltered S ingle C ell M otility ................................................... 84 Discussion .............................................................................................................. 84 Conclusion .............................................................................................................. 85 7 A STAMPWOUND ASSAY TO STUDY COUPLED CELL PEELING AND MIGRATION: TOWARD A REALISTIC WOUND HEALING ASSAY ...................... 90 Introduction ............................................................................................................. 90 Materials and Methods ............................................................................................ 91 Fabrication of P oly(dimethyl)siloxane (PDMS) M olds ....................................... 91 Cell C ulture and S oft I mprinting with the PDMS M old ...................................... 91 Time L apse M icroscopy ................................................................................... 92 Cell V iability A ssay ........................................................................................... 92 Blebbistatin A ssay ............................................................................................ 92 Results .................................................................................................................... 92 Discussion .............................................................................................................. 94 Conclusions ............................................................................................................ 95 8 CONCLUSIONS ................................................................................................... 102 LIST OF REFERENCES ............................................................................................. 107 BIOGRAPHICAL SKETCH .......................................................................................... 122

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9 LIST OF TABLES Table page 3 1 Average area of cell spreading on ZnO flat substrate and ZnO nanorods .............. 42

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10 LIST OF FIGURES Figure page 1 1 Cellsubstrat e a dhesion .......................................................................................... 22 3 1 The morphology of ZnO nanorods and flat substrate .............................................. 37 3 2 Cells do not assemble stress fibers or focal adhesions on nanorods. Fluorescent micrographs of NIH 3T3, HUVEC, and BCE cells stained for vinculin (green) and F actin (red) on glass, ZnO flat substrate and ZnO nanorods ........................................................................................................... 38 3 3 Total cell number and number of live adherent cells are reduced on nanorods ...... 39 3 4 Cells cannot assemble lamellipodia on nanorods. Representative SEM images of NIH 3T3 fibroblasts on ZnO nanorods ............................................................ 40 3 5 Dynamic cell spreading is altered on nanorods ....................................................... 41 4 1 The morphology of nanorods .................................................................................. 52 4 2 Fluorescent microscopic images of HUVEC and NIH 3T3 on glass and nanorods ............................................................................................................ 53 4 3 The average area of cell spreading on glass and nanorods .................................... 54 4 4 Contact angles of water on glass and SiO2 coated nanorods ................................. 55 4 5 Fluorescent images and intensity of rhodamine fibronectin coated glass and nanorods ............................................................................................................ 56 4 6 S EM images of patterned nanorods ........................................................................ 57 4 7 NIH 3T3 fibroblasts on patterned SiO2 coated nanorods ......................................... 58 4 8 NIH 3T3 fibrob lasts on patterned ZnO nanorods .................................................... 59 4 9 Time lapse phase microscope images of NIH 3T3 on patterned ZnO nanorods. Black arrows indicate the direction of cell motility ............................................... 60 4 10 Cell attachment and viability on nanorods ............................................................. 61 4 11 Material toxicity test ............................................................................................... 62 5 1 Esophageal epithelial tumor cell adhesion was decreased on nanorods ................ 72 5 2 Nanorods are not toxic to tumor cells ...................................................................... 73

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11 5 3 Individual tumor cells in colonies were rounded on nanorods unlike cells on glass ................................................................................................................... 74 5 4 Confocal microscopic images of OE33 on glass and nanorods .............................. 75 5 5 Non muscle myosin II activity is significantly reduced in cells on nanorods compared to cells on glass ................................................................................. 76 5 6 Tumor cells are softer on nanorods compared to glass .......................................... 77 5 7 OE33 cell motility is altered on nanorods ................................................................ 78 6 1 Average cell spreading area ................................................................................... 86 6 2 Fluorescent microscopy images of vinculin and F actin .......................................... 87 6 3 Representative mean square displacement (MSD) calculated for SHUVEC and NKHUVEC on PAAms of v arying stiffness and model fits .................................. 88 6 4 Cell speed, persistence time and persistence length of NKHUVEC and SHUVEC on PAAms ........................................................................................... 89 7 1 Schematic diagram of stamping wound assa y ........................................................ 96 7 2 Time lapse microscope images of stamped Het1A cells ......................................... 97 7 3 Wound healing ratio with Het1A cells ...................................................................... 98 7 4 Epithelial cell migrates to the wound after peeling off dead cells ............................ 99 7 5 Time lapse images of stamped Het1A cells with blebbistatin treatment ................ 100 7 6 Wound healing ratio with nonmuscle myosin II inhibiti on ..................................... 101

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12 LIST OF ABBREVIATION S AFM Atomic force microscopy APS A mmonium persulfate APTMS ( 3 aminopropyl)trimethoxysilane BCE Bovine capillary endothelial cell BrdU 5 bromo 2 deoxyuridine BS Blebbistatin CPD Critical point drying DAPI 4' 6 diamidino2 phenylindole DBS Donor bovine serum DMEM Dulbecco/Vogt modified Eagle's minimal essential medium ECM Extracellular matrix EthD 1 E thidium homodimer 1 FBS Fetal b ovine serum FCS Fetal calf serum FITC Fluorescein isothiocyanate FN Fibronectin FTIR Fo urier transform infrared spectroscopy GAPDH Glyceraldehyde3 phosphate dehydrogenase GFP Green fluorescent protein HCl Hydrochloride Het1A Human esophageal epithelial cells HUVEC Human umbilical cord vein endothelial cells LINC Linker of nucleoskeleton to the cytoskeleton MEM M inimum essential medium eagle

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13 MSC Nave mesenchymal stem cell MSD Mean square displacement NaOH Sodium hydroxide NKHUVEC Nesprin 1 knockdown siRN A transfected HUVEC NMR N uclear magnetic resonance PAAm Polyacrylamide PBS Phosphate buffered saline PCL P oly( caprolactone) PCU P oly(carbonate urethane) PDMS Poly(dimethyl)siloxane PECVD P lasma enhanced chemical vapor deposition PR Photoresist PU Pellethane PVDF P olyvinylidene fluoride RGD Arginine Glycine Aspartic acid SD Standard deviation SDS Sodium dodecyl sulfate SEM Scanning electron microscopy SEM Standard error of the mean SHUVEC C ontrol smartpool siRNAs transfected HUVEC SiO2 Silicon dioxide TBST Tris buffered saline with Tween 20 TEM Transmission electron microscopy TEMED T etramethylethylenediamine TiO2 Titanium dioxide

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14 TIRF T otal internal reflectance fluorescence UV Ultraviolet ZnO Zinc oxide

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15 Abstract of Dissertation Presented to t he Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE CONTROL OF NORMAL AND TUMOR CELL BEHAVIOR USING NOVEL MATERIALS By Jiyeon Lee May 2010 Chair: Tanmay P. Lele Major: Chemical Engineering Cell interactions with nanostructured biomaterials are of broad interest because of applications in controlling tissue response to biomedical implants. In particular, mammalian cell adhesion to and proliferation on implanted devices is desirable for preventing the blockage of cardiovascular stents and tumor stents. Current methods to prevent stent blockage include coating with slow release chemicals, but these have a finite lifetime requiring stent removal which caus es clinical complications. The potential of densely coated nanorod monolayers for controlling normal mammalian cell adhesion involved in cardiovascular stent blockage was first explored. Densely packed n anorods were fabricated with a solutionbased techni que. NIH 3T3 fibroblasts and vascular endothelial cells were unable to adhere on dense nanorods. Cells could not assemble focal adhesions, and were poorly spread. Cell survival in adherent cells was reduced by more than 100 fold on nanorods. This reduction was not due to changes in protein adsorption nor due to toxicity of unknown dissolving material into the solution.

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16 The adhesion of tumor epithelial cells on nanorods was investigated. The morphology of tumor epithelial cells cultured on nanorods was roun ded compared to flat surfaces and was associated with decreased cellular stiffness and nonmuscle myosin II phosphorylation. Tumor cell number was decreased by nearly 50 %, although proliferation and survival in adherent cells was unaltered. Single tumor ce ll motility was significantly increased on nanorods compared to flat surfaces coupled with a decrease in cell adhesion. Collectively, the results appear to support a model in which nanorods interfere with integrin clustering at the nanoscale, preventing cell adhesion and spreading and resulting in decreased cell survival. Nanostructured surfaces may be a promising approach to decrease cell adhesion. The role of nuclear cytoskeletal connections in mediating cell mechanosensitivity was investigated. E ndothelial cell adhesion and motility was found to be significantly perturbed in the absence of nesprin1 a nuclear cytoskeletal linker. Cells lacking nesprin1 were observed to lose the ability to sense substrate rigidity. The results suggest a new role for n esprin1 in mediating cell mechanosensing A new method for studying wound healing under realistic conditions in vitro was developed. The method involves creating defined patterns of damaged, necrotic cells with PDMS stamping. This novel assay permitted the quantification of wound healing rates in the presence of cell debris. Experimental results with this assay suggest that cell migration in the presence of cell debris is a two step process requiring migration and myosin dependent phagocytosis.

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17 CHAPTER 1 INTRODUCTION An important application of tailoring the surface of biomedical implants is preventing mammalian cell adhesion to the implant surface An example of this is anti fouling tumor stents. Tumor s tents are used to prevent the collapse of gastrointestinal, pancreatic and biliary ducts that have tumors surgically r emoved from them. However, a persistent problem that interferes with normal stent function is the adhesion and growth of tumor cells on the stent surface [1, 2]. A s the stent blockage increases morbidity and mortality in patients there is a critical need for strategies for preventing tumor cell adhesion to the stent surface. Similarly, preventing macrophage adhesion to titanium surfaces is crucial for the success of bone prosthesis [3]. Platelet adhesion and subsequent clogging of cardiovascular stents is another problem that needs anti fouling materials [4, 5] Anchoragedependent cells (typically involved in the blockage of stents) require adhesion to the solid extracellular matrix (ECM) for normal function [6 9] The ECM consists of fibrous networks composed of proteins like fibronectin, laminin, vitronectin, collagen and elastin [10] These structures provide physical and chemical signals at the nanometer length scale that control cell functions such as migration, proliferation, and apoptosis [8, 11, 12] The ECM in the body can be mimicked in vitro by fabricating materials that present defined chemical and physical signals to the cell [13 17] In particular, manipulating cell adhesion by fabricating material surface s with nanoscale structures has emerged as a promising approach to control cell function both in vivo [18, 19] and in vitro [20 24] Hence, f abricating biomedic al implants with nanostructur ed

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18 surfaces can allow the selective control of cellular interactions with the implant. The mechanisms of cell adhesion at the nanoscale to solid substrates are discussed below. Cell S ubstrate A dhesion Cell adhes ion to the solid substrat e occurs through specific binding interactions between transmembrane receptors called integrins and their ligands (e.g. fibronectin, collagen, laminin and vitronectin) whic h are adsorbed on the substrat e [25 27] The integrin receptor is a heterodimer consisting of and subunits. In mammals 18 subunits and 8 subunits have been so far discovered [28] The different subunits make it possible to form different types of integrin heterodimers which bind selectively to specific ligands. Many integrin receptors recognize ArginineGlycine Aspartic aci d (RGD) motifs which are present in ECM ligands such as fibronectin and vitronectin. The ligation of integrin receptors causes a change in their conformation. This triggers the binding of cytoplasmic proteins such as talin to the cytoplasmic tails of inte grins. Talin binding is thought to cross link neighboring integrin receptors, giving rise to spatial clustering of ligated integrins [29] The clustering of integrins causes the recruitment of several different types of proteins including e nzymes like focal adhesion kinase and src kinase [30] adaptor molecules like paxillin [31] that are known to bind to several other proteins and actinin 1 [32] that directly link integrins and the actin cytoskeleton [27, 33] The resulting multimolec ular assembly of proteins is collectively called the focal adhesion. Focal adhesions measure roughly ~60 nanometers in thickness [34] and extend out a few square micrometers in the plane of the cell membrane (Fig ure 1 1 ) Tension generated through the action of the motor protein myosin as it walks along actin filaments is transmitted through connector proteins like -

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19 actinin to integrins, and hence to the substrateadsorbed ligands [3538] This connection between tensed actomyosin filaments (that are crosslinked further into tensed structures called stress fibers, see Figure 1 1) and focal adhesion proteins allows a continuous mechanical link between the inside of the cell and the external substrat e In this mann e r the focal adhesion establishes a physical path for mechanical force transfer between the cell and the substrat e Importantly, focal adhesions are not static structures but dynamic multi molecular assemblies that start out as dot like focal complexes at the leading edge of the motile cell, and eventually mature into focal adhesions [39 41] In addition to acting as locations where cellular tensile forces are in balance with compressive forces in the substrate, focal adhesions are also signaling complexes. For example, integrin ligation can trigger signaling cascades that contro l gene expression, differentiation and apoptosis [42] Interfering with focal adhesion assembly controls the degree of cell spreading, cell shape as well as cell fate [43] In fact, control of focal adhesion assembly at the nanoscale is a key mechanism by which nanostructured surfaces control cell function. Nanostructured S urfaces for A nti fouling A pplications A class of nanostructures that have sho wn promise for reducing mammalian cell adhesion is a surface covered with upright nanoscale cylinders, variously referred to as nanorods, nanoposts and nanoislands [44 48] A recent paper showed that fibroblast numbers were reduced to approximately 30% on needlelike silicon nanoposts compar ed to the smooth substrate [44] Cells on nanoposts extended filopodia structure along the sharp tips and spread much less than cells on the smooth surface. The authors of this study also suggest that upright nanoposts may be useful as an anti -

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20 fouling surface [44] Other studies have demonstrated that nanoscale island structures can reduce cell adhesion [47 49] These studies have showed that the dimension of the nanoisland play s an important role in modulating cell adhesion. Similarly, in osteo blasts, cell number s on nanoislands with height 85 nm were reduced by ~ 80% compared to cells o n the flat surface [49] A ligned and densely packed carbon nanotubes have also been reported as an excellent anti f ouling surface toward platelet adhesion [4] Few platelets could adhere on fluorinated poly(carbonate urethane) ( PCU) treated carbon nanotubes (these are superhydrophobic films which potentially interfere with ECM protein adsorption) Moreover, activation of platelets was decreased by 50% on nanotubes The above studies offer strong support to the hypothesis that upright cylindrical structures with nanoscale dimensions can be used to decrease cell adhesion. The mechanisms of how this may occur at the molecular level are less clear. As discussed in the r eported literatures [9, 17, 18, 50] a ltered matrix protein adsorption may be an important factor in determining cell response. Other possibilities include the disruption of the molecular processes that participate in cell adhesion to surfaces such as clustering integrins (see Chapter 2) T his dissertation explores the potential of upright nanorods for achieving a significant reduction in cell adhesion. We show that nanorods covered surface can be used as effective anti fouling surface (see Chapter 3 and Chapter 4) toward fibroblasts and vascular endothelial cells We also show that the adhesion of malignant human tumor cells can be modulated with nanorod monolayers (see Chapter 5) We suggest that the decrease in adhesion is not due to altered protein adsorption, but rather due to

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21 a disruption of integrin clustering at the nanoscale. Together, our results suggest a novel, promising approach that could be potentially used to prevent blockage of stents. Organization of t his D ocument In Chapter 2, current literature on the nanoscale control of cell substrate adhesion is reviewed. In Chapter 3, the potential of zinc oxide ( ZnO ) nanorods grown on substrates to reduce cell substrate adhesion is investigated. Because of the potential toxicity of ZnO to some cell types silicon dioxide ( SiO2) coated nanorods are investigated in Chapter 4. Finally, in Chapter 5, tumor cell interactions with SiO2 coated nanorod surfaces are studied. In Chapter 6, t he role of nuclear cytoskeletal connections in mediating cell mechanosensitivity is explored. In Chapter 7, a new wound healing assay is presented to study coupled cell peeling and migration under realistic conditions in vitro

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22 Figure 1 1. Cellsubstrat e a dhesion. A) Fluorescent microscope image of a single bovine capillary endothelial cell cultured on a smooth glass substrate. Cell was stained for vinculin (green) and F actin (red). Focal adhesions (green) are located at the tips o f stress fibers (inset im age). B) A simplified diagram of ed to the ECM and intracellular proteins that link integrins to the cytoskeleton.

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23 CHAPTER 2 NANO SCALE CONTROL OF CEL L SUBSTRATE ADHESION Introduction At the nanoscale, cells have been found to be sensitive to a variety of surface topologies, include nanopits [51] nanoposts [52] nanocracks [53 ] nanotubes [54] and nanoislands [55] While it is clear that cells ar e exquisitely sensitive to nanostructured surfaces, the molecular mechanisms that determine this sensitivity are less clear. This chapter focuses on current understanding of the mechanism of nano scale control of cellsubstrate adhesion. C ontrol of Cell A dhesion by Intermolecular S pacing Evidence that focal adhesion assembly can be directly controlled at the nanometer scale has come from recent studies with gold nanodot arrays [8, 56 60] The approach relies on fabricating ordered arrays of nanodots, each with diameter comparable to the size of a single integrin receptor ( an integrin cylinder is ~ 160 long and ~ 20 in diameter [61] a head of an integrin is roughly 5.6~7.2 nm in diameter [62] ) and the nanodots are 5~8 nm in diameter [56] The nanodots are fabricated with dibock copolymer nanolithography, which allows precise control between nanodot spacing. An RGD peptide is conjugated to an organic molecule containing a thiol group that is bonded to the gold nanodot. Areas between the nanodots are passivated with adsorption of the linear poly( ethylene glycol) mPEG2000urea polymer which prevents any protein adsorption from serum. Owing to the size of the nanodot ( 5 ~ 8 nm), only one RGD peptide is presented per nanodot, and only one integrin receptor can be conjugated per nanodot. Therefore, t he systematic variation of nanodot spacing at the

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24 nanoscale results in a control of the spacing between ligated integrins at the nanoscale. This model system has been used to understand some key features of cell adhesion at the nanoscale. An important finding is that spacings larger than 70 nm cause a significant decrease in cell adhesion [56] The number of attached osteoblasts on 73 nm nanodots was decreased by 8 0 % compared with that on 28 nm nanodots. Cells were unable to assemble f ocal adhesions and stress fibers on 73 nm spaced nanodots. The dynamic turnover of focal adhesions was observed to be significantly higher on nanodots with spacing of 108 nm [59] The levels of the adhesion protein paxillin were found to be significantly decreased, and stress fiber formation and cell adhesion were decreased on the nanodots with spacing 108 nm These studies provide clear evidence that nano scale p resentation of ligands alters focal adhesion assembly. It is hypothesized [59] that forcing ligated integrins to be more than 70 nm apart potentially prevents cr osslinking on the cytoplasmic side and prevents adequate coupling between the actomyosin cytoskeleton and the substrate. This results in decreased adhesive forces on the substrate, a lack of cell spreading and altered cell signaling The nanoscale topogr aphy of the surface can also profoundly control cell adhesion and cell migration, a process called contact guidance. The molecular mechanisms of how nanotopographical features alter cell adhesion are less clear. Multiple possibilities exist which are discu ssed below. Protein Adsorption on N anostructures The ECM protein adsorption can be substantially different on nanorough materials compared to smooth materials. Materials which are rough at the nanoscale display

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25 large changes in their surface hydrophilicity or hydrophobicity [63] which can potentially influence not only the amount of matrix protein adsorption, but also the confor mation of the adsorbed protein. Interestingly, nanorough materials exhibit an increase in hydrophilicity if the corresponding smooth material is hydrophilic and an increase in hydrophobicity i f the corresponding smooth material is hydrophobic [63 66] Webster et al [67] have show n that vitronectin adsorption i s significantly enhanced on nanoscale alumina c ompared to conventional alumina. Vitron ectin on nanoscale alumina was observed to be in a more unfolded structure, suggesting that binding motifs in the ligand may be more easily available on nanoscale alumina. The mechanism for increased vitronectin adsorption is less clear, but is attributable either to increased hydrophilicity of nanoscale alumina or increased adsorption of other molecules (such as calcium) that promote vitronectin adsorption [67] The adsorption of fibronectin has similarly been shown to increase with increased nanoscale roughness on composites made of carbon nanotubes and PCU [68] The increased adsorption was shown to correlate with increased surface energy of the nanoscale structures. Fibronectin adsorption has also been shown to increase on titanium surfaces with increasing roughness at the nano scale which is attributable to increased surface energy [69] Increasing hydrophobicity by creating nano rough surfaces has been proposed as an approach to create anti fouling protein surfaces [4, 70] For example, FeCo Ni metal alloy nanowires are more hydrophobic than the corresponding smooth surface [70] Bovine serum albumin adsorption on these nanowires decreased significantly compared to the flat surface. Superhydrophobic surface s have also been created by

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26 grafting aligned carbon nanotubes with fluorinated PCU [4] and abnormal platelet adhesion to these structures has been attributed to altered protein adsorption. While these studies clearly indicate th e importance of protein adsorption in cell nanoscale interactions, considerable more work remains to be done for obtaining a clear atomic level understanding of how protein adsorption is influenced by nanostructure. Alterations in F ocal A dhesions A ssembly by N anoscale T opography As discussed in Chapter 1, the assembly of focal adhesions and the transfer of intracellular tension to the nanostructured material can be altered by nanoscale presentation of ligand molecules. In a similar manner, nanoscale topography could alter the assembly of focal adhesions. It has been shown, for example, that epithelial cells align parallel along nanoscale ridges of width 70 nm created on silicon substrates [71] Focal adhesions and stress fibers aligned parallel to the nano scale ridges and the width of focal adhesions was observed to be controlled by the ridge width [71] Interestingly, cell and focal adhesion alignment could be made perpendicular to the nanoscale ridges by changing soluble factors in the culture medium, indicating a n interplay between soluble signaling pathways and adhesion assembly at the nanoscale [ 24] Similar l y, fibroblasts have been shown to align on the surface that presents nanoscale grooves [72, 73] Alignment was observed only when the depth of patterns was over 35 nm and the ridge widths were bigger than 100 nm [72, 73] Electrospun nanofibers have been examined for creating cell align ing scaffolds [74, 75] Human schwann cells cultured on aligned poly( caprolactone) (PCL) fibers had aligned nuclei and stress fibers along the fiber axes [74] On random PCL fibers, cells were randomly oriented but still aligned along the random fiber axes Similarly,

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27 h uman ligament fibroblasts have been observed to align along electrospun pellethane (PU) nanofibers [75] Th e se result s clearly show that nanoscale topography guides cell alignment and elongation. Conclusions Cells are sensitive to the nanostructure on the surface of the substrat e The mechani sms of how cells sense nanostructure are less clear. ECM ligands on a nanorough material could influence the degree of integrin clustering by virtue of their threedimensional presentation. This could feedback to regulate cell tension, cell shape and henc e cell fate. Such effects in combination with altered matrix protein conformation or local surface concentration on nanorough materials could alter adhesion assembly. Parsing out the degree to which these factors influence cell adhesion remains a formidable challenge, but it is the key to a clear understanding of cell sensing of nanostructure. Such understanding can greatly promote the rational design of nanostructured biomedical implants for applications such as the development of anti fouling stents.

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28 CHAPTER 3 T HE CONTROL OF CELL A DHESION AND VIA B ILITY BY ZINC OXIDE NANORODS Introduction The success of implanted devices such as orthopedic implants, cardiovascular prosthesis and neural electrodes is affected by the ability of cells to interact with the exposed device material. Because properties such as surface topology are stable features of the surface, compared to chemical modifications which may be degraded over time, there has been immense interest in directing cell behavior by controlling the topology of materials [24, 52, 7678] Cells have been found to respond di fferently to smooth surfaces compared to materials with micro or nanoscale roughness in a cell type dependent manner [78 81] One class of nanostructu res that has received recent attention in the literature is a surface covered with upright slender cylinders, variously referred to as nanoposts, nanorods and nanocolumns [51, 52, 76, 82] A recent study showed that cell numbers and proliferation in fibroblasts are greatly reduced on needlelike silicon nanoposts [52] This study suggests that nanoposts may be useful as anti fouling materials. Such surfaces could potentially be used for modulating the fibrotic response around implanted biomaterials. W e developed a strategy to reduce cell adhesion and survival on surfaces by culturing cells of three different cell types on a monolayer of upright ZnO nanorods of 50 nm diameter and 500 nm height i n this chapter A large number of nanorods were exposed to the cell (~ 60,000 to 150,000 per cell). Owing to the uniform distribution of the nanorod monolayer, the cells were not able to attach to any flat portion of the substrate. Our results indicate that initial adhesion, lamellipodia formation, dynamic cell

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29 spreading and cell survival at 24 hours is greatly reduced on nanorod covered substrates in three different cell types. Materials and Methods Fabrication of ZnO N anorods ZnO nanorods were made by a solutionbased hydrothermal growth method [83] First, ZnO nanoparticle s were prepared by mixing 10 mM zinc acetate dehydrate (Sigma Aldrich, St. Louis, MO) with 30 mM of sodium hydroxide ( NaOH) (Sigma Aldrich, St. Louis, MO) at 58 coated onto the substrate several times and then p ost baked on a hot plate at 150 The substrate with these seeds was then suspended upside down in a Pyrex glass dish filled with an aqueous nutrient solution. The growth rate was approximately 1 m per hour with 100 ml aqueous solution containing 20 mM zinc nitrate hexahydrate and 20 mM hexamethylenetriamine (Sigma Aldrich, St. Louis, MO). To arrest the nanorod growth, the substrates were removed from solution, rinsed with deionized water and dried in air at room temperature. Prep aration of Substrates for Cell C ulture For control substrate, we used 22 mm square glass cover slips (Corning, Inc., Lowell, MA) and ZnO flat substrates (Cermet Inc., Atlanta, GA). Before use, each substrate was sterilized with ultraviolet ( UV ) for 5 min and cleaned in 70% ethanol and deionized water. After drying substrates in air at room temperature, they were treated with 5 g/ml human fibronectin (FN) (BD biosciences, Bedford, MA). After overnight incubation with FN at 4 ere washed twice with phosphate buffered saline ( PBS) Cell suspensions of the same concentration and volume (i.e. same number of cells) were then seeded on each substrate.

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30 Cell Culture and A dhesion Cells of three different types were seeded on FN coated substrates. NIH 3T3 fibroblasts were cultured in Dulbecco/Vogt modified Eagle's minimal essential medium ( DMEM) (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). Human umbilical cord vein endothelial cells (HUVECs) were cultured in EBM 2 Basal Medium and EGM 2 SingleQuot Kit (Lonza, Walkersville, MD). Bovine capillary endothelial cells (BCEs) were cultured in low gl ucose DMEM supplemented with 10 % fetal calf serum (FCS) (Hyclone, Logan, UT). Immunostaining After 24 hours of cell seeding, nonadherent cells were removed with two gentle washes with PBS The samples were fixed with 4% paraformaldehyde for 20 min and washed several times with PBS. Fixed cells were immunostained for vinculin and stained for act in using our previously reported methods [84, 85] B riefly, cells were fixed with 4 % paraformaldehyde, permeabilized with 0.2% Triton X 100, and treated with mouse monoclonal anti vinculin antibody (Sigma Aldrich, St. Louis, MO), followed by goat anti mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Eugene, OR). Acti n was stained with phalloidin conjugated with Alexa Fluor 594 (Invitrogen, Eugene, OR). Cells were then imaged on a Nikon TE 2000 epifluorescence microscope using fluorescein isothiocyanate ( FITC ) and Texas Red filters. All images were collected using the NIS Elements program (Nikon). Cell Viability A ssay The live/dead viability/cytotoxicity kit for mammalian cells (Invitrogen, Eugene, OR) was used for quantifying adherent cell viability on each substrate. Cells were incubated at 30 45 minutes with calcein AM (2 M for fibroblast, 5 M for endothelial cells) and

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31 ethidium homodimer 1 (EthD 1) (4 M for fibroblast, 1.5 M for endothelial cells) [86] Next, epifluorescence images of five random fields were collected on a Nikon TE 2000 inverted microscope using a 10 X lens. The average number of cells adherent on each substrate, the number of adherent live cell s (stained green with calcein AM) and adherent dead cells (stained red with EthD 1) were quantified from these images using the NIS Elements program (Nikon). The experimental data was pooled and used for statistical comparisons using the Students T test. Scanning Electron M icroscopy (SEM) Cells were prepared for SEM by fixation with 2% glutaraldehyde buff ered in PBS and post fixed in 1% osmium tetroxide. Samples were next dehydrated in graded ethanol concentrations. Critical point drying (CPD) was perform ed on a Bal Tec 030 instrument (ICBR Electron Microscopy Core Lab, University of Florida) followed by ebeam metal deposition (Ti/Au, 10/50 ). SEM was performed on a Hitachi S 4000 FE SEM (ICBR Electron Microscopy Core Lab, University of Florida). Images of samples were taken at 1.8 8.0 kX magnifications. Time Lapse I maging Cells which had been cultured as mentioned above were trypsinized and resuspended in bicarbonatefree optically clear medium containing Hanks balanced salts (Sigma Aldrich, St. Lou is, MO), L glutamine (2.0 mM), 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid ( HEPES) (20.0 mM), minimum essential medium eagle ( MEM) and nonessential amino acids (Sigma Aldrich, St. Louis, MO), and 10% FCS [87] Cells were passed onto FN coated glass or ZnO nanorods, and phase contrast imaging performed overnight for 10 hours on t he Nikon TE 2000 microscope. Images were collected every 1 minute, using a 20 X objective.

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32 Results Formation of Uniform ZnO Nanorod M onolayers Shown in Figure 31A are SEM images of <001> vertically aligned ZnO nanorod arrays. Such nanorods could be grown over areas on the order of 1 cm2 and thus ZnO nanorods could be grown in uniform monolayers over very long distances compared to cellular length scales. The nanorods were approximately 50 nm in diameter, 500 nm in height and the density of nanorods was ap proximately 126 rods per square micron. Based on measured cell spreading areas, this number corresponds to approximately 60,000 nanorods per fibroblast and approximately 75,000150,000 nanorods per endothelial cell. Because of our focus on the effect of t opology on cells, it was important to choose an appropriate control for statistically comparing effects of nanorods on cells. As the material itself can have effects on protein adsorption and cell adhesion, we chose a topologically smooth substrate made of ZnO which is a thin film commercially available from Cermet Inc. An AFM image of t his substrate is shown in Fig ure 31 B. The flat substrate is smooth over long length scales with an average roughness of 1.33 nm. Interestingly, similar results were obtained for glass (average roughness of 1.34 nm, not shown), which allowed us to compare the performance of the ZnO flat substrate and ZnO nanorods with glass, a well established substrate for cell culture. Decreased C ell S preading and F ocal A dhesion F ormation on ZnO N anorods We next investigated the influence of ZnO nanorods on cell spreading. Cells in vitro spread by assembling focal adhesions and stress fibers. Fig ure 3 2 shows fluorescence images of three different cell types NIH 3T3s, HUVECs, and BCEs on glass, ZnO flat substrate, and ZnO nanorods. Cells on ZnO flat substrates and glass

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33 cover slips assembled clear focal adhesions and stress fibers. Focal adhesions and stress fibers were not visible in cells on nanorods. The average area of cell spr eading was decreased significantly on nanorods compared with ZnO flat substrates (a reduction of 6070%, Table 3 1). These trends were observed in each of the three cell types. Lack of Lamellipodia and Filopodia Formation on ZnO N anorods A recent study showed that cells on needlelike nanostructures only assemble filopodia [52] To investigate this possibility for ZnO nanorods, we performed SEM studies on NIH 3T3 fibroblasts cultured on ZnO nanorods ( Fig ure 34). Most cells on ZnO nanorods were rounded (Fig ure 34A). Instead of flat sheet like lamellipodia, some cells formed thi n processes (black arrow in Fig ure 34B) and thin filopodialike structures (white arrows in Fig ure 34B) that appeared to attach to the ZnO nanorods. Therefore, while cells can attach to the ZnO nanorods using filopodialike structures, they are not able to spread on the nanorods. Decreased Initial Cell Spreading on ZnO N anorods In our studies, a large number of nanorods were exposed to cells. The results of Kim and coworkers showed that Si nanowires with diameter similar to our nanorods are engulfed by cells [82] This raises the possibility that cells may spread initially on the nanorods but undergo apoptosis due to engulfment of nanorods at longer times. To clarify this, we performed timelapse imaging for studying dynamic cell spreading on nanorods (Fig ure 35) After seeding, initial adhesion of HUVECs on glass occurred in the first hour (Fig ure 35A). Lamellipodia formation could be seen from 2 hours onward followed by complete spreading at approximately 5 hours (white arrows in Fig ure 35 A). Conversely, on nan orods, little initial spreading occurred and cells remained rounded

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34 over several hours (Figure 35B). No lamellipodia formation was visible. These results show that nanorods did not support initial cell spreading. While these results alone do not rule out long term toxicity of nanorods due to engulfment, they provide evidence that cells are not able to initially spread on nanorods, which may contribute to decreased survival at long times. Discussion ZnO nanorods, nanowires, and nanotubes have attracted considerable attention for biosensing applications owing to their chemical stability, high specific surface area, and electrochemical activity [88 90] ZnO nanoplaforms have been developed f or highly sensitive and specific detection of biological samples [91, 92] It is easy to control the aspect ratio and spacing of ZnO nanorods which is desirable for eng ineered materials [93] However, before the promise of ZnO nanostructures for in vivo applications can be r ealized, it is crucial to prevent cell adhesion to these structures. In this chapter we found that the adhesion and viability of fibroblasts, umbilical vein endothelial cells, and capillary endothelial cells are greatly altered on ZnO nanorods. Cells adh ered less and spread less on ZnO nanorods than the corresponding ZnO flat substrate. Scanning electron microscopy indicated that cells were not able to assemble lamellipodia on nanorods. Timelapse phase contrast imaging showed that cells initially adherent to nanorods are unable to spread. This suggests that the lack of initial spreading on ZnO nanorods may cause cell death. Our results indicate a lack of focal adhesion assembly in cells cultured on ZnO nanorods. The spacing between the ZnO nanorods is approximately 100 nm. Recent work by Arnold et al. showed that focal adhesion assembly requires that the spacing between ligated integrins be less than 70 nm [94 ] Local integrin clustering probably can

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35 occur on single nanorods as their diameter is on the order of 50 nm. However, focal adhesions extend over several microns. It is possible that integrin clustering does not occur over contiguous lengths of micron length scales, preventing focal adhesion assembly. Cells on nanorods also have no visible lamellipodia. As initial adhesion is required to polymerize actin filaments [95] the lack of lamellipodia is probably due to an inability of cells to establish strong initial adhesion to the substrate, thereby altering the dynamics of cell spreading. Our observations of altered cell spreading dynamics are consistent with observations by Cavalcanti Adam et al. who observed similar behavior on RGD (Arginine Glycine Aspartic acid) nanopatterned substrates [59] Our results can therefore be explained by a mechanism in which abnormal assembly of focal adhesions due to an inability to cluster integrins contributes to decreased cell spreading on nanorods. Because a lack of cell spreading can cause cell death in each of the cell types studied here [9, 17] decreased spreading may explain the observed decrease in cell survival on nanorods. It is interesting to contrast our results with the work of Kim et al. [82] They found that nanowires are engulfed by cells, but do not induce apoptosis. Because the nanowires were sparse in this study (2030 nanowires exposed to each cell), it is likely that cells attach to the flat portions of the substrate and therefore survived. In our experiments, each cell was exposed to ~60,000 to 150,000 nanorods. Thus, we cannot rule out the possibility that a large number of nanorods are engulfed by our cells. If this is the case, then toxicity due to nanorod engulfment may cause cell death. Indeed, phagocytosed ZnO nanoparticles have been reported to be cytotoxic in vascular endothelial cells [96] More detailed studies are needed to investigate this possibility. If

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36 ZnO nanorods are engulfed by cells, then an interesting avenue for future investigation is the delivery of toxic material into cells. For example, the work by Kim et al showed that DNA immobilized on Si nanowires could be delivered into cells [88] Thus, the efficiency of ZnO nanorods in preventing cel l survival may be further enhanced by chemically conjugating toxins to the surface, and delivering these into the cell through penetration and subsequent cleavage. The nanorod aspect ratio probably plays an important role in the observed response. For example, Curtis and coworkers do not report a large decrease in cell survival, although they also observed decreased cell spreading on nanoposts [88] The diameter in these studies was 100 nm and the height was 160 nm. Curtis et al. report that nanocolumns are not engulfed by cells. As our aspect ratio is more similar to Kim and coworkers [82] where the nanowires were engulfed by cells, this could be another reason for the decreased cell survival in our experiments. Additionally, our observations of re duced cell adhesion and survival on nanorods are consistent with at least one recent study which employed an aspect ratio similar to the one used in this chapter [52] Conclusions Collectively, cell adhesion and viability were greatly decreased on ZnO nanorods. O ur results indicate that ZnO nanorods can be used as an adhesion resi stant biomaterial capable of inducing death in anchoragedependent cells. A better understanding of the mechanisms for the observed effects will be a key for designing optimal nanorod based substrates for minimizing cell adhesion and survival.

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37 Fig ure 3 1. The morphology of ZnO nanorods and flat substrate. A) SEM images of ZnO nanorods indicating a uniform monolayer of ZnO (left, scale bar is 2 m), and the upright growth of nanorods (right, scale bar is 500 nm). The diameter of nanorods was ~50 nm and the height was ~500 nm. B) AFM image of ZnO flat substrate. The surface roughness was approximately 1.33 nm indicating that this substrate is much smoother than the nanorods and can be used for comparisons of cell behavior between nanorods and smooth surf aces.

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38 Fig ure 32 Cells do not assemble stress fibers or focal adhesions on nanorods. Fluorescent micrographs of NIH 3T3, HUVEC, and BCE cells stained for vinculin (green) and F actin (red) on glass, ZnO flat substrate and ZnO nanorods The cell spr eading area is greatly reduced, and focal adhesions and stress fibers are not visible in cells cultured on the nanorods. Scale bar is 20 m.

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39 Fig ure 33 Total cell number and number of live adherent cells are reduced on nanorods. The average number of cells adherent on each substrate, the number of adherent live cells (stained with calcein AM) and adherent dead cells (stained with EthD 1) were quantified in three cell types A C) by pooling data from five different images per cell type and condition. Bars indicate standard error of the mean (SEM). indicates statistically significant differences with p<0.01 between the number of cells on ZnO nanorods and ZnO flat substrates (n>50 for HUVEC, n>30 for BCE, n>300 for fibroblasts, where n is total number of cells). D) The number of attached and live cells on ZnO nanorods normalized by the number of attached and live cells on ZnO flat substrates respectively. T he results show that the ratio of attached cells on ZnO nanorods to that on ZnO flat substrates is approximately same for all three types of cells. The decrease in the number of live adherent cells on the nanorods is robust across three different cell types, with a larger effect demonstrated in endothelial cells (HUVEC, BCE) than fibroblasts (NIH 3T3).

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40 Fig ure 34. Cells cannot assemble lamellipodia on nanorods. Representative SEM images of NIH 3T3 fib roblasts on ZnO nanorods. A) Most of cells on ZnO nanorods were round and they did not form lamellipodia. Scale bars in left image and inset are 3 m and 1 m respectively B) Filopodialike structures were observed in some cells on nanorods (white arrows in inset) along with thin processes (black arrows). Scale bars in left image and inset are 5 m and 2 m respectively.

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41 Fig ure 35. Dynamic cell spreading is altered on nanorods. Phase contrast imaging of HUVECs spread ing on glass and ZnO nanorods. A) Cell spreading HUVECs is accompanied by lamellipodia formation (white arrows) and is complete in approximately five hours. B) Cells on nanorods do not spread, and do not develop any lamellipodia. Scale bar is 20 m.

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42 Table 31. Average area of cell spreading on ZnO flat substrate and ZnO nanorods (Average area Standard Error of the Mean (m)) Section name NIH 3T3 HUVEC BCE ZnO flat substrate (1.44E+03) (2.76E+02) (1.73E+03) (2.35E+02) (4.18E+03) (7.49E+02) ZnO nanorods (4.73E+02) (7.44E+01) (6.02E+02) (6.94E+01) (1.22E+03) (1.17E+02) The differences of cell spreading area on ZnO flat substrate versus ZnO nanorods were statistically significant. (n=10, for NIH 3T3 and BCE, p<0.005 and for HUVEC p<0.0005)

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43 CHAPTER 4 RANDOMLY ORIENTED, UPRIGHT SIO2 COATED NANORODS FOR REDUCED ADHESION OF MAMMALIAN CELLS Introduction In the previous chapter we have shown that endothelial cells and fibroblasts are unable to ad here and survive on ZnO nanorods compared to flat ZnO substrates The advantage of ZnO nanorods is that they can be grown with solution based crystallization techniques at low temperature. Thus, the nanorods can be coated on surfaces of irregular geometries, and temperature sensitive materials such as stents. However, it is unclear if the dr amatic decrease in cell adhesion and survival observed on ZnO nanorods is reproducible with similar nanorods but of a different material. The chemical nature of the nanorod surface is clearly important given that it can potentially influence protein adsorption. In addition, ZnO has the potential for having long term toxicity to cells due t o leaching into solution [97 99] SiO2 based nanowires and nanoneedles have received recent attention for modulating cell adhesion [44, 82] Previous studies have shown that stem cells can survive for long periods of time on surfaces sparsely coated with SiO2 nanowires [82] Conversely, on comparatively denser SiO2 nanoneedles, cell adhesion is decreased, suggesting their potential for anti fouling surfaces [44] However, the decrease in cell adhesion on nanoneedles was not observed to be as dramatic [44] as previously reported with ZnO nanorods (see Chapter 3) Therefore, in this chapter we explored if SiO2 nanorods with similar morphologies as the previously used ZnO nanorods can result in a similar dra matic decrease in mammalian cell adhesion and survival. Our observations provide further evidence that

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44 densely packed upright nanorods can be used to develop surfaces resistive to mammalian cell adhesion. Materials and Methods Fabrication of N anorods ZnO nanorods were made by a solutionbas ed hydrothermal growth method [83] First, ZnO nanocrystal seed solutions were prepared by mixing 15 mM zinc acetate dihydrate (Sigma Aldrich, St. Louis, MO) with 30 mM of NaOH (Sigm a Aldrich, St. Louis, MO) at 60 C for 2 h Next, ZnO nanocrystals were spincoated onto the substrate and then post baked on a hot pl ate at 200 C for better adhesion. The substrate with these seeds was then suspended upside down in a Pyrex glass dish filled with an aqueous nutrient solution. The growth rate was approximately 1 m per hour with 100 ml aqueous solution containing 20 mM zinc nitrate hexahydrate and 20 mM hexamethylenetriamine (Sigma Aldrich, St. Louis, MO). To arrest the nanorod growth, the substrates were removed from solution, rins ed with deionized water and dried in air at room temperature. SiO2 was deposited with a Unaxis 790 plasma enhanced chemical vapor deposition (PECVD) system at 50C using N2O and 2% SiH4 balanced by nitrogen as the pr ecursors as reported before [100] Patterned nanorods were fabricated by conventional photoresist (PR ) lithography [83] A glass cover slide was processed with negative PR (SU 8 2007, Microchem) so that a pattern with 50 micron cir cles was formed on the surface. The subst rate was then post baked at 110 C for 30 min. The processed substrate was spincoated with ZnO nanocrystals as seed materials and nanorods were grown on the substrate with an aqueous nutrient solution. The negative PR was removed by PG remover in a warm bath at 60 C for 30 min.

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45 Contact Angle M easurements The contact angle of deionized water with surfaces was measured with a RamHart Goniometer and RamHart DROPimage Advanced Software using the sessile drop techniq ue. Cell C ulture For control substrate, we used 22 mm square glass cover slips (Corning, Inc., Lowell, MA). Before use, each substrate was sterilized with UV for 5 min and cleaned in 70% ethanol and deionized water. After drying substrates in air at room temperature, they were treated with 5 g/ml human fibronectin (FN) (BD biosciences, Bedford, MA). After ov ernight incubation with FN at 4 C, the substrates were washed twice with PBS. NIH 3T3 fibroblasts were cultured in DMEM (Mediatech, Inc., Herndon, VA) supplemented with 10 % donor bovine serum (DBS) (Hyclone, Logan, UT). Human umbilical cord vein endothelial cells (HUVECs) were cultured in EBM 2 Basal Medium and EGM 2 Single Quot Kit (Lonza, Walkersville, MD). Cell suspensions of the same concentration a nd volume (i.e. same number of cells) were then seeded on each substrate. Immunostaining and Cell Viability A ssay After 24 hours of cell seeding, nonadherent cells were removed with two gentle washes with PBS The samples were fixed with 4% paraformaldehyde for 20 min and washed several times with PBS. Fixed cells were immunostained for vinculin and stained for actin and nucleus using our previously reported methods [46] B riefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X 100, and treated with mouse monoclonal anti vinculin antibody (Sigma Aldrich, St. Louis, MO), followed by goat anti mouse secondary antibody conjugated with Alexa Fluor 488

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46 (Invitrogen, Eugene, OR). Actin was stained with phalloidin conjugated with Alexa Fluor 594 (Invitrogen, Eugene, OR) and nucleus was stained with 4' 6 diamidino 2 phenylindole (DAPI) (Sigma Aldrich, St. Louis, MO). Cells were then imaged on a Nikon TE 2000 epifluorescence microscope using green fluorescent protein ( GFP ) Texas Red and DAPI filters. All images were collected using the NIS Elements program (Nikon). The live/dead viability/cytotoxicity kit for mammalian cells (Invitrogen, Eugene, OR) was used for quantifying adherent cell viability on each substrate. Cells were incubated at 30 45 minutes with calcein AM (2 M for fibroblast and 4 M for endothelial cells) and ethidium homodimer 1 (EthD 1) (4 M for all types of the cells). Next, epifluorescence images of six to ten random fields were collected on a Nikon TE 2000 inverted microscope using a 10 X lens for NIH 3T3 and HUVEC. The average number of cells adherent on each substrate, the number of adherent live cells (stained green with calcein AM) and adherent dead cells (stained red with EthD 1) were quantified from these images using the NIS Elements program (Nikon). Three independent experiments of cell viability were performed and the data were pooled. The average area of cell spreading was determined from three independent experiments with statistical comparison using the Students T test. Time Lapse I maging Cells were precultured on the patterned nanor ods for 24 hours as mentioned above. Before taking a movie, nonadherent cells were removed with two gentle washes with PBS and new media was added to the dish. Phase contrast imaging was performed for 6 hours on the Nikon TE 2000 microscope with humidifie d incubator (In Vivo Scientific, St. Louis, MO). Images were collected every 5 minutes using a 10 X objective.

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47 Protein Adsorption on Nanorods and G lass Sterilized SiO2 coated nanorod and glass substrates were prepared as outlined above. Both of the substr ates were incubated with 10 g/ml rhodamine fibronection (Cytoskeleton, CO) diluted in PBS overnight, and these dishes were washed with PBS several times. Five randomly taken 20 X fluorescent images were collected with identical illumination and exposure t ime and the fluorescent intensity was analyzed by the NIS Element program (Nikon). Results and Discussion Fabrication of SiO2 Coated N anorods Many biomedical implants are made of temperaturesensitive materials such as plastic. Hence, it is necessary to g row nanorods with techniques that do not require high temperature. Densely packed ZnO nanorods were fabric ated with a low temperature (95 C) hydrothermal, solutionbased growth method [83] We next deposited nanothin films of SiO2 with controlled thickness, 50 using PECVD at 50 C according to our previously published methods [100] Transmission electron microscopy (TEM) images of the resulting nanorods with 50 thickn ess of SiO2 nanofilms deposited are shown in Fig ure 41A The nanorods were randomly oriented in the upright direction, approximately 40~50 nm in diameter, 500 nm in height. The average spacing between nanorods was approximately 80 to 100 nm (Fig ure 41 B white arrows). Importantly, the SiO2 coatings were deposited uniformly on each nanorod free of any local defects, which was confirmed with TEM, local electrical conductance measurements, chemical wet etching and photoluminescence intensity measurements [100] Our technique thus resulted in randomly oriented, upright SiO2 deposited nanorods that cover the surface

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48 with densely packed monolayers without any defects over cm length scales (Fi g ure 41 C) Decreased Cell A dhesion on SiO2 Coated N anorods As mentioned in Chapter 1 and Chapter 2, c ell adhesion and spreading occurs by the ligation of trans membrane integrins to ligands (such as fibronectin) immobilized on the surface. This is followed by clust ering of the integrins at the nanoscale, and subsequent formation of multi protein, micron scale assemblies called focal adhesions [101] Focal adhesions allow force transfer from the contractile actomyosin cytoskeleton inside the cell to the outside surface, and this allows cells to adhere to and spread on the surface. If focal adhesions are not allowed to assemble in cel ls that depend on anchorage for survival, this leads to weak attachment to the surface, lack of cell spreading and subsequent apoptosis [9, 17] Therefore, the assembly of focal adhesions was next studied using immunofluorescence microscopy. Human umbilical vein endothelial cells (HUVECs) and NIH 3T3 fibroblasts were cultured on SiO2 nanorods which were preincubated with fibronectin overnight. Cells were fixed with paraformaldehyde and stained for vinculin, actin stress fibers and the nucleus. Both HUVECs and NIH 3T3 fibroblasts assembled vinculinlabeled focal adhesions on glass (Fi g ure 42). On the nanorodcoated surfaces, focal adhesions were not visible and cells were rounded and poorly spread (Fig ure 42). Cells on nanorods were also unable to assemble contractile stress fibers. Consequently, the average area of cell spreading on nanorods was significantly decreased (Fig ure 43) with a lack of focal adhesion and stress fiber formation. This result suggests that cells are unable to spread and assemble focal adhesions on nanorods, which may cause apoptosis in these adhesiondependent cells [9, 17]

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49 Protein Adsorption on N anorods Recent work by Spatz and coworkers showed that focal adhesion assembly requires the spacing between ligated integrins to be less than 70 nm [8, 56] A spacing of more than 73 nm between ligated integrins limits attachment, spreading, and actin stress fiber formation in fibroblasts. As the diameter of the SiO2 nanorods is approximately 40~50 nm, local integrin clustering may occur but to a very limited extent given the vertical nature and small length (500 nm) of the nanorods. Due to the spacing of 80~100 nm, integrin clustering may not occur over mult iple nanorods, preventing the assembly of contiguous focal adhesions on the micron length scale (Fig ure 42). Other possible explanations for the fact that cells cannot s pread on nanorods are the super hydrophobic nature of nanostructured surfaces such as ZnO nanorods [4, 102] Protein adsorption is decreased on super hydrophobic surfaces which potentially can explain decreased adhesion. To address this question, we first measured contact angles of SiO2 coated nanorods and compared the contact angle with glass. We found that SiO2 coated nanorods were hydrophilic (Fig ure 44: contact angle of 6.93+/ 1.27o compared to glass of 42.1 +/ 1.14o). As f ibronectin is known to adsorb successfully on hydrophilic surfaces [103] this result suggests that reduced matrix protein adsorption is likely not the reason for decreased adhesion. To confirm this, we next measured the extent of fibronectin adsorption on nanorods (Fig ure 45) Rhodaminelabeled fibronectin was deposited overnight on SiO2 coated nanorods and flat glass substrates. Fluorescent images of the rhodamine fibronectin adsorbed surface were captured and analyzed for differences in intensity. Interestingly, we found that fibronectin adsorption as measured by fluorescence intensity was increased twofold on SiO2 coated nanorods compared to glass. An

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50 increase in protein adsorption is to be expected given the increased surface area of the nanorods. The increase in fibronecti n adsorption argues against large decreases in protein adsorption as being responsible for the observed reduction in cell adhesion. Importantly, fibronectin is known to adsorb in an active conformation on hydrophilic surfaces [103] Given that all our experiments were carried out in 10% ser um which allows the adsorption of other matrix proteins on the hydrophilic surface, and also promotes the secretion of fibronectin by the cells themselves, it is unlikely that decreased or abnormal matrix protein adsorption plays a significant role in the observed response. Spatial Patterning of Cell Adhesion with N anorods To investigate if it is feasible to pattern cell adhesion with nanorods, we spatially patterned nanorods using a low temperature, and patterned growth method [83] This method results in pa tterned nanorods that are not present inside circles, and are present outside in dense monolayers (Fi gure 46 ameter and 500 nm in height. Similar patterning was also observed with ZnO nanorods without SiO2 coating (Fig ure 4 8 ). Moreo v er, w hile the cells were confined to the circular regions on average, cells were frequently able to migrate from circle to circle by spanning the intervening nanorods ( Figure 49 ). This result suggests th at spatially patterned nanorods provide a new way of dynamically patterning cells and therefore creating complex tissues. Decreased Cell Survival on N anorods The number of cells adherent on SiO2 coated nanorods was significantly reduced (a reduction of 98% in fibroblasts, 82% in HUVECs) compared to cells on glass (Fig ure

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51 4 10A) after 24 hour culture. Next, a live/dead viability/cytotoxicity kit for mammalian cells was used for quantifying adheren t cell viability. The decrease in viability in cells on nanorods compared to that on glass was dramatic (Fig ure 410 B) with only one or two cells surviving on the SiO2 nanorods for every 100 viable cells on glass. By culturing cells on glass in media that was incubated for 1 day, 3 days and 7 days with the nanorods, we confirmed that the cell death was not due to toxicity of unknown dissolving material fr om the nanorods ( Figure 411) Therefore, t hese results suggest that densely packed nanorods have excel lent anti fouling potential by virtue of their topology. Conclusions In this chapter we show ed that cell adhesion was significantly decreased on SiO2 coated nanorods. None of the cells were able to assemble vinculinmarked focal adhesions. When cultured on a patterned surface where flat circular areas were surrounded by nanorods, cells were able to migrate to adjacent flat areas by spanning the nanorods. Taken together the results of this chapter indicated again that nanorods can be an anti fouling surface to reduce anchorage cell adhesion as shown in Chapter3 Regardless of material composition of nanorods or protein adsorption, topology of nanorods plays an important role for reducing cell adhesion.

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52 Figure 4 1. The morphology of nanorods. A ) TEM image of SiO2 deposited ZnO nanorods. Black arrows indicate SiO2 thin film with a 50 thickness ZnO nanorods are encapsulated by SiO2. B ) SEM image of nanorods on glas s. White arrows indicate the spacing between nanorods. The spacing between nanorods ranges from 80 to 100 nm. C ) SEM image of a monolayer of nanorods. Upright nanorods were covered on the underlying glass substrate uniformly.

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53 Figure 4 2. Fluorescent microscopic images of HUVEC and NIH 3T3 on glass and nanorods. HUVEC and NIH 3T3 on glass assemble focal adhesions stained with vinculin (green) and actin stress fibers (red). N ucle i were stained with DAPI (blue). HUVEC and NIH 3T3 on nanorods are unable t o spread and assemble focal adhesions and stress fibers

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54 Figure 4 3. The average area of cell spreading on glass and nanorods. A) HUVEC on glass and nanorods (n > 170). B ) NIH 3T3 on glass and nanorods (n > 110). indicates p < 0.005. Bar indicat es standard error of the mean (SEM). The data were pooled from three independent experiments.

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55 Figure 4 4. Contact angles of water on glass and SiO2 coated nanorods.

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56 Figure 4 5. Fluorescent images and intensity of rhodamine fibronectin coated glass and nanorods. A) Representative fluorescent images of rhodamine fibronectin on glass and SiO2 coated nanorods B) Plots show the average intensity profile pooled from five randomly taken images. Bar indicates the standard deviation (SD).

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57 Figure 4 6. SEM images of patterned nanorods. A) Optical microscope image with 400 X objective. B) SEM image.

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58 Figure 4 7 NIH 3T3 fibroblasts on patterned SiO2 coated nanorods. F luorescent microscopic images showing that NIH 3T3 fibroblasts preferabl y attached on glass. Cells are stained for actin (red), vinculin (green) and nucleus (blue). Cells were confined on the flat circular regions. Dashed lines indicate the edge of patterns.

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59 Figure 4 8 NIH 3T3 fibroblasts on patterned ZnO nanorods. A ) Phase contrast and fluorescent microscopic images showing that NIH 3T3 fibroblasts preferably attached on glass. Cells are stained for actin (red), vincul in (green) and nucleus (blue). B ) Differential interference contrast and fluorescent microscope images Cells were confined on the flat circular regions. Dashed lines indicate the edge of patterns.

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60 Figure 4 9 Time lapse phase microscope images of NIH 3T3 on patterned ZnO nanorods. Black arrows indicate the direction of cell motility. Cells are obser ved to move from glass to glass by spanning intervening nanorods (top panel), or continuously explore the nanorod environment at the edges of the circle (middle and bottom panel). Arrows indicate direction of motion.

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61 Figure 4 10. Cell attachment and viability on nanorods. A ) The ratio of the number of attached cells on nanorods to that on glass. B ) The ratio of the number of live cells on nanorods to that on glass. (n > 2500 for HUVECs, n > 1500 for NIH 3T3. Bar indicates SEM. Cells are considerably reduced in numbers on nanorods.

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62 Figure 4 11. M aterial toxicity test. Media incubated with nanorods (or pure glass) for 1 day 3 days, and 7 days was added to cells. Cells were cultured in the nanorod treated media for one day. No obvious differences in cell numbers or morphology were observed when cultured with nanorod treated media. This argues against solution toxicity as being responsible for c ell death on nanorods day incubated media with nanorods and glass were added to 1 day pre cultured HUVEC and NIH 3T3 dishes.

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63 CHAPTER 5 MODULATING MALIGNANT EPITHELIAL TUMOR CEL L ADHESION, MIGRATION AND MECHANICS WITH NANOROD SURFACES Introduction Tumor s that occlude the gastrointestinal, pancreatic and biliary ducts are typically surgically removed, and metallic stents are placed in the ducts for preventing subsequent collapse of the injured tissue [1, 104] However, tumor cell adhesion, migration and proliferation on the installed stents cause stent re blockage. This requires further surgical interventions for stent removal and causes severe complications in the management of malignancy [105] To overcome such recurrent problem s with metallic stent s, plastic covered stents have been developed [2, 106, 107] However, plastic covered stent s tend to migrate to other organ s [104, 108] and have poor performance compared to metallic stent s [108, 109] Thus, combining the advantages of metal stents with prevention of tumor cell adhesion to the stent surface remains a key challenge [2, 105109] One approach to reduce the blockage of stents is to fabricate nanostructured features on the cell surface that will interfere with tumor cell adhesion. A number of studies have shown that cell adhesion [44, 56, 110] assembly [111] and migration [21] are sensitive to the micro and nanoscale topography of the culture substrate [112] These studies have been carried out for cells of nontumor origin, but there are relatively few studies on tumor cell interactions with nanostructured materials. One study showed that the spreading and proliferation of human osteosarcoma cells decreases on microgrid titatium coated silicon surfaces with increasing surface roughness [113] Similarly the adherent human hepatocellular carcinoma cell number on a silicon nanowire surface was decreased by 60.5% compared to a bare silicon

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64 wafer [114] These studies support the promise of using structured surfaces for controlling tumor cell adhesion. We recently reported that coating surfaces with dense monolayers of randomly oriented, upright nanorods significantly reduces adhesion and viability of fibroblasts and endothelial cells [45] Cells on nanorods were unable to assemble focal adhesions a nd stress fibers, which we hypothesized to be due to disruption of integrin clustering on nanorod substrates [45] However, it is unclear if a similar approach can be used to modulate the adhesion and viability of tumor cells responsible for stent occlusion. In this chapter we investigated the effect of nanorod coatings on the adhesion, motility, and mechanics of malignant, human esophageal epithelial cells. Malignant tumor cells cultured on nanoroadcoated surfaces had significantly decreased nonmuscle myosin II activity, decreased stiffness and increased motility. The lack of firm adhesion correlated with an overall decrease in tumor cell n umber. Materials and Methods Growth o f N anorods A solution based hydrothermal growth method was used for fabricating ZnO nanorods on the substrates [83] Briefly, ZnO nanocrystal seed solutions composed with 15 mM zinc acetate dihydrate (Sigma Aldrich, St. Louis, MO) and 30 mM of NaOH (Sigma Aldr ich, St. Louis, MO) were prepared at 60C for 2 h and spincoated onto the substrates. Nanorods were grown by placing seedcoated substrate s upside down in an aqueous nutrient solution of 20 mM zinc nitrate hexahydrate and 20 mM hexamethylenetriamine (Sigm a Aldrich, St. Louis, MO). A U naxis 790 plasma enhanced chemical vapor deposition (PECVD) system was used to deposi t SiO2 on the ZnO

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65 nanorods at 50 C using N2O and 2 % SiH4 balanced by nitrogen as the precursors as reported before [100] Cell C ulture 22 mm square glass cover slips (Corning, Inc., Lowell, MA) were used as control substrates. All of the substrates were sterilized with UV for 5 min, and washed with 70 % ethanol and deionized water. Before cell culture, the substrates were treated with 5 g/ml human fibronectin (FN) (BD biosciences, Bedford, MA) at 4C overnight. OE33 (human esophageal epithelial tumor cells) cells were cultured in RPMI supplemented with 10% donor bovine serum ( DBS ) and 200 mM LGlutamine (Sigma, St. Louis, MO ). Cell Viability A ssay Cells cultured for 24 hours on each substrate were stained with t he live/dead viability/cytotoxicity kit for mammalian cells (Invitrogen, Eugene, OR) for quantifying adherent cell viability. The number of OE33 on glass and nanorods was counted from ten fluorescent images taken randomly using a 20 X objective. T hree independent experiments of cell viability were performed and the data were pooled. To check f or solution toxicity of nanorods, OE 33 media w as incubated with sterilized nanorods or with glass for 1 day, 3 days and 7 days in incubator. The conditioned media was next used to culture cells for 24 hours Cell morphology and numbers with nanorodincubat ed media was compared to that with glass incubated media. BrdU S taining 10 M 5bromo 2 deoxyuridine (BrdU) (Sigma Aldrich, St. Louis, MO) was added to cells on glass and nanorods [115] After 20 hour s of incubation, cells were fix ed with 4 % paraformaldehyde for 20 min and washed several times with PBS. 2 M hydrochloride ( HCl) was added to the cells and incubated for 20 min at room

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66 temperature. Cells were permeabilized with 0.2% Triton X 100 supplemented with bovine serum albumin. C ells were stained with primary anti BrdU IgG (Sigma Aldrich, St. Louis, MO) and goat anti mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Eugene, OR ). The number of attached and proliferated cells on glass and nanorods was counted with ten randomly taken images using a 20X objective. Three independent experiments were performed, and the data were pooled. A similar fixation and staining protocol was followed for immunostaining of adhesion proteins [45, 46] Scanning E lectron M icroscopy (SEM) After 24 ho ur s of culture, c ells were prepared for SEM by fixation with 2% glutaraldehyde buffered in PBS and post fixed in 1% osmium tetroxide and dehydrated in graded ethanol concentrations. Critical point drying (CPD) was performed on a Bal Tec 030 instrument (ICB R Electron Microscopy Core Lab, University of Florida) and Au /Pd ( 50 ) was deposited on the substrate. SEM was performed on a Hitachi S 4000 FE SEM (ICBR Electron Microscopy Core Lab, University of Florida). Images of samples were taken at 1. 0 2 0 kX mag nifications. Cell Motility A ssay Phase contrast imaging was performed for 12 hours on a Nikon TE 2000 microscope with a humidified incubator (In Vivo Scientific, St. Louis, MO). Images were collected every 10 minutes using a 10X objective. The images were then analyzed using a Matlab program that tracked the position of the centroid of cells in (x,y) coordinates vs. time. The mean squared displacement was calculated from the data using nonoverlapping time int ervals [116] The speed of each cell was determined from the average displacement by the tracking interval 10 min. The persistence time of each

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67 cell w as obtained from fit using nonlinear least square regression with the measured speed to a persist ent random walk model for cell migration as reported elsewhere [117] Cell Stiffness Measurement by Atomic Force M icroscopy (AFM) Cells were cultured on FN coated glass and nanorods for 20 hours. Detailed method was reported elsewhere [118] Briefly, Asylum MFP3D AFM (Asylum Research, CA) coupled to a Nikon TE2000U epifluorescence microscope was used for measuring cell stiffness. The pyram id tip had a spring constant of 60 pN/nm, and tip half angle was 37 degrees. 122 cells on glass and 87 cells on nanorods were measured from glasses and nanorods. Each profile was fit with a modified Hertzian model. Western B lotting Cells cultured on 76.2 mm x 2 5.4 mm glass and same size of nanorods were washed with cold PBS and lysed with cell lysis buffer (Cell Signaling Technology, Inc. MA ) for 10 minutes on ice. Cells were then collected and centrifuged at 10,000 rpm for 10 minutes at 4 C. The supernatant was then collected and sodium dodecyl sulfate ( SDS ) sample buffer was added and stored at 20 C until used. The samples were separated on 10% SDS polyacrylamide gels and then transferred onto a p olyvinylidene fluoride ( PVDF ) membrane. The membr anes were blocked with 5% milk in Tris buffered saline with Tween 20 ( TBST ) at room temperature for 30 min. The membranes were treated with phosphor myosin light chain 2 antibody (Cell Signaling Technology, Inc. MA ) at 1: 1000 dilutions in 5% mi lk overnight at 4 C. The membranes were then washed three times in TBST and treated with peroxidase conjugated secondary antibody at 1:10000 in 5 % milk in TBST for 2 hours. Blots were developed using SuperSignal West Pico Chemiluminescent reagent (Pierce Biotechnolog y, IL ) and exposed to X OMAT film ( Eastman Kodak Inc., NY ).

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68 Results and Discussion Decrease in A dhesion of Esophageal Epithelial Cells on N anorods We used a previously developed method to grow SiO2 coated nanorods on glass surfaces (Fig ure 51 A and 5 1B) [100] Transmission electron microscopy (TEM) and electrical conductance measurements confirmed that the nanorods were uniformly covered by SiO2 without any defect s [100] Nanorod surfaces were then coated with fibronectin and tumor epithelial cells were cultured on the substrates. After 24 hours of culture, the number of adherent tumor cells was observed to be nearly 5 0 % lower on nanorod surfaces compared to glass (Fig ure 51 C) This decrease was not due to toxicity of materials leached from the nanorods themselves (Fig ure 52 ). We have also previously shown that the SiO2 coated nanorod surfaces are hydrophilic, and fibronectin adsorption is unaltered on these surfaces in Chapter 4. This argues against altered matrix protein adsorption as a potential cause of the decrease in tumor cell numbers. Viability and Proliferation is Unchanged in Tumor Cells on N anorods Staining with calcein AM (4 M) and ethidium homodimer 1 (EthD 1) showed that adherent tumor cells on glass and nanorods were both equally viable (data not shown) BrdU staining revealed that tumor cell proliferation on nanorods was similar to that on glass at 24 hours (Fig ure 51D ). Together, these results suggest that the decrease of adherent tumor cells on nanorods is due to weakened adhesion rather than a decrease in proliferation rate or cell viability The fact the proliferation and viability is unchanged although weak tumor cell adhesion is consistent with the fact that malignant tumor cells lose their dependence on firm adhesion for survival [119, 120]

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69 Tumor Cell C ultured on Nanorods have Decreased C ontractility While tumor cells were able to form colonies on nanorods, individual cells in colonies were rounded on nanorods compared to glass ( Fig ure 5 3 ). We next stained cells for vinculin and imaged cells with confocal fluorescence microscopy, but clearly defined focal adhesions proved difficult to detect on both glass and nanorods (Fig ure 54 ). Cells at the periphe ry of the colonies were observed to form lamellipodial structures on glass, but similar structures were absent on nanorods (Fig ure 53 arrows). These results raised the possibility that nanorods could potentially decrease intracellular tension in the tumor cells. To evaluate this possibility directly, we next measured the levels of phosphorylated nonmuscle myosin II as a measure of intracellular contractility in tumor cells. As seen in Fig ure 5 5 the level of phosphorylated myosin II is significantly dec reased in tumor cells adherent to nanorods compared to flat surfaces. These results provide an explanation for the rounded cell morphologies seen in tumor cell colonies on nanorods. It is known that the levels of phosphorylated nonmuscle myosin II correlate with the stiffness of the cortical actomyosin cytoskeleton in adherent cells [121, 122] We therefore measured stiffness of the adherent tumor cell cortex using atomic force microscopy ( AFM ) [123, 124] As cell tension is proportional to cortical stiffness [123] the stiffness can be consi dered an indirect readout of cell tension. Our measurements revealed that t he stiffness of single tumor cells on nanorods was decreased by nearly 50% of that on glass (Figure 56 ). Together, these results suggest that tumor epithelial cells have reduced tension on nanorods compared to glass substrates, which leads to weak adhesion, decreased cell numbers and rounded cell morphologies.

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70 The mechanism of how nanorods alter tumor cell adhesion and mechanics is at least in part due to the nanoscale control of i ntegrin clustering as mentioned in the previous chapters The clustering of integrins occurs through crosslinking by intracellular proteins like talin [125] which causes the formation of stable adhesions that are p hysically linked to the intracellular actomyosin cytoskeleton. Interfering with integrin clustering interferes with focal adhesion assembly [8, 59, 94, 126] and feeds back to change actomyosin contractility [6]. Work by Spatz and co workers has shown that integrin clustering requires that adjacent ligated integrin molecules be at a distance of less than 70 nm [8, 59, 94, 126] Distances higher than these reduce clustering and focal adhes ion formation. A similar mechanism may be responsible for our results because the inter nanorod spacing is on the order of 50100 nm [45] which likely interferes with normal integr in clustering Single Tumor Cell Motility is Increased on N anorods Cell motility has been previously shown to be sensitive to micro and nanoscale surface topology [127129] For example, f ibroblasts migrate faster on surfaces with 500 nm nanoholes compared to the corresponding flat glass surfac e [128] Similarly, on titanium dioxide ( TiO2) nanotube surface s, mesenchymal stem cells and fibroblasts moved faster on 15 nm nanotubes compared with the smooth surface [127, 129] However, it is not clear if tumor cell motility is similarly sensitive to nanostructure. We therefore measured the single tumor ce ll migration speed and persistence time on nanorods. T he average cell speed of single tumor cells and the mean persistence time were both found to be increased on nanorods compared with glass (Fig ure 57A and 5 7 B). Given our observation that myosin II bas ed contractility is decreased in tumor cells

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71 on nanorods, (Fig ure 5 6 ) it is possible that the observed increase in motility on nanorods is due to weakened adhesion [117, 130] Conclusions In summary, our results suggest that it is possible to modulate malignant tumor cell adhesion, migration and mechanics with nanorod surfaces. The weakened adhesion raises the possibility that increased tumor cell detachment may occur under shear forces whi ch are commonly encountered in the body (although not studied here). Our results suggest that nanostructurebased approaches may be a powerful yet simple approach to modulate tumor cell adhesion.

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72 Fig ure 51. Esophageal epithelial tumor cell adhes ion was decreased on nanorods. A), B) SEM images of nanorod morphology. Upright nanorods were covered on the underlying glass substrate uniformly. C) Numbers of attached OE33 cells were reduced by 50% on nanorods compared to the flat glass surface after 24 hour culture. D) Cell proliferation is unchanged on nanorods compared to glass as measured by Br d U incorporation. Bars indicate the standard error of the mean (SEM). indicates statistically significant difference (p<0.05).

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73 Figure 5 2 Nanorods are not toxic to tumor cells. Media was first incubated with either nanorods or glass for the times indicated. The media was then added to cells cultured at identical s eeding densities for 24 hours. A) Phase contrast images of OE33 cells cultured for 24 hours with media incubated either with glass or nanorods for the times indicated. Images show no significant change in the adherent cell number on culturing cells with nanorod or glass incubated media. B) Quantification of data from A suggesting that the nanorod inc ubated media does not cause a decrease in cell numbers compared to glass incubated media. Number of cells was quantified from five images and divided by the total measured area.

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74 Fig ure 53. Individual tumor cells in colonies were rounded on nanorods unlike cells on glass. A) Phase contrast images of OE33 colony on nanorods and glass. B) SEM images of colony on nanorods and glass. Arrows point to lamellipodial structures on glass surface; similar structures were not detected on nanorods.

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75 Figure 5 4 Confocal microscopic images of OE33 on glass and nanorods. Cells were stained with vinculin (green), and actin stress fibers (red). Clearly defined focal adhesions are difficult to detect both on glass and on nanorods.

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76 Fig ure 55. Non muscle myosin II activity is significantly reduced in cells on nanorods compared to cells on glass. Western Blot of phosphorylated myosin shows decreased levels in tumor cells on nanorods. The comparison was made for identical levels of GAPDH to account for the decrease in cell number on nanorods.

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77 Fig ure 56. Tumor cells are softer on nanorods compared to glass. A) Histograms of single cell stiffnesses measured by AFM on nanor ods and glass. B) The mean cell stiffness on nanorods was decreased by 50% compared to that on glass.

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78 Fig ure 5 7. OE33 cell motility is altered on nanorods. A) The average speed of OE33 on nanorods was higher than that on glass (n=15 for glass, n=16 for nanorods). B) The mean persistence time is longer on nanorods than on glass (n=9 for glass, n=11 for nanorods). Bars indicate SEM. indicates a statistically significant difference (p<0.05).

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79 CHAPTER 6 THE ROLE OF NUCLEARCYTOSKELETAL LINKAGES IN CELL MECHANO SENSING Introduction Recent studies suggest that the physical connection between the nucleus and the actomyosin cytoskeleton may be important in cell sensing of mechanical cues [131 134] The different proteins that propagate cytoskeletal forces from outside the nucleus to the inside form a complex referred to as the LINC complex (for Linker of Nucleoskeleton to the Cytoskeleton). This complex consists of lamin A/C, a protein that forms a rigid, elastic network under the inner nuclear membrane, SUN family of proteins that bind to lamin A/C and reside in the inner nuclear membrane, and nesprin family of proteins that reside in the outer nuclear membrane. The primary evidence that these proteins are important in cell mechanosensing comes from studies with lamin A/C deficient cells [135 137] Unlike normal cells, lamin A/C deficient cells exhibit defects in transducing mechanical forces into signaling pathways [135] Similar effects have been observed in cells lacking other inner nuclear membrane proteins. For example, emerin deficient fibroblasts have irregular nuclear shape, exhibit an increase in cell death in response to mechanical strain and exhibit abnormal mechanotransduction [138] Even though nesprin1 is a direct linker between F actin and the nucleus, the extent to which it influences cell mechanosensing has not been determined. In addition to sensing externally applied mechanical forces, cells also are sensitive to substrate rigidity. For example, decreasing the stiffness of soft substrate composed of polyacrylamide (PAAm) hydrogel decreased neurite outgrowth and branching of PC12 cells [139] Depending on the stiffness of substrates, nave mesenchymal stem cells

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80 (MSCs) specified lineage and committed to phenotypes differently [140] However, the molecular mechanism s by which cells sense substrate stiffness are unclear Recently, Chancellor and co w orkers have discovered a novel role for nesprin1 in endothelial sensing of mechanical forces [14 1] They showed that nesprin1 deficient cells assemble more focal adhesions and exert gre ater traction on the substrate. The altered traction and adhesion assembly suggests that nesprin1 may also be necessary for cellular sensing of substrate stiffnes s. In this chapter, we explored this possibility. Materials and M ethods Cell S ulture and siRNA K nock D own of N esprin 1 Human umbilical vascular endothelial cells (HUVECs) were cultured with DMEM high glucose (Cellgro, Manassa, VA) supplemented with 10% donor bovine serum (DBS) (Gibco, Grand Island, NY). Cells were transfected with 100 nM of smartpool siRNAs (Dharmacon, Lafayette, CO) against human nesprin1 using siLentFect lipid transfection reagent (BioRad, Hercules, CA ) [141] Non target ing siRNAs served as controls. After 4872 hours of transfection, cells were passed onto polyacrylamide sub strates and glass Fabrication of P olyacrylamide S ubstrates Polyacr ylamide (PAAm) substrates were fabricated as reported elsewhere [16, 142] Briefly, 0.1 M NaOH was dropped on the cover glass portion of glass bottom ed dishes (MatTek, Ashland, MA) and dried overnight at room temperature. 10% pre polymerized (3aminopropyl)trimethoxysilane (APTMS) (Sigma, St. Louis, MO) was added onto the glass portion of dishes and incubated for 5 min. DI water was dropped on top of the APTMS layer and incubated for 10 min. After washing the solution with water, the dishes were shaken for 10 min. 0.5 % Glutaraldehyde solution was added to

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81 the dishes for 30 min. The dishes were washed with DI water several times and dried overnight. Four different ratios of acrylamide and bis acrylamide (Fisher Scientific, Pittsburgh, PA), i.e. 50:1, 40:1, 20:1, and 12.5:1 were chosen to make gels with Young s modulus of 1.0 7.8 kPa, 21.6 6.17 kPa, 45.8 3.31 kPa, and 308.0 14.9 kPa as reported elsew here [143] The polyacrylamide solution was degassed, and 10% ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) (Sigma, St. Louis, MO) was added. This solution was dropped on the chemically modified glass portion of glass bottom ed dishes. The drop was covered by a cleaned circle cover glass, and allowed to polymerize at room temperature for 10 min. PBS was added before peeling th e cover glass with tweezers. The gel surface was treated with 200 mM sulfoSANPAH (Thermo Fisher Scientific, Waltham, MA) and then incubated with 5 g/ml fibronectin (FN) overnight. The coverslip glass was treated by FN as a control. Immunostaining After 24 hours of cell culture, nonadherent cells were removed by PBS washes Cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS several times. Then, the samples were permeablized with 0.2% Triton X 100 and treated by mouse monoclonal anti vinculin antibody (Sigma Aldrich, St. Louis, MO), followed by goat anti mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Eugene, OR) Actin was stained with phalloidin (Invitrogen, Eugene, OR) and nucleus was stained with Hoechst 3325 8 (Sigma Aldrich, St. Louis, MO) Using GFP, Texas Red and DAPI filters on a Nikon TE 2000 microscope, cells were imaged. Measurement of C ell S preading A rea 1 day cultured cells were stained with phalloidin and Hoechst as reported above. Cell spreading im age was taken with a 20X lens. Individual cell s which w ere sufficiently

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82 distant from each other (> 50 m ) w ere measured using Nikon Element program. The average spreading area of 40 cells for each condition was pooled from two independent experiments. Cel l M otility A ssay Nesprin 1 knockdown siRNA transfected HUVEC (NKHUVEC) and control smartpool siRNAs transfected HUVEC (SHUVEC) were cultured on FN coated PAAms for 6 hours. Cell movement was observed with a 10X phase contrast lens on the Nikon TE 2000 micr oscope with humidified incubator (In Vivo Scientific, St. Louis, MO). Images were taken every 10 minutes for 10 hours. The images were then analyzed using a Matlab program that tracked the position of the centroid of nuclei in (x, y) coordinates vs. time. The mean square displacement (MSD ) w as calculated from the data using nonoverlapping time intervals (t) [144] The average cell speed (s) was calculated as (displacement in 10 min)/( 10 min) The rationale for this calculation is that cells undergo persistent motion at short time intervals of 10 min. The persistence time (p) and length (p multiplied by s) of each cell were obtained from fit ting using nonlinear least square regression with the measured speed to a persistent random walk model for cell migration as reported elsewhere [145] (6 1 ) 22/()2[(1e)]tpdtsptp

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83 Results The R igidity D ependence of C ell S preading is A ltered in N esprin 1 D eficient C ells A simple method to quantify rigidity sensing in cells is to measure the area of cell spreading on substrates of varying rigidities. Typically, cells on soft substrates are poorly spread, while cells on stiff substrates are well spread. NKHUVEC and SHUVEC were cultured on PAAms with varying Young s modulus ( 1 kPa (1.0 7.8 kPa), 22 kPa (21.6 6.17 kPa), 46 kPa (45.8 3.31 kPa), and 308 kPa (308.0 14.9 kPa) [143] ) for 1 day. Figure 6 1 show s average cell spreading area pooled from two independent experiments. The spreading area of SHUVEC increased with increasi ng stiffness in agreement with previous reports [143, 146, 147] However, NKHUVEC spreading area did not increase from 22 kPa to 46 kPa, and increased much less on 308 kPa compared to SKHUVEC. Also, the spreading area between SHUVEC and NKHUVEC was significantly different on 1 kPa, 22 kPa and 308 kPa. This suggest s that there is at least some disruption of cell sensing of substrate rigidity. Focal A dhesion and S tress F iber A ssembly on PAAms Focal adhesion assembly and stress fiber formation was next explored. It has been previously shown for normal rat kidney epithelial cells that f ocal adhesions appear as irregular punctuate structures on the soft PAAm surface (Young s modulus is approximately 15 kPa) but cells formed stable focal adhesions at the high stiffness PAAm (Young s modulus is approximately 70 kPa) [142] As seen in Fig ure 62 s tress fibers and adhesions are visible in SHUVECs on 46 kPa and 308 kPa PAAms, while these structures are less clear in NKHUVECs on all stiffnesses

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84 NKHUVECs have A ltered S ingle C ell M otility Next, the single cell motility of NKHUVEC and SHUVEC was monitored every 10 minutes for 10 hours. The centroid of nuclei in ( x, y) coordinates was tracked in time Non overlapping MSD using x, y positions was quantified and fit to a random walk model [144, 145] Figure 6 3 show the model fits to representative time dependent MSD Cell speed has been to shown to depend on stiffness in a bimodal fashion [143] We observed a very small peak for S HUVECs on 22 kPa and a bigger peak for NKHUVECs ( arrows in Figure 64A ). It is possible that the HUVECs exhibit a bimodal dependence; however, we do not have data on PA Am gels with intermediate stiffnesses between 0, 22 and 46 kPa to be conclusive. Qualitatively the speed dependence on stiffness is not significantly different between NKHUVEC and SHUVEC. What is surprising is that the dependence of persistence length on stiffness of NKHUVECs is bell shaped compared to the monotonic increase for SHUVECs (Figu re 6 4B and 64C) This may be due to the drastic decrease in the persistence length (or time) in NKHUVECs on high stiffnesses, a finding that is already supported by the results of Chancellor et al on glass surfaces [141] Together, these results suggest that nesprin1 may play a key role in cell ular sensing of matrix rigidity Discussion Recent research by Chancellor et al has shown an important role for nesprin1 in endothelial mechanosensing [141] This study proposed that actomyosin tension normally balanced by the nucleus is balanced in nesprin1 deficient cells by the substrate. As a consequence, in the absence of nesprin1, cells form ed more focal adhesions and exert ed increased traction on the substrate. Interestingly, there wa s no change in nonmuscle myosin II activity.

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85 The decrease in directional persistence observed in our experiments on 308 kPa is similar to the observation made by Chancellor et al on glass surfaces [141] One possible explanation for this observation is the fact that nesprin1 deficient cells form mo re adhesions on 308 kPa, leading to decreased frequency of detachment. Our images of focal adhesions on 308 kPa do not support this explanation as adhesions were less visible with nesprin1 deficient cells on 308 kPa. Another possi ble explanation is that a ctin polymerization is inhibited in nesprin1 deficient cells on 308 kPa. This possibility is supported by our observation that cell spreading area is significantly decreased on 308 kPa in nesprin1 deficient cells A ctin polymerization is known to decreas e on soft substrates and this has been shown to correlate with decreased spreading [148, 149] More experiments with live cell imaging are needed to quantify the rates of actin polymerization in cells on different stiffnesses to support this prel iminary conclusion It is possible that Rho pathways that control actin polymerization are altered in nesprin1 deficient cells these include Rac and Cdc42 signaling. Western blotting to assay for differences in GTPase activity of these proteins in cells on different stiffnesses would greatly aid in better understanding the mechanisms that cause altered stiffness sensing in nesprin1 deficient cells. Conclusion Endothelial sensing of substrate rigidity is altered in the absence of nuclear cytoskeletal con nections mediated by nesprin1 The relationships most influenced by nesprin1 deficiency are stiffness dependence of cell spreading area and stiffness dependence of directional persistence. More experiments that measure the rates of actin polymerization and the levels of Rac/Cdc42 signaling pathways are needed to understand the mechanism for our observations.

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86 Figure 6 1. Average cell spreading area. Error bars represent the standard error of the mean ( SEM ) from t wo different experiments and **, *** indicate p < 0.0 5

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87 Figure 6 2. Fluorescent microscopy images of vinculin and F actin

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88 Figure 6 3. Representative mean square displacement (MSD) calculated for SHUVEC and NKHUVEC on PAAms of varying stiffness and m o del fits.

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89 Figure 6 4. Cell speed, persistence time and persistence length of NKHUVEC and SHUVEC on PAAms. A ) Arrows indicate a n apparent peak in speed on 22 kPa PAAm. B) Persistence time and C) persistence length increase with stiffness for SHUVEC, whereas a bell shaped trend of persistence time and length is observed for NKHUVEC. Error bars represent SEM and indicates p < 0.0 5 (n=17~34 for PAAm, n=6~9 for glass ).

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90 CHAPTER 7 A STAMPWOUND ASSAY TO STUDY COUPLED CELL PEELING AND MIGRATION: T OWARD A REALISTIC WO UND HEALING ASSAY Introduction Wound healing is a complex process that is critical for preserving the integrity of multicellular organisms and tissue homeostasis [150] Wound healing involves the migration of cells of different types directed by chemotactic signals into the wound. In vitro models of wound healing traditionally [151 153] involve scratching a confluent cell mono layer with a microneedle or micropipette tip, and capturing the time dependent closure of the wound with microscopy. Such studies have allowed the discovery of key signaling pathways that control the migration of cells during wound closure [154 156] One limitation of the scratch wound assay is that it lacks precision for creating a contr olled wound. Alternative assays to create wounds have been recently reported that use laser photoablation [157] or masks to prevent cell adhesion to defined wound areas [158160] While these traditional techniques offer more reliable models to study wound healing, they all share the common feature that the wound area is devoid of any cells. However, wound healing in the body involves not only the migration of cells into the wound, but also the simultaneous clearing of cell debris by the process of phagocytosis [161 165] Assays that allow study of the coupled process of wound healing and phagocytosis are therefore desirable for realizing realistic in vitro wound healing assays. Here, we report a new technique to make more wounds on epithelial cell monolayer using a stamping technique. The method involves the physical contact of a soft mold with raised features onto confluent epithelial cells With this method, we successfully create d well defined wounds with dead cell debris in the wound area.

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91 Imaging over several hours showed that the cells migrate into the wound after first clearing the wound area of cell debris. The rate of wound closure was found to be faster than the scratch wound assay. Interestingly, the inhibition of nonmuscle myosin II with blebbistatin interfered with the heal ing of the wound in the presence of cell debris, but was unable to inhibit wound healing without dead cells These results suggest that the proposed stampwound assay can be used to provide novel insight into the mechanisms of wound healing. Materials and M ethods Fabrication of P oly(dimethyl)siloxane (PDMS) M olds A master mold was fabricated by a conventional photolithography method. Prepolymers of soft PDMS (Sylgard 184, Dow Corning, MI) were poured over the photoresist master mold and degassed for 20 mi n in a vacuum, then cured at 60 C in an oven for 2 hours. After peel off, the PDMS mold was sterilized by 70% ethanol and washed several times with sterilized DI water. For removing any remaining solvent and prepolymer, the PDMS mold attach ed to a glass slide was baked at 120 C in an oven for 2 hours. Cell C ulture and S oft I mprint ing with the PDMS M old Human esophageal epithelial cells (Het1A) were cultured in LHC 9 Medium (Invitrogen, Eugene, OR) supplemented with 5% donor bovine serum (DBS) (Gibco, Gr and Island, NY). Cells were passed to glass bottom dishes (MatTek, Ashland, MA) or normal petri dishes, and cultured. When cells were confluent, fabricated PDMS mold was placed on the top of cell monolayer with an 86 3 g weight. After 20 min, the weight and the PDMS mold was carefully removed from the cell culture dish.

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92 Time L apse M icroscopy After imprinting the cell monolayer with a PDMS mold, cell culture dishes were washed with PBS once for removing nonadherent cells and new media was added to the di sh. Phase contrast imaging was performed for 18 hours on the Nikon TE 2000 microscope with humidified incubator (In Vivo Scientific, St. Louis, MO). Images were collected every 5 min or 10 min using 10x, 20x, and 60x objectives. Cell V iabil ity A ssay The l ive/dead viability/cytotoxicity kit for mammalian cells (Invitrogen, Eugene, OR) was used for observing live and dead cells. Imprinted cells were washed with PBS once and incubated with 4 M calcein AM and 4 M ethidium homodimer 1 (EthD 1) for 3045 min. After washing with PBS, new media was added to the dish. Cells were imaged with time lapse microscopy using phase contrast, GFP and Texas R e d filters as mentioned above. Blebbistatin A ssay Imprinted cells were washed with PBS once, and 5 M blebbistatin ( Calbiochem, San Diego, CA) in cell media was added to the dishes. Images were collected every 10 min using a 10x objective for 18 hours on the Nikon TE 2000 microscope. The area of wound was measured with I mage J, and the time dependent wound closure ratio was pooled and averaged. Results We utilize d the soft imprint technology to stamp a wound on a confluent monolayer of cultured cells. Figure 7 1 shows the experimental scheme. Briefly, PDMS prepolymers were poured on the photoresist master mold and then cured. After PDMS mold was carefully peeled off, they were sterilized and baked again at 120C to remove

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93 any remaining solvent and prepolymer. The raised features on the PDMS were square in shape with a size of 250 m. I mprinting the PDM S mold on the confluent human epithelial cell monolayer caused cells to have a flattened morphology in the stamped area ( Figure 7 2A ) The flattened cells were completely replaced by normal cells after 15 hours (Figure 7 2A ) When the PDMS mold was shifted laterally before pee l ing it off from the confluent cells, the flattened cells were removed and clean wounds could be created (Figure 72B). Interestingly, the rate o f wound closure in this cleaned wound without dead cells was faster than that measured in the stamped wound with dead cells (Fig ure 7 3 ). We next stained cells to test viability of the stamped cells All the cells in the stamped area were found to be dead with predominant nuclei, suggesting that the stamping caused subst antial damage to the cell (Fig ure 7 4 A ). T ime lapse images further revealed that the dead cell debris was cleaned by inwardly migrating cells (Figure 7 4 B). Cells were observed to efficiently peel off dead cell debris first and then migrated further into the wound (Figure 7 4 B). Overlaid images of phase contrast and fluorescent images shows that peeled off dead cells were engulfed by neighboring live cells (white arrows in Figure 7 4 C). Given that wound closure in the stampwound assay requires the peel ing off of dead cell debris, we hypothesized that this process may require contractile forces generated by migrating cells. We therefore inhibited nonmuscle myosin II using blebbistatin ( 5 M ). During the experiment, blebbistatin was not washed out to prevent possible recover y of myosin II activity during the slow healing process Interestingly, cells were not peeled off after blebbistatin treatment even after 15 hours (Figure 7 5 A).

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94 However, wound healing was only slightly slowed in the presence of blebbistatin without dead cells in the cleaned wound suggesting a fundamental requirement for actomyosin contractility in wound closure in the stamped wound (Figure 7 5 B and 76 ) Discussion S oft imprint ing technology is widely used for fabricating patterns at the nano and micron scale [166] This method has been used to pattern extracellular matrix prot ein s ( such as fibronectin) on the substrate and confining cell adhesion on the individual protein islands [167 169] Here, we used a similar soft imprint ing method to create wounds with dead cell debris. This stamping wound assay can be used to study both the migration of cells into the wound and simultaneous phagocytosis in the wound site. Alternative methods to achieve dead cells surrounded by live cells include electric pulses to kill cells locally [170] We showed that the rate of wound closure in the stamped wound without dead cells was slower than that measured in the cleaned wound with dead cells This is probably due to the fact that migrating cells were observed to first peel off dead cells before moving forward. On e interesting finding in our study is that the wound closure rate with nonmuscle myosin II inhibition is faster when no dead cells or debris remain at the site of wound. We attribute this to the fact that cells cannot phagocytose the dead cell debris in t he absence of myosin activity [171173] Due to the inability of cells to peel off debris cells are unable to migrate inw ard due to the presence of cell debris resulting in decreased rates of wound healing The fact that this assay allows us to study wound healing in the presence of cell debris is significant because local necrosis of cells cause s the release of chemotactic factors locally that influence the directionality of cell migration [174 176] Therefore

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95 wound healing occurs not only because of the disruption of cell cell junctions due to the generation of a clean surface but also because of a local burst in chemotactic factors. As a result, this assay can be used to quantify the extent to which local chemotactic factors influence cell migration during wound healing. The mechanism for migration involves a complex interplay between chemotaxis, migr ation due to disruption of cell cell junctions and myosindependent cell peel off. The efficiency with which cells peel off cells is surprising no structures are evident on the dish surface once peel off occurs. The peeling process itself appears similar to phagocytosis but with important differences. Our confocal images demonstrate that the debris is not internalized entirely (data not shown here) which is intriguing given that cells contin ue to migrate with this extra payload without any noticeable decrease in speed. Conclusions A new method for creating realistic wounds in adherent cell monolayers was proposed. Successful removal of migration of healthy cells toward dead cells was obser ved and the partial engulfment of dead cells by live cells was shown. The migration of cells into the wound was shown to require myosin activity only in the presence of debris. Collectively, this novel wound assay is expected to result in better understan ding of the process of wound healing.

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96 Figure 7 1. Schematic diagram of stamping wound assay.

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97 Figure 7 2 Time lapse microscope images of stamped Het1A cells. Neighboring cells (outside of dashed line) started migrating into patterned cells (inside of dashed line) after removing the stamp. A) Stamping caused flattened cells (stamped wound) B) Shifting the sta mp created a clean wound After 15 hours, neighboring cells occupied the wound site.

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98 Figure 7 3 Wound healing ratio with Het1A cells Average wound healing ratio of control (not treated by any drug) cells with dead cells (stamped wound) and without dead cells (cleaned wound) Wound healing rate without dead cells was a little bit faster than that with dead cells, but both of wounds w ere completely closed in both of cases after 15 hours. Bars indicate the standard error of the mean (SEM) (n=3 for each condition).

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99 Figure 7 4 Epithelial cell migrates to the wound after peeling off dead cells. A) Overlaid image of phase contrast and fluorescent microscope. Stamped cells were dead (red fluorescent), whereas neighboring cells were live (green fluorescent). B) Phase contrast images of stamped Het1A cells by time. Dead cells were peeled off by neighboring live cells. Dashed line indicates the boundary of stamped cells. C) Overlaid images of phase contrast and fluorescent m icroscope. White arrows show live cell ingest dead cell debris (red fluorescent).

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100 Figure 7 5 Time lapse images of stamped Het1A cells with blebbistatin treatment. A) Stamped wound closure was not completed even after 15 hours. B) Approximately 70% of cleaned wound was closed after 15 hours.

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101 Figure 7 6 Wound healing ratio with nonmuscle myosin II inhibition. Average wound healing ratio of blebbistatin (BS) treated cells with dead cells (stamped wound) and without dead cells (cleaned wound) After 15 hours, BS treated cells with dead cells did not close wound entirely (only 30% ), while BS treated cells without dead cells completed 80 % of wound healing. Bars indicate SEM (n=4 for each condition).

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102 CHAPTER 8 CONCLUSIONS Cell interactions with nanostructured biomaterials have received increasing attention because of applications in controlling tissue response to biomedical implants. Biomedical implants such as stents that are designed to keep lumenal organs open (e.g. cardiovascular stents or tumor stents) are frequently blocked and require removal and replacement. This increases morbidity and mortality and is a serious challenge to the success of therapies. The blockage occurs in part due to mammalian cell adhesion and proliferation on the impla nted stent surface. Because f undamental cell behaviors such as adhesion, proliferation, and migration are exquisitely sensitive to nanoscale topography we investigated the potential of upright randomly oriented monolayers of nanorods for anti fouling. Th is work resulted in the following contributions 1. NIH 3T3 fibroblasts, human umbilical vein endothelial cells, and bovine capillary endothelial cells were found to adhere far less to ZnO nanorods than the corresponding ZnO flat substrate. The decrease in numbers was associated with a lack of focal adhesion and stress fibers assembly, decrease in spreading area and a lack of lamellipodia formation. 2. To account for potential toxicity issues due to ZnO leaching, the above experiments were repeated wi th SiO2 coated ZnO nanorods. The SiO2 coating was confirmed to be conformal and solution toxicity assays showed that chemical dissolved material from the nanorods did not reduce cell number. Cell adhesion in NIH 3T3 fibroblasts and endothelial cells was dr amatically decreased on SiO2 coated nanorods The lack of adhesion wa s not due to a decrease i n matrix protein adsorption on t he nanostructures (as confirmed by fibronectin adsorption) but rather an inability of cells to assemble focal adhesions. 3. The adhesion of e s ophageal epithelial tumor cells to SiO2 coated nanorod monolayers was next investigated. The morphology of tumor epithelial cells cultured on nanorods was rounded compared to flat s urfac es and was associated with decreased cellular stiffness and non muscle myosin II phosphorylation. Single tumor cell motility was significantly increased on nanorods compared to flat surfaces w hile cell adhesion was reduced. Tumor cell number was decreased by nearly 50%, al though proliferation and survival i n adherent cells w as unchanged. Collectively these results support ed the conc l usion that tumor epithelial cells are unable to adhere firmly to nanorod monolayers.

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103 4. A new stamp wound healing assay was developed that allowed the quantification of wound healing rates in the presence of cell debris in the wound area. Significant differences were found between wound healing rates with and without cell debris in the wound area. The wound healing was found to depend on myosin activity only when cell debris was present. Finally, the role of nesprin1 in cell sensing of substrate stiffness was investigated. Nesprin1 depletion abolished cellular response to stiffness, sugges ting a new role for nuclear cytoskeletal connections in intracellular tension generation. Together, the experiments with four different cell types on nanorods of two different materials support a model in which interfering with the nanoscale spacing of ligated integrins results in reduced cell ad hesion and for the case of normal cells, cell death. These findings are significant because they suggest a novel approach to interfere with cell adhesion based on surface topology. The key advantage is that the surface topology is permanent compared to other strategies that rely on coating with soluble chemicals The work in this dissertation clearly indicates the promise of upright nanorods for modulating cell adhesion and behavior. However, much work needs to be done before the technology can be tested for clinical applications. First the mechanism underlying cellnanorod interactions needs further investigation. Our initial conclusion that the cell adhesion is decreased on nanorods owing to the spacing between neighboring nanorods is based on the observations that ( 1) adhesion assembly and initial cell spreading are disrupted on nanorods ; ( 2) fibronectin adsorption is unchanged; and ( 3) decreased cell numbers are not due to the leaching of toxic chemicals from the nanorods. The conclusion is supported by findings in the literature [58, 59, 177] that the critical spacing for integrin clustering is ~70 nm, which is comparable to than the average inter nanorod spacing (~80100 nm). Finally, cells spread well on electrospun

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104 nanofibers [74, 178] which are horizontal, thereby providing a mesh like continuous surface for integrin li gation. However, this conclusion has not yet been directly tested in our experiments. Systematic experiments that will control the inter nanorod spacing while at the same time preventing adhesion in between the nanorods are necessary to unequivocally answer this question. We showed that more fibronectin was a d sorbed on nanorods than glass However, it is not clear if fibronectin conformation on the nanorods is conducive to cell adhesion [179] Protein adsorption is a dynamic process relating to hydrophobic interactions, electrostatic forces, hydrogen bonding, and van der Waals forces [180] and hence protein parameters such as primary structure, size, stability, and conformation can be altered by surface energy, roughness, and chemistry [181] Currently, protein adsorption and its partial unfolding structure on the surface can be determined by surface analysis measurements inc luding Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), AFM, and total internal reflectance fluorescence (TIRF) spectroscopy [182185] Analyzing unfolding fibronectin conformation on nanorods u sing these surface analys i s measurements is required for better understanding the mechanism In this work, w e investigated two different mat e rials ZnO and SiO2 coated ZnO S imilar studies need to be repeated with other materials with similar geometries to firmly establish the observed effect as well as to establish feasibility with a broad spectrum of materials. Gold, titanium, and silicon have been used as biocompatible materials [52, 79, 177] which do not induce an activation of the immune system and are nontoxic Further experiment s with these materials could promote o ther possible applications of upright

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105 nanorods structures, such as bone prosthes e s and biosensors Another potential application is the delivery of drug s immobilized on the nanorods into the cell. S ilicon nanowires have been recently used to deliver genes in to cells [82]. For successful use of nanorod coated substrates in tumor stents, a number of questions need to be addressed. First, strategies are needed to decrease tumor cell numbers to values lower than the 50% reduction reported here. One strategy is to coat the nanorods with proteinre sistant polymers this would be a synergistic approach that incorporates both topology and chemistry into the stent surface. Also, our results suggest that the tumor cells adhere only weak ly to the nanorods. Based on these results m echanical forces as are commonly seen in the body could detach the tumor cells and further reduce their numbers. Therefore, studies that include hydrodynamic flows are crucial. Also, the nanorods need to be very resistant to mechanical forces tests based on hydrodynamic shear f low or frictional forces due to solid abrasion need to be performed and strategies to increase the adhesive strength between the nanorods and the underlying surface need to be explored. While nanorods have recently been coated on curv ed surfaces [186] it remains to be seen if the nanorods can withstand the expansioncompression that inevitably occurs with preexpanding stents. If locally, the nanorods get removed, this could allow cells to adhere and more importantly, the removed nanorods could cause wear to surrounding tissue or cause toxicity, which is highly undesirable. Finally, the adhesion of other cell types (part icularly macrophages) to nanorods should be studied, as this will help understand the inflammatory response that the body could potentially have to implanted nanorodcoated devices. Also,

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106 n anorods may be useful for preventing bacterial adhesionthis could be an inexpensive approach for maintaining sterility of surfaces.

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107 LIST OF REFERENCES [1] Dormann A, Meisner S, Verin N, Lang AW. Self expanding metal stents for gastroduodenal malignancies: Systematic review of their clinical effectiveness. Endoscopy 2004;36(6):543550. [2] Togawa O, Kawabe T, Isayama H, Nakai Y, Sasaki T, Arizumi T, et al. Management of occluded uncovered metallic stents in patients with malignant distal b iliary obstructions using covered metallic stents. Journal of Clinical Gastroenterology 2008;42(5):546549. [3] Refai AK, Textor M, Brunette DM, Waterfield JD. Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines. J Biomed Mater Res A 2004;70(2):194205. [4] Sun T, Tan H, Han D, Fu Q, Jiang L. No platelet can adhere--largely improved blood compatibility on nanostructured superhydrophobic surfaces. Small 2005;1(10):959963. [5] Peppas NA, Langer R. New challenges in biomaterials. Science 1994;263(5154):17151720. [6] Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J 2006;20(7):811827. [7] Benzeev A, Farmer SR, Penman S. Protei n Synthesis Requires Cell Surface Contact While Nuclear Events Respond to Cell Shape in AnchorageDependent Fibroblasts. Cell 1980;21(2):365372. [8] Girard PP, Cavalcanti Adam EA, Kemkemer R, Spatz JP. Cellular chemomechanics at interfaces: sensing, integration and response. Soft Matter 2007;3(3):307326. [9] Re F, Zanetti A, Sironi M, Polentarutti N, Lanfrancone L, Dejana E, et al. Inhibition of anchoragedependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Bio l 1994;127(2):537546. [10] Vakonakis I, Campbell ID. Extracellular matrix: from atomic resolution to ultrastructure. Curr Opin Cell Biol 2007;19(5):578583. [11] Stevens MM, George JH. Exploring and engineering the cell surface interface. Science 20 05;310(5751):11351138. [12] Ingber DE. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc Natl Acad Sci U S A 1990;87(9):35793583.

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122 BIOGRAPHICAL SKETCH Jiyeon Lee was born in Sokcho City, Gangwon Province, South Korea, in 1981. She graduated from Chuncheon Girls High School in Chuncheon City, Gangwon Province, South Korea, in 2000. In 2004, she received a Bachelor of Science in chemical engineering from Seoul National University with honors. She joined Dr. Hong H. Lee s research group in 2004 and studied nanopatterning, electronic materials and organic devices, specifically studying modification and application of electronic materials for two years. In 2006, she received a Master of Science in chemical and b iological e ngineering from Seoul National University. She started her Ph.D course at the University of Florida in chemical Engineering in 2006 and joined Dr. Tanmay Lele s group in 2007. She obtained her Doctor of Philosophy in chemical engineering from University of Florida in 2010.