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Fabrication of Coated Biodegradable Polymer Scaffolds and Their Effects on Murine Embryonic Stem Cells

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Title:
Fabrication of Coated Biodegradable Polymer Scaffolds and Their Effects on Murine Embryonic Stem Cells
Creator:
TOLLON, MICHAEL H. ( Author, Primary )
Copyright Date:
2008

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Biodegradation ( jstor )
Cells ( jstor )
Gelatins ( jstor )
Images ( jstor )
In vitro fertilization ( jstor )
Microscopy ( jstor )
Molecular weight ( jstor )
Polymers ( jstor )
Scaffolds ( jstor )
Tissue engineering ( jstor )

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University of Florida
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University of Florida
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Copyright Michael H. Tollon. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/1/2005
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FABRICATION OF COATED BIODEGRADABLE POLYMER SCAFFOLDS AND
THEIR EFFECTS ON MURINE EMBRYONIC STEM CELLS

















By

MICHAEL H. TOLLON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Michael H. Tollon















ACKNOWLEDGMENTS

I would like to thank Dr. Christopher Batich, Brad Willenberg, and Dr. Takashi

Hamazaki for their help in my research.
















TABLE OF CONTENTS

page

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

LIST OF TABLES ................................. .... ...................... .. vii

LIST OF FIGU RE S ......... .. ... ........ .................. .... .............. ............. viii

A B STR A C T ................................................. ..................................... .. x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

S ig n ific a n c e .......................................................... ................ 1
Sp ecific A im s ..................................................... ..................... 2
Sp ecific A im 1 ................................................ ........................ 2
Specific A im 2 ............................................... ........................ 2
Stu dy D design ................................................. 2

2 B A C K G R O U N D .................... .... ................................ ........ ........ .......... .. ....

Scaffolding in Tissue Engineering.......................................... ........... ............... 4
Poly(D L-lactic-co-glycolic acid) ........................................................ ............... 5
Poly(E-caprolactone) .................. ...................................... ................ .9
E m bryonic Stem cells............ .......................................................... ......... .. 10

3 M ATERIALS AND M ETHOD S ........................................ ......................... 12

Scaffold Synthesis ............................................ .. .. ... ......... ..... .. ... 12
Salt-leached Scaffold ............ .... ................................................ .. .... .... ... ... 12
M materials .................................................................. ............... 12
M holding ............................................................... .. ... ......... 12
Bathing ................................... ................................ ........13
D trying and storage ....................................... .................................14
Fibrous Phase Separated Scaffold ............................................ ............... 14
M materials ................................................ ............. ......... ... 14
M holding ..................................................................... ......... 14
L a y e rin g ................................................................................................. 1 5
D ry in g an d Storag e ................................................................................. 15









Scaffold C haracterization ............................................. ........................................ 15
G general O observations ............................................ ............ .. ........... ..15
Scanning Electron M icroscopy....................................... ......................... 15
In vitro D egradation Study ........................................... ........................................ 16
In vitro m urine E S C ell Studies ...................................................................... .. .... 16
Sterilization .......................................................................................... .......17
Preparation of Coatings ................................................................ .............17
C oating Procedure ........................................................ .. .... ... ........... 18
Murine ES Cell Preparation and Seeding..........................................................18
P re p aratio n .................................................. ................ 1 8
Seeding ................................... .......................... ................ 19
m ES Cell Study A analysis ............................................................................. 19
F luorescent m icroscopy ........................................... ............................... 19
F low cytom etry ........................ .. ...................... .. ...... .... ..... ...... 20
FE SE M ................................... ............................20
Reverse transcriptase-polymerase chain reaction (RT-PCR).....................21

4 RE SU L TS A N D D ISCU SSION ...................................................... .....................22

Scaffold C haracterization ........................................ ............................................22
Salt-leached Scaffold ............ ..................................................... .... .... .... 22
G general observations ......................................................... ............. 22
F E S E M ................... ...................................... ......................... 2 3
Fibrous Phase Separated Scaffold ............................................ ............... 24
G general observations ......................................................... ............. 24
F E S E M .................................................................. ...............................2 4
C o a tin g s ................... ...................2...................5..........
In vitro D egradation Study .............................................................. .....................26
G general O observations ........................ .. ................................ .... ...... ...... 26
Mass Analysis................................. ........... 28
Molecular Weight (MW) Analysis................................ ........................30
pH A naly sis ............. ......... ..... ............................................................... 3 1
FESEM (W eek 1,3,5,7,9) ............................................................................ 33
P L G A ..................................................................................................... 3 3
P C L ......................................................................................................... 3 3
In vitro m E S C ell Studies ...................................................... .......... ............... 35
PCL Salt-leached Scaffolds (Study 1)...................................... ...............36
PCL Fibrous Phase Separated Scaffold (Study 2) ............................................37
F luorescent m icroscopy .......................................... ................................ 37
F low cytom etry ........................ .. ...................... .. ...... .... ..... ...... 4 1
FE SE M ................................... ............................4 1
Reverse transcriptase-polymerase chain reaction (RT-PCR).....................43

5 CONCLUSIONS ..................................... .......... ...............45

Scaffold Fabrication and Characterization ...................................... ............... 45
In vitro D egradation Study .............................................................. .....................45


v









In vitro m E S C ell Stu dies ........................................ ............................................46
S tu d y 1 ........................................................................................................... 4 6
S tu d y 2 ........................................................................................................... 4 6
Final Thought. ......................... ....... ......................................... 46

LIST OF REFERENCES ................ ........ ......... ........48

BIOGRAPHICAL SKETCH ................ ........ .................51
















LIST OF TABLES

Table pge

4-1 General observations of PLGA scaffolds in PBS................................ ............... 27

4-2 General observations of PCL scaffolds in PBS.....................................................27
















LIST OF FIGURES


Figure p

3-1 Schematic drawing and picture of Teflon mold for salt-leached scaffold
fabrication with m old release. ..... .....................................................................13

4-1 FESEM images illustrating the difference in washed and unwashed scaffolds.......23

4-2 FESEM im ages of scaffold ............................................. ............................. 25

4-3 FESEM images comparing the salt-leached and fibrous phase separated scaffold
m orphologies ............................................................... ... .... ........ 25

4-4 FESEM images of Matrigel and gelatin coated PCL scaffolds.............................26

4-5 PLGA average percent mass loss during degradation study ..................................29

4-6 PCL average percent mass loss during degradation study ....................................29

4-7 PLGA molecular weight change during degradation study ...................................30

4-8 PCL molecular weight change during degradation study .....................................31

4-9 Graph presenting pH change of PBS for PLGA scaffolds during degradation
stu d y ............................................................................. 3 2

4-10 Graph presenting pH change of PBS for PCL scaffolds during degradation study .32

4-11 FESEM images of degrading PLGA scaffolds at. A) Week 1, B) Week 3, C)
W eek 5, D) W eek 7 and E) W eek 9 .............................................. ............... 34

4-12 FESEM images of degrading PCL scaffolds at. A) Week 1, B) Week 3, C) Week
5, D) W eek 7 and E) W eek 9 .............................................................................35

4-13 Optical and fluorescent microscopy images of PCL salt-leached scaffold on day
6 ................................................................................ 3 7

4-14 Optical and fluorescent microscopy images on Day 9 of PCL phase separated
m E S cell study .................................................. ................. 39

4-15 Optical and fluorescent microscopy images on Day 24 of PCL phase separated
m E S cell study .................................................. ................. 40









4-16 Fluorescent microscopy showing cells on multiple layers of the scaffold..............41

4-17 Flow cytometry analysis at Day 24 of PCL phase separated mES cell study..........41

4-18 FESEM images of cellular attachment on Matrigel coated PCL scaffold ..............42

4-19 FESEM images of cellular attachment on gelatin coated PCL scaffolds.................42

4-20 RT-PCR analysis at Day 24 of PCL phase separated mES cell study ...................44















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

FABRICATION OF COATED BIODEGRADABLE POLYMER SCAFFOLDS AND
THEIR EFFECTS ON MURINE EMBRYONIC STEM CELLS

By

Michael H. Tollon

May 2005

Chair: Christopher Batich
Major Department: Materials Science and Engineering

In the past decade, tissue engineering has become a great interest in materials

science research. Embryonic stem (ES) cell transplantation has become one of the most

researched therapies for restoring tissue and organ function. Many studies have

investigated the use of porous biodegradable scaffolds to promote cell adhesion, growth,

proliferation, differentiation, and to help steer the course of tissue development.

Research has shown that extracellular matrices and the basement membranes affect

various cell types and cellular behaviors. However, the effects of these materials on ES

cell behavior are currently understudied and poorly understood.

In this study, the synthetic biodegradable polymers polycaprolactone (PCL) and

poly(lactic-co-glycolic acid) (PLGA) were chosen to create an interconnected foam

structure. A phase separated scaffold method was developed and compared to previously

designed salt-leached scaffolds. In vitro degradation studies were conducted on salt-

leached scaffolds at 370C, while submerged in a phosphate buffered saline solution.









These scaffolds were analyzed for mass loss, pH, molecular weight, and morphology.

The PCL scaffolds maintained their structure and functionality during the 9 week

degradation study when compared to PLGA. Multiple coating techniques were explored

to modify cellular results using these scaffolds. When the phase separated PCL scaffolds

were coated with Matrigel and gelatin solutions murine ES (mES) cells attached, spread,

and differentiated within the scaffolds. Comparisons of the coating's effect on the mES

cells were analyzed using fluorescent microscopy, flow cytometry, scanning electron

microscopy, and reverse-transcriptase polymerase chain reaction. In conclusion it was

found that a scaffold's coating/extracellular matrix affects a mES cell's morphology and

differentiation.














CHAPTER 1
INTRODUCTION

Significance

Tissue engineering has become of great interest to materials science research in the

past decade. It has become one of the most researched therapies for tissue and organ

revival [1,2]. The use of tissue engineered devices has the potential to reduce the annual

cost of tissue loss and end-stage organ failure, which is estimated to reach over $10

billion in the U.S. alone [3]. Liver regeneration, in recent years, has become one of the

most sought after goals in tissue engineering [4]. Liver diseases, such as viral hepatitis

(A, B, and C), cirrhosis, and cancer, have created this major necessity for success in

tissue engineering. It has been shown that acute liver failure has a mortality rate of more

than 80% and it is estimated that one in 10 Americans, or 25 million people in this

country suffer from a form of liver disease [4,5]. Approximately 30,000 individuals in the

U.S. die each year due to the lack of a cure for this disease and a shortage of donor

organs [4-6].

One of the challenges in tissue engineering concerns introducing and controlling

the stem cells into a specific lineage and to fit them into the desired tissue architecture.

Embryonic stem cells (ES cells) can be cultivated in an undifferentiated state indefinitely

in vitro and have the potential to differentiate into multiple cell lineages. The most

widely used method to differentiate ES cells in vitro begins with cell aggregation in a

suspension culture [7,8]. However these cell aggregates, also known as embryoid bodies,

have poor ability to generate the target cellular phenotypes, such as hepatocytes.









Therefore, bioengineered scaffolds have been fabricated to help cellular attachment,

proliferation and to avoid embryoid body formation.

Porous, biodegradable polymers have been found to play a vital role by creating a

scaffold structure for tissue regeneration [9-11]. The scaffold's structural integrity is a

key attribute during the course of tissue formation [2,12]. Scaffolds supply the necessary

foundation for cell attachment, proliferation and maintaining differentiated functions,

while limiting embryoid body formation [7,9,10]. Other important factors for cell

scaffolding success are the addition of growth factors and/or other protein coatings

[2,12,13]. Presently there is no ideal scaffold developed to achieve the ultimate goal of

ES cell differentiation towards a hepatocyte lineage.

Specific Aims

Specific Aim 1

Fabricate biodegradable poly(s-caprolactone) scaffolds with porous morphologies

for murine embryonic stem cell study.

Specific Aim 2

Investigate differentiation of murine embryonic stem cells on the constructed

biodegradable polymer scaffolds coated with Matrigel or gelatin.

Study Design

In this study, poly(s-caprolactone) (PCL) was investigated for biodegradable

embryonic stem cell scaffold research. Two methods of fabrication: salt-leaching and

phase separation, were considered to create an open, interconnected foam structure for

murine embryonic stem (mES) cell evaluation. The PCL salt-leached method was

compared to previously constructed poly(lactic-co-glycolic acid) scaffolds described in

the literature [2,12,14]. In vitro degradation studies were conducted on the salt-leached









scaffolds at 370C while submerged in a phosphate buffered solution (PBS) with a pH of

7.4. The scaffolds were analyzed and compared for mass loss, molecular weight,

morphology, and pH change of the PBS throughout the nine week study.

The salt-leached and phase separated PCL scaffolds were then analyzed for

biological assessment with the help of Dr. Naohiro Terada and Dr. Takashi Hamazaki,

from the Department of Pathology, Immunology and Laboratory Medicine at the

University of Florida. Murine ES cells were seeded on both scaffold designs with various

coating materials applied. Next, the coatings were investigated for the possible significant

differences in cellular results. The biological assessments were analyzed using

fluorescent microscopy, flow cytometry, field emission scanning electron microscopy

(FESEM), and reverse transcriptase-polymerase chain reaction (RT-PCR),














CHAPTER 2
BACKGROUND

Scaffolding in Tissue Engineering

Tissue engineering using cell transplantation has become one of the most

researched therapies for restoring tissue and organ function [1,2]. Already, research

groups are investigating possible tissue engineered products such as cartilage, bone, heart

valves, nerves and muscle [15]. In this technique, the scaffold plays the fundamental role

of promoting cell: adhesion, growth, proliferation, differentiation, and will help steer the

course for tissue development [2,12]. The 3D architecture of the scaffold will ultimately

define the newly formed soft or hard tissue [15]. The crucial function of the scaffold

design is to produce an ideal structure that can act as the natural extracellular matrix

(ECM) until host cells can repopulate and resynthesize a new natural matrix. The

scaffolding material and design must be carefully selected to assure that the seeded cells

are biocompatible with the fabricated scaffold [16].

Porous scaffolds with an open pore system are often desired for maximizing cell

seeding, attachment, growth, and ECM production [9,10]. A scaffold's pore size,

porosity, and the global continuity of the pores are used to characterize the pore structure

[2]. Several successful methods to fabricate highly porous polymer scaffolds include

particulate-leaching, gas foaming, phase separation, emulsion freeze drying, and 3D

printing methods. These techniques can be used to fabricate sponge-like scaffolds or

combined to develop more complex superstructures [2,12,14].









Biodegradable polymers have also shown their importance in tissue engineering

scaffolds, since the scaffold material should not remain in the final engineered tissue.

The scaffold's degradation rate may affect many cellular processes including cell growth,

tissue regeneration, and host response. The degradation of the scaffold should either be

similar to or slower than the rate of tissue formation for optimum results [9-11].

Scaffold coatings have also been experimented with lately in tissue engineering

research. It is believed that with a scaffold's physical support, the coating can provide

chemical cues which could support an environment for ES cells to organize and

differentiate [13]. Coatings can range from biodegradable polymers to growth factors that

may help promote cell adhesion, growth, proliferation, differentiation, and steer the

course for tissue development [2,12,13].

According to Ma et al., the understanding of the principles of scaffolding is far

from satisfactory, and the "ideal" scaffolds are yet to be developed [12].

Poly(DL-lactic-co-glycolic acid)

For the last two decades, synthetic biodegradable polymers have been increasingly

used in the medical field, compared to natural polymers [17-19]. Amongst them, the

thermoplastic poly(esters) like poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and

especially poly(lactic-co-glycolic acid) (PLGA) have generated a tremendous interest due

to their excellent biocompatibility and biodegradability [17-20].

The discovery and the synthetic work on low molecular weight oligomeric forms of

lactide and/or glycolide polymers was first carried out several decades ago [18,19]. The

methods to synthesize high molecular weights of these polymers were first reported

during the late 1960s and early 1970s. A number of groups have published pioneering

work on the utility of these polymers to make sutures/fibers [18,19]. These fibers had









several advantages like good mechanical properties, low immunogenicity and toxicity,

excellent biocompatibility, and predictable biodegradation kinetics [18,19]. The wide

acceptance of the lactide/glycolide polymers as suture materials, made them attractive

candidates for biomedical applications like ligament reconstruction, tracheal replacement,

surgical dressings, vascular grafts, nerve, dental, and fracture repairs [18-20].

Many researchers have investigated and documented the biodegradation,

biocompatibility, and tissue reaction of PLGA [19]. Various polymeric devices like

microspheres, microcapsules, nanoparticles, pellets, implants, and films have been

fabricated using these polymers for the delivery of a variety of drug classes. They are

also easy to formulate into drug carrying devices for various applications which have

been approved by the FDA for drug delivery use [17-20].

Understanding the chemical, physical and biological properties of a polymer is

helpful, before formulating a tissue scaffold device. The various properties of the PLGA

co-polymer are better understood by considering PLA and PGA as individual polymers.

The polymer PLA can exist in an optically active stereoregular form (L-PLA) and

in an optically inactive racemic form (D,L-PLA) [17,19,20]. L-PLA is found to be semi-

crystalline in nature due to high regularity of its polymer chain while D,L-PLA is an

amorphous polymer because of irregularities in its polymer chain structure [18,20].

Hence the use of D,L-PLA is usually preferred over L-PLA as it enables more

homogeneous dispersion of the drug in the polymer matrix [20]. PGA is highly

crystalline because it lacks the methyl side groups of the PLA [18,20]. Lactic acid is

more hydrophobic than glycolic acid and therefore lactide-rich PLGA copolymers are

less hydrophilic, absorb less water, and subsequently degrade more slowly [17,18].









The physical properties such as the molecular weight and the polydispersity index

affect the mechanical strength of the polymer and its ability to be formulated as a drug

delivery device [18,19]. Also these properties may control the polymer biodegradation

rate and hydrolysis [18]. The commercially available PLGA polymers are usually

characterized in terms of intrinsic viscosity, which is directly related to their molecular

weights [18].

The mechanical strength, swelling behavior, capacity to undergo hydrolysis, and

subsequently the biodegradation rate are directly influenced by the crystallinity of the

PLGA polymer [18]. The resultant crystallinity of the PLGA copolymer is dependent on

the type and the molar ratio of the individual monomer components (lactide and

glycolide) in the copolymer chain [17]. PLGA polymers containing 50:50 ratio of lactic

and glycolic acids are hydrolyzed much faster than those containing higher proportion of

either of the two monomers [19]. PLGAs prepared from L-PLA and PGA are crystalline

copolymers while those from D,L-PLA and PGA are amorphous in nature [18,19]. It has

been found that PLGAs containing less than 70% glycolide are amorphous in nature [21].

The degree of crystallinity and the melting point of the polymers are directly related to

the molecular weight of the polymer [18,19].

The glass transition temperatures (Tg) of the PLGA copolymers are above the

physiological temperature of 370C and hence they are normally glassy in nature [18,19].

Thus, they have a fairly rigid chain structure, which gives them significant mechanical

strength to be formulated as a degradable device [18,19]. It has been reported that the

Tg's of PLGA decrease with the decrease of lactide content in the copolymer composition

and with decreasing molecular weight [22].









The PLGA polymers chosen should also have considerable mechanical strength,

since tissue scaffolds are subjected to significant physical stress. Different factors like the

molecular weight, copolymer composition (lactide/glycolide ratio), crystallinity, and

geometric regularity of individual chains significantly affect the mechanical strength of

the particular polymer [17-19].

For both in vitro and in vivo the PLGA copolymer undergoes degradation in an

aqueous environment (hydrolytic degradation or biodegradation) through cleavage of its

backbone ester linkages [17-19]. The polymer chains undergo bulk degradation and the

degradation generally occurs at a uniform rate throughout the PLGA matrix [18]. It has

been recorded that the PLGA biodegradation occurs through random hydrolytic chain

scissions of the swollen polymer [18,19]. The carboxylic end groups present in the PLGA

chains increase in number during the biodegradation process as the individual polymer

chains are cleaved; these are known to catalyze the biodegradation process [18,19]. It has

also been reported that large fragments are degraded faster internally and amorphous

regions degrade faster than crystalline. The biodegradation rate of the PLGA copolymers

are dependent on the molar ratio of the lactic and glycolic acids in the polymer chain,

molecular weight of the polymer, the degree of crystallinity, and the Tg of the polymer

[18,19]. A three-phase mechanism for the PLGA biodegradation has been proposed [23]:

1. Random chain scission process. The molecular weight of the polymer decreases
significantly, but no appreciable weight loss and no soluble monomer products are
formed.

2. In the middle phase a decrease in molecular weight accompanied by rapid loss of
mass and soluble oligomeric and monomer products are formed.

3. Soluble monomer products formed from soluble oligomeric fragments. This phase
is that of complete polymer solubilization.









The role of enzymes in any PLGA biodegradation is unclear [18,19]. Most of the

literature indicates that the PLGA biodegradation does not involve any enzymatic activity

and is purely through hydrolysis [18]. However, some findings have suggested an

enzymatic role in PLGA breakdown based upon the difference in the in vitro and in vivo

degradation rates. It has also been found that motion and buffers may affect their rate

differences [19].

However, it is known that PLGA biodegrades into lactic and glycolic acids [17-19].

Lactic acid enters the tricarboxylic acid cycle and is metabolized and subsequently

eliminated from the body as carbon dioxide and water [17-20]. Glycolic acid is either

excreted unchanged in the kidney or it enters the tricarboxylic acid cycle and eventually

eliminated as carbon dioxide and water [18].

Poly(E-caprolactone)

Synthetic biodegradable polymers over the years have gradually taken over the

medical field, compared to natural polymers [17-19]. Polycaprolactone (PCL), a

polyester biodegradable polymer, is becoming one of these biomedical materials of

interest [24]. Polycaprolactone is synthesized by a ring opening polymerization of the g-

caprolactone monomer.

PCL is a semi-crystalline polymer which exhibits a low melting point (57C) and a

low glass transition temperature (-62C) [24-26]. It is considered a soft and hard-tissue

biocompatible, non-toxic polymer [24-26]. The rubbery characteristics of PCL have been

utilized in low molecular weight drug delivery, resorbable sutures, and bone graft

substitutes. It has been found that PCL demonstrates a lower tensile modulus and

strength than PLA, but higher extensibility, which is important in tissue scaffolding [27].









Like other polyesters, PCL will undergo auto-catalyzed bulk hydrolysis

degradation because of the susceptibility of its aliphatic ester linkage. However, the

hydrophobic, semi-crystalline polymer retards degradation and resorption kinetics when

compared to other aliphatic polyesters such as PLGA, which makes it more suitable for

long term implantable devices [24-26]. Bulk hydrolysis breaks the ester linkage, which

creates fragmentation and the release of oligomeric species. Low molecular-weight

fragments are eventually engrossed by giant cells and macrophages. The byproduct e-

hydroxycaproic acid, is either metabolized via the tricarboxylic acid (TCA) cycle or

removed by direct renal secretion [24,25,28]. It is also possible for PCL to enzymatically

degrade (enzymatic surface erosion) by lipases and esterases, though this is rare [24,29].

Embryonic Stem cells

Embryonic stem (ES) cells have shown potential success for cell transplantation in

tissue scaffolding because of their ability to differentiate into multiple somatic cell

linkages [13]. The most widely used method to differentiate ES cells in vitro initiates

with cell aggregation in suspension culture [7]. The cell aggregates, called embryoid

bodies, are made up of multicellular, multi-differentiated structures replicating an in vivo

developmental program [7]. These embryoid bodies are complicated to study and to

determine their differentiation state within the embryoid bodies. Once embryoid bodies

form, their reproducibility and function towards creating useful tissue structures diminish

[7].

However, it has also been found that porous biodegradable polymer scaffolds

support ES cells during the formation of 3D tissues. These scaffolds have the opportunity

to prevent embryoid bodies from forming. The scaffold's porosity and biodegradability

provides space for cell adhesion, growth, proliferation, and differentiation [2,9,11,13].






11


Hence, these dimensional scaffolds are expected to be useful to carry stem cells and to

allow them to be more useful (than without scaffolds) for regenerative medicine. This

has been demonstrated for PLGA sponges and for collagen gels for instance. However

these structures are weak or brittle, and there is a need for a longer lasting, tougher

scaffolds.














CHAPTER 3
MATERIALS AND METHODS

Scaffold Synthesis

Salt-leached Scaffold

Materials

The polymers used for this scaffold fabrication were polycaprolactone (PCL) (Mw

~ 120,000) provided by Sigma Aldrich and poly (DL-lactic-co-glycolic acid) (PLGA) in

a 50:50 mole ratio (Mw 85,000) provided by Birmingham Polymers. Polymer samples

were first dissolved with pesticide grade methylene chloride (Fisher) to get the desired

weight/volume of 5% (PCL) and 10% (PLGA).

Molding

A Teflon mold was fabricated by Analytical Research Systems (Gainesville, FL)

for this scaffold fabrication method. It has spaces for 8 casting cavities at 1cm x Icm per

cavity. An L-shaped mold release was placed in each cavity to help free the scaffold from

the cavity (Figure 3-1). Recipes for polymer solution and NaCl (250-500[tm) particles

were established to fill all 8 cavities to maximize results per mold. For the 10% PLGA

solution a mixture of 10g NaCl and 3mL polymer solution was used. As for the 5% PCL,

a blend of 10g NaCl and 4mL of polymer solution was used. Once NaCl particles are

combined and thoroughly mixed in with the appropriate amount of polymer solution, the

mixture is dispensed and packed into each cavity. The Teflon mold was then placed into

a vacuum oven for 45 minutes to evaporate off the solvent. Once removed from the

vacuum oven, each scaffold was detached from its cavity by freeing the edges of the









scaffold and pulling up on the mold release. Deionized (DI) water may also help free the

scaffold from the mold. The stainless steel mold release was removed from the scaffold

and the bathing process begins.

D Polymer solution
MIold release
D NaClparticlesold grease






Icin



Ic/ l

Figure 3-1. Schematic drawing and picture of Teflon mold for salt-leached scaffold
fabrication with mold release.

Bathing

Once the scaffolds are extracted from the mold, they were placed in a 90mm

diameter Petri dish. The dish was then filled with 45mL of DI water and rotated at 50rpm

on an orbital shaker (Bellco Biotechnology) for 7 days. For the first 2 hours, DI water

was exchanged every 30 minutes to help extract the NaCl from the scaffold. The

following exchanges occur daily. Every time the DI water is exchanged, each scaffold

was removed from the DI water bath and squeezed while excess water was siphoned off

using a transfer pipette. The scaffolds are then placed back into a fresh DI water bath.

The bathing process leached out the salt particles to create voids in the scaffold's

morphology.









Drying and storage

After the total bathing process, the scaffolds are removed and are squeezed and

siphoned off using a transfer pipette. Next, the scaffolds were air dried over night and

then dried in a vacuum oven for 45 minutes. After being dried, they were stored under

vacuum until needed for future experiments.

Fibrous Phase Separated Scaffold

Materials

The polymers used for this scaffold fabrication were polycaprolactone (PCL) (Mw

~ 120,000) provided by Sigma Aldrich. The polymer was first dissolved using

histological grade acetone (Fisher) to get the desired 5% weight/volume. The solutions

were then refrigerated until needed.

Molding

First, pipette ImL of 5% (wt/vol) PCL onto a 75mm diameter watch glass. While

holding the glass in one hand, rotate it in a slow circular pattern. Next, spray 2mL of DI

water at the glass approximately 4 to 6 inches away with a sterile syringe and needle (22

gauge) in a left to right fashion covering the entire watch glass. This generates a phase

separation forcing the polymer out of the solution creating fibrous webbing. Once the

phase separation starts to occur, pour the excess "cloudy" solution into the waste storage.

Once the webbing has formed, thoroughly rinse the fibrous web with DI water. Rotate

the watch glass 90 and repeat the above steps on top of the newly formed fibrous web to

create another polymer deposit. Once the second application has been applied, the web is

soaked with methanol to help free it from the glass and then dried in a vacuum oven for

30 minutes. After dried, this web is now considered one layer of the final scaffold.









Multiple layers are attached together to create the full scaffold. Layers are stored under

vacuum until needed for the layering process.

Layering

Four layers are first placed on top of each other in a 75mm diameter watch glass.

Next, the layers are united together by spraying ImL of the polymer solution with a

sterile syringe and needle (22 gauge) in a left to right fashion covering the entire layer.

Quickly spraying DI water over the layers to cause a phase separation once again, which

fastens the four layers together. Once the phase separation is visible, pour the "cloudy"

solution into waste storage which was discarded at a later date. Thoroughly rinse the

layers with DI water to clean out any excess solution. Next, flip over the newly formed

scaffold and repeat the above steps to bond the layers again from the other side.

Drying and Storage

After the layers have been connected, the scaffold is soaked in methanol, removed

and then dried in a vacuum oven for 30 minutes. The scaffolds are subsequently stored

under vacuum until needed for future experiments.

Scaffold Characterization

General Observations

After fabrication, the scaffolds were characterized. The scaffolds were weighed and

basic handling properties were explored. Comparisons were made for each polymer and

scaffold fabrication method used, and with the literature as far as possible.

Scanning Electron Microscopy

The scaffolds morphology was examined by field emission scanning electron

microscopy (FESEM) (JEOL 6335F). After the scaffolds were dried, they were mounted

on an aluminum stub and a AuPd sputter coating was applied. The samples were imaged









at 10kV with a working distance of 15mm. Images were taken at low magnifications to

identify the scaffolds structure.

In vitro Degradation Study

A nine week in vitro degradation study was conducted on both the PCL and PLGA

salt-leached scaffolds to study their molecular weight (MW) loss, mass loss and

morphology changes. For both polymers, 12 groups were divided up with 6 scaffolds per

group (144 total samples). Each group of 6 scaffolds was placed in 21x70mm, 4DR vial

(Fisher) with 12mL of phosphate buffer saline (PBS) (IX) (Sigma) with a pH of 7.4.

Each group was then placed into a rotating incubator (Robbins Scientific) at 37C

rotating at 10rpm. The PBS was exchanged at 12 hours, 24 hours, 36 hours, day 2, day 3,

day 4, day 5, day 6, week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8,

and week 9 for each group. Groups were extracted from the study at the following

designated times: 12 hours, day 2, week 1, day 10, week 2, week 3, week 4, week 5, week

6, week 7, week 8, and week 9. After each group was extracted, it was rotated at 25 rpm

in a bath of DI water for 15 minutes to clean the scaffolds of PBS. At this point the

scaffolds were air dried overnight and then vacuum dried for 2 hours the following day.

For each group, all six samples were weighed dry, five samples were used for gel

permeation chromatography (GPC), and one sample was used for FESEM analysis.

In vitro murine ES Cell Studies

Once scaffold fabrication was complete, the scaffolds were evaluated for their

possible significance in tissue engineering. Dr. Naohiro Terada and Dr. Takashi

Hamazaki, from the Department of Pathology, Immunology and Laboratory Medicine at

the University of Florida, greatly assisted in the cellular studies. Both PCL salt-leached

and fibrous phase separated scaffolds were used, with main emphasis on the fibrous









phase separated scaffold. The interest of the studies was to compare the salt-leached

method to the novel, fibrous phase separation procedure, while also judging the

difference in coating materials. The first study involved Matrigel coated and uncoated

salt-leached PCL scaffolds which were analyzed by fluorescent microscopy. The second

study involved Matrigel coated, gelatin coated, and uncoated fibrous phase separated

scaffolds that were analyzed using fluorescent microscopy, reverse transcriptase-

polymerase chain reaction (RT-PCR), flow cytometry and FESEM.

Sterilization

The scaffolds were sectioned into lin x lin samples (thickness was dependent on

original scaffold) and placed in a bath overnight of 70/30 solution of ethanol and DI

water respectively. The scaffolds were dried, washed with PBS twice and then dried once

again. The scaffolds were kept sterile until coated.

Preparation of Coatings

The two coating solutions were prepared as listed below (700LtL):

* 10% (vol/vol) Matrigel Matrix (BD Bioscience) mixed with D-MEM/F-12 serum
free media

* 0.1% gelatin solution (Specialty Media) in ultra pure water

The Matrigel Matrix is a solubilized basement membrane preparation extracted

from the Englbreth-Holm-Swarm (EHS) mouse sarcoma, which is a tumor rich in

extracellular matrix proteins [30]. Its main components are laminin, collagen IV,

entactin, and heparan sulfate proteoglycan. The matrix also contains TGF-P, fibroblast

growth factor (FGF), tissue plasminogen activator (TPA), and other growth factors which

are found naturally in EHS tumors [31]. At room temperature, the Matrigel Matrix

polymerizes to produce a biologically active matrix material that resembles the









mammalian basement membrane. Matrigel is a viscous gel and must be kept frozen for

storage purposes.

Gelatin is a pure protein obtained by the partial hydrolysis of collagen. This

collagneous material has been used from food substitutes to scientific studies. It also

provides a basement membrane in cellular studies and can increase a surface's

hydrophilicity [4]. The gelatin solution has a viscosity close to water and differs greatly

from the Matrigel's hydrogel properties.

Coating Procedure

After the preparations have been completed, the coating procedure is started

immediately. For each coating material, the same procedure is followed. The scaffold is

first submerged in the coating solution for 2 minutes. Next, the scaffold is removed with

excess coating solution being siphoned off. The scaffold was then incubated for 5

minutes at 37C. The process is repeated until 3 coatings have been applied and

incubated, respectively.

Murine ES Cell Preparation and Seeding

Preparation

The cell line used for the studies was murine Alpha fetoprotein/green fluorescent

protein (Afp/GFP) ES stem cells. This cell line can be used to monitor primitive

endoderm differentiation [7,8]. The mES cells were maintained prior to the study in an

undifferentiated state on gelatin-coated dishes in Knock-out Dulbecco's Modified Eagle

Medium (DMEM) (Gibco) containing 10% knockout serum replacement (Gibco), 1%

fetal bovine serum (FBS) (Atlanta biologicals), 2mM L-glutamine, 100units/mL

penicillin, 100[tg/mL streptomycin, 25mM HEPES (Gibco), 300[tM monothioglyercol

(Sigma), and 1000unit/mL recombinant mouse LIF media (Chemicon).









The cells were suspended before seeding them on the scaffold. The media was

siphoned out of the culture dish trying not to disturb the cells. A wash of PBS was

applied to the Petri dish and cells, and then siphoned off. While rotating the dish in a

circular pattern, ImL of 0.25% Trypsin/EDTA (Gibco) was added to dissociate the cells

from the dish. The mES cells were then suspended in the mES Differentiated Media:

500mL of IMDM (Gibco), 5mL of penicillin/streptomycin (Gibco), 16[tL of

monothiglycerol (Sigma).

Next, 100mL were removed from the prepared solution and stored for later use.

Finally, 100mL of 20% FBS is added to the remaining solution. The mES Differentiated

Media was then stored for future cell experiments.

Seeding

The sectioned and coated scaffolds were first placed in its individual 12 well

culture dish. NextlOOLL of mES cell suspension (2.17x106 cells/mL) was added in each

well. The suspension was placed on top of the scaffold to help cellular adhesion. The

culture wells were then incubated for 30 minutes at 37C. Once removed from the

incubator, ImL of mES Differentiated Media was added to each culture dishes. The

scaffolds were incubated at 37C again until needed for observation. The scaffolds were

observed with an optical fluorescent microscope on day 0, 2, 4, 7, 9, 21, 24 for cellular

analysis.

mES Cell Study Analysis

Fluorescent microscopy

The cells were observed under a fluorescence microscope (Olympus IX70) and

three fluorescent labels were used. The Afp/GFP cells will express a green fluorescent

protein after they have differentiated to visceral endoderm. Hoechst 33342 is a blue









fluorescent stain used to mark the nucleus of cells, both dead and alive. Propidium iodide

(PI), a red fluorescent stain, was also used because it's only permeable to non-living

membranes, and is used for detecting dead cells. By comparing GFP, Hoechst and PI

presence, one can distinguish live cells from dead ones and identify if the embryonic

stem cells have differentiated into the more mature endodermal cells [7].

Flow cytometry

Cells were dissociated with 0.25% trypsin/EDTA after day 24 of the experiment.

The flow cytometry was performed on FACS Sort (BD Biosciences) on a data set of

30,000 cells, which was then recorded using CellQuest Acquisition software (BD

Biosciences). Flow cytometry helps illustrate the separation of GFP-positive and negative

cells and the cell's size [7].

FESEM

The scaffold's morphology and cellular attachment was examined by FESEM

(JEOL-6335F). Once the cell study was completed, the cells were fixed in a 2.5%

glutaraldehyde/PBS fixation made from 50% glutaraldehyde (Electron Microscopy

Sciences) and PBS (IX) with a pH of 7.4 (Gibco). The scaffolds were rinsed with DI

water and then dehydrated with ten minute baths of 70%, 80%, 95%, and 100% (three

times) of ethanol/DI water solutions [32]. After the scaffolds were fixed and dehydrated,

they were mounted on an aluminum stub and a C evaporation coating was applied. The

samples were imaged at 10kV with a working distance of 15mm. Low acceleration

voltages were used initially to ensure no beam damage. Images were taken at high and

low magnifications to identify the scaffold's structure and any possible cellular

attachment.






21


Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted by using RNA aqueous kit (Ambion). Then cDNA was

synthesized by using SuperScript II first-strand synthesis system with oligo(dT) (Gibco).

PCR was performed by using Taq DNA polymerase (Eppendorf). For each gene, the

DNA primers were originated from different exons to ensure that the PCR product

represents the specific mRNA species and not geomic DNA [7]. Gene markers are then

compared to determine possible cells lineages.














CHAPTER 4
RESULTS AND DISCUSSION

Scaffold Characterization

Salt-leached Scaffold

General observations

PLGA. During fabrication, the PLGA scaffolds were extracted from the mold

fairly easy using the L-shaped mold release mentioned earlier. However, there was a

slight indentation from the mold release but this did not cause any significant effects on

the overall scaffold. After fabrication, the PLGA salt-leached scaffolds were weighed and

observed for general handling properties. The weights of the final scaffolds were 12.0

2.51mg and exemplified a rigid character after drying. Their dimensions were 9 Imm in

diameter with a thickness of 6 2mm. The scaffolds were very brittle and difficult to

work with. The PLGA scaffolds compared well with similar particle leaching methods

used in prior work [10,12,14].

PCL. During fabrication, the PCL scaffolds were extracted from the mold

effortlessly from the mold. Again, there was a slight indentation from the mold release,

but no affects to the overall scaffolds structure. After fabrication, the PCL salt-leached

scaffolds were weighed and observed for general handling properties. The weights of the

scaffolds were 13.6 .85mg and illustrated an elastic quality when compared to the

PLGA scaffolds. Their dimensions were 9 Imm in diameter with a thickness of 4 +

2mm. During literature searches, nothing was found on salt-leached scaffolds using PCL,

but the scaffolds did compare morphologically to other salt-leached scaffolds.









FESEM

The scaffolds were characterized using FESEM to compare morphologies. Images

were taken before and after washing to show the salt-leaching process. The results are

shown in Figure 4-1. The figure also displays the porous structure of both PLGA and

PCL scaffolds. It should also be noted that the salt-leaching can create "pockets" in the

scaffolds instead of the desired interconnected porous microstructure. As noted before, an

interconnected porous structure helps provide for maximizing cell seeding, attachment,

and growth [9,10]. Therefore this method may not be the most desirable for mES cell

studies.











A B


C D

Figure 4-1. FESEM images illustrating the difference in washed and unwashed scaffolds.
A) PCL unwashed, B) PCL unwashed, C) PLGA washed and D) PCL washed.
(Scale bar 100[tm)









Fibrous Phase Separated Scaffold

General observations

After fabrication, the PCL salt-leached scaffolds were weighed and observed for

general handling properties. The weights of the scaffolds layers were 136 8.56mg and

demonstrated an elastic quality when handled in the lab. Once the layers were bonded

together, the final scaffold's weight was 661 8.55mg. Also the scaffold became more

rigid after the final bonding of the layers was complete, but still had elastic handling

qualities. The scaffold's final dimensions were 50 3mm in diameter with a thickness of

4 2mm. During literature searches, nothing was found on using a PCL/acetone phase

separation system to create multi-layered cellular scaffolds.

FESEM

The scaffolds were characterized using FESEM to compare morphologies of the

phase separation scaffolds. Images were taken of both layers and completed scaffolds to

present the microstructure. The results show an interconnected porous scaffold as seen in

Figure 4-2. As stated earlier, it should be noted that the salt-leaching can create

"pockets" in the scaffolds, as where this novel procedure has a more open morphology.

A comparison image is shown in Figure 4-3 of the morphologies for both methods of

fabrication. It is important that the scaffold has an interconnected porous structure, which

helps provide for maximizing cell seeding, attachment, and growth [9,10]. Therefore this

method may be more desirable for mES cell studies when compared to the salt-leaching

method.



















A B

Figure 4-2. FESEM images of scaffold. A) 100x and B) 2500x. (Scale marker bar 100ltm
and 10tm respectively)


Figure 4-3. FESEM images comparing the salt-leached and fibrous phase separated
scaffold morphologies. (Scale marker bar 100[tm)

Coatings

Figure 4-4 displays the effect of coatings on the fibrous phase separated PCL

coated scaffolds. The Matrigel coated scaffold had a much rougher, bumpy surface after

it had been dried and prepared for FESEM analysis. This is probably a result of fact that

the Matrigel coating is much more viscous and globular then the gelatin coating. The

gelatin coating seen in Figure 4-4 (B) looks more like thin sheet coating over the scaffold.

It was hypothesized that the morphologies of these coatings may affect the cell's

morphology on the coating surface. However, the true morphologies of these coatings









cannot be represented using FESEM imagining and is believed that the coatings would be

hydrated and swollen in solution.











A B

Figure 4-4. FESEM images of Matrigel and gelatin coated PCL scaffolds. A) Matrigel
and B) gelatin.

In vitro Degradation Study

During the nine week in vitro degradation study, general visible observations were

conducted on both the PCL and PLGA salt-leached scaffolds. For both of the polymers

studied, their respective groups were extracted from the study at the following designated

times: 12 hours, day 2, week 1, day 10, week 2, week 3, week 4, week 5, week 6, week 7,

week 8, and week 9. Upcoming sections will discuss mass, molecular weight, pH, and

morphology analysis for both PLGA and PCL scaffolds for each extraction date.

General Observations

General, visible observations were recorded on the scaffold's degradation, which

are displayed in Table 4-1 and Table 4-2. Each week the samples were inspected for any

obvious changes in the scaffolds in the PBS (pH 7.4) solution during the experiment.









Table 4-1. General observations of PLGA scaffolds in PBS.
General Observations during degradation
Week 1 No visible change
Week 2 Light particulates
Week 3 Light particulates
Week 4 Medium/light particulates
Scaffolds have become softer and more delicate to handle
Week 5 Medium/light particulates
Extracted samples stuck to drying plate
Week 6 Light particulates
Scaffolds are starting to conglomerate
Week 7 Light/no particulates
Scaffolds are conglomerating
Week 8 Light/no particulates
Scaffolds are conglomerated
After being dry, scaffolds turned brown
Week 9 Scaffolds have conglomerated into a shiny, grey clump



Table 4-2. General observations of PCL scaffolds in PBS.
General Observations during degradation
Week 1 No visible change
Week 2 No visible change
Week 3 No visible change
Week 4 Very fine particulates
Scaffolds seem to be flattening out
Week 5 Light particulates
Porosity is becoming to increase
Week 6 Light particulates
Porosity slightly increases
Week 7 Fine, light particulates
Week 8 Fine, light particulates
Week 9 Fine, light particulates









To the visible eye, the PCL scaffolds preserved their shape and morphology when

compared to the PLGA scaffolds. The PLGA scaffolds seemed to lose its shape by week

3 and by week 5 had lost its scaffold function. The PLGA scaffolds also seemed to give

off particulates at an earlier stage of the degradation study. The PCL scaffolds also did

not conglomerate in later stages of the study like the PLGA scaffolds. Finally, no visible

color change occurred in the PCL scaffolds during the study, unlike the grey/brown color

the observed for the PLGA scaffolds.

Mass Analysis

Throughout the nine week in vitro degradation study, the average percent mass loss

was recorded for both the PLGA and PCL salt-leached scaffolds. Each group of scaffolds

were extracted and evaluated from the study at their designated point in time. Figures 4-5

and 4-6 display the results of the average percent mass remaining at extraction, during the

in vitro degradation study for both PLGA and PCL respectively. Trendlines were applied

to help evaluate the data.

Discussion. As noted above, the PLGA scaffolds loss a significant amount of mass

during the study when compared to their PCL counterpart. The PLGA scaffolds mass

started to drop drastically after day 28 and continued to plummet after day 49. However,

the PCL scaffolds kept a slightly consistent linear slope throughout the nine week study.

As noted earlier in Table 4-1, PLGA scaffolds on week 5 stuck to the drying plate and

broke into pieces when trying to remove for weighing, causing the discrepancy in Figure

4-5. After this incident, scaffolds were dried on a Teflon plate to avoid the mishap again.

The large error bars after day 49 on Figure 4-5, are because the PLGA scaffolds had

conglomerated together distorting the scaffold's weights.














100.00

90.00

o 80.00
E
E 70.00

2 60.00

S 50.00
5 o.oo

40.00

c 30.00

20.00

10.00

0.00
0 10 20 30 40 50 60 70
Degradation time (days)

Figure 4-5. PLGA average percent mass loss during degradation study. (Polynomial
trendline)


100.00


95.00

'E
90.00


ci 85.00


80.00


75.00


70.00
0 10 20 30 40 50 60
Degradation time (days)


Figure 4-6. PCL average percent mass loss during degradation study. (Polynomial
trendline)










Molecular Weight (MW) Analysis

For the duration of the nine week in vitro degradation study, the molecular weight

was analyzed for both the PLGA and PCL salt-leached scaffolds. Each group of scaffolds

were extracted and evaluated from the study at their designated point in time. Figures 4-7

and 4-8 display the results of the molecular weight change during the in vitro degradation

study for both PLGA and PCL respectively. Trendlines were applied to help evaluate the

data.

Discussion. Figures 4-7 and 4-8 display the obvious dissimilarities in the two

polymers MW loss. The PLGA scaffolds exponentially decreased in MW during the in

vitro degradation study, where again the PCL scaffolds had a slight linear slope. By day

28, the PLGA scaffolds had lost nearly 87% of their original MW; where as the PCL

scaffolds had retained almost 98% of their original MW. However, from day 28 till the

end of the study, the MW loss of the PGLA scaffolds leveled out.



90000
80000
70000
S60000
50000
S40000 77301-00565x
y 77301e
30000
20000
10000 *
0
0 10 20 30 40 50 60 70
Degradation time (days)


Figure 4-7. PLGA molecular weight change during degradation study. (Exponential
trendline)












125000


120000


S115000


S110000

0
o
105000


100000
0 10 20 30 40 50 60 70
Degradation time (days)



Figure 4-8. PCL molecular weight change during degradation study. (Polynomial
trendline)

pH Analysis

During the in vitro degradation study, the buffer's pH was evaluated from both the

PLGA and PCL salt-leached scaffolds. At each designated PBS exchange time point,

each vial was analyzed using a pH meter. Figures 4-9 and 4-10 display the results of the

PBS solution's pH change during the in vitro degradation study for both PLGA and PCL

respectively. Trendlines were applied to help evaluate the data.

Discussion. As Figure 4-9 notes, the pH trendline severely dives to very acidic

values after day 14, implying that the PLGA scaffolds are beginning to degrade. By day

63, the pH had risen slightly, indicating that the polymer's degradation rate had tapered

off [33]. Figure 4-10 illustrates that the PCL scaffolds' buffer solution hardly shifted in

pH during the nine week study, with no significant drop off.














7.40

7.10
6.80

6.50
6.20

1 5.90
5.60

5.30
5.00

4.70
4.40


0 10 20 30 40
Degradation time (days)


50 60 70


Figure 4-9. Graph presenting pH change of PBS for PLGA scaffolds during degradation
study. (Polynomial trendline)



7.40


7.20 -


7.00


6.80


6.60


6.40
0 10 20 30 40
Degradation time (days)


50 60 70


Figure 4-10. Graph presenting pH change of PBS for PCL scaffolds during degradation
study. (Polynomial trendline)









FESEM (Week 1,3,5,7,9)

Throughout the study, the scaffold's morphology was analyzed by SEM for both

the PLGA and PCL salt-leached scaffolds. Each group of scaffolds were extracted from

solution and evaluated from the study at their designated point in time. Figures 4-11 and

4-12 display the morphology change of the scaffolds during the in vitro degradation study

for both PLGA and PCL respectively.

PLGA

During the degradation study, the PLGA scaffolds underwent vast morphological

changes as seen in Figure 4-11. From week 1 to week 3, the porosity of the scaffold

seemed to increase slightly during the study. However, by week 5 the scaffolds had lost

all of their functional scaffolding properties needed for tissue engineering, as described

earlier in the background. The scaffolds were no longer porous and had developed into a

rough surface mass. By week 9, the scaffolds had greatly degraded and all that was left

was a smooth mass.

PCL

The PCL scaffolds morphology varied little during the in vitro degradation study.

However it was noted that the visible porosity of the scaffolds did increase after week 6.

After the nine week study, the PCL scaffolds still withheld their vital scaffold properties

of porosity and interconnected pores, needed for tissue engineering purposes. In fact, the

PCL scaffolds could be considered a more likely candidate after the study than before,

since the scaffolds seemed to become more porous after some degradation. These results

can be seen below in Figure 4-12.



















A B











C











D E

Figure 4-11. FESEM images of degrading PLGA scaffolds at. A) Week 1, B) Week 3, C)
Week 5, D) Week 7 and E) Week 9. (Scale marker bar 100[tm)










































D E
Figure 4-12. FESEM images of degrading PCL scaffolds at. A) Week 1, B) Week 3, C)
Week 5, D) Week 7 and E) Week 9. (Scale marker bar 100[tm)

In vitro mES Cell Studies

Once scaffold fabrication was complete, the scaffolds were evaluated for in vitro mES

cell differentiation. Dr. Naohiro Terada and Dr. Takashi Hamazaki, from the Department

of Pathology, Immunology and Laboratory Medicine at the University of Florida, greatly

assisted in the cellular studies. Both coated and uncoated PCL salt-leached and fibrous

phase separated scaffolds were used in the studies. The interests of these studies were to









evaluate the difference between the salt-leached and the novel, fibrous phase-separation

procedures, while also considering the various coating materials.

PCL Salt-leached Scaffolds (Study 1)

The scaffolds were prepared and examined to determine the cell's growth, survival

and differentiation state. Under fluorescent microscopy, Afp-GFP/mES cells express a

green fluorescent protein once the cells have differentiated toward endodermal cells.

Figure 4-13 illustrates the differences in how the Afp/GFP mES cells interacted with the

uncoated and coated scaffolds.

Day 6. Figures 4-13 [A-B] are representative areas showing that some of the mES

cells had aggregated forming embryoid bodies. The outer layer of the embryoid bodies

had also differentiated into endodermal cells shown by the GFP expression marker. After

looking at the uncoated scaffolds, it seems the mES had not attached to the scaffold and

are aggregating without interacting with the scaffold. Figures 4-13 [C-D] are

representative areas demonstrating how some of the cells have attached to the Matrigel

coated scaffold and differentiated into endodermal cells. However, it was noted that the

cellular attachment was limited by the scaffolds morphology. The cells seemed to exist

only in "pockets" of the scaffold and were not found through the entire structure. A few

embryoid bodies were also found with the Matrigel coated scaffolds. These embryoid

bodies are believed not to have attached to the scaffold.



















pA B










C D
Figure 4-13. Optical and fluorescent microscopy images of PCL salt-leached scaffold on
day 6. A) Optical image of uncoated PCL, B) GFP image of uncoated PCL, C)
optical image of Matrigel coated PCL and D) GFP image of Matrigel coated
PCL. (Scale marker bar 200[tm)

PCL Fibrous Phase Separated Scaffold (Study 2)

Fluorescent microscopy

The scaffolds were prepared and examined to determine cellular growth, survival

and differentiation fate. As noted before, Afp-GFP mES cells express green fluorescent

protein when the cells differentiate toward endodermal cells. Hoechst 33342, a blue

fluorescent dye specifically staining cell nuclei (both dead and alive), and PI, a red

fluorescent dye staining only dead cell nuclei, where used to help illuminate the cellular

results. Figures 4-14 and 4-15 illustrate representative areas of the mES cell-scaffold

interactions with the various scaffold and coating types.

Day 9. After observing the scaffolds with an optical/fluorescent microscope, cells

were mainly located on corners of the scaffolds. Figure 4-14 displays representative









areas of were cells were found on each specific scaffold coating. The uncoated scaffolds

exhibited unattached embryoid bodies with the outer layers differentiating to endodermal

cells. Some cells attached and had also differentiated into endoderm. By comparing the

nuclei stains, most cells seemed dead on the scaffold. The gelatin coated scaffolds

formed did not seem to form embryoid bodies or differentiated into endodermal cells.

However, the cells that did attach seemed alive and spreading onto the scaffold. The

Matrigel coated scaffolds showed little evidence of embryoid bodies forming in the

fluorescent microscopy. The mES cells seemed to have attached, spread and

differentiated endodermally in locations in the scaffold's interconnected structure. After

monitoring the Matrigel coated scaffolds, cells seemed to be alive rather than dead.

Day 24. At Day 24, mES cells were discovered throughout the scaffolds and

representative areas were selected for fluorescent microscopy seen in Figure 4-15. The

uncoated PCL scaffolds had some cellular attachment and differentiation into endoderm.

Some embryoid bodies were also visible in the culture well. The gelatin coated scaffold

displayed both cellular attachment and spreading. Some differentiations into endodermal

cells were found, along with some embryoid bodies. Also, few dead cells were spotted

using PI and fluorescent microscopy. The Matrigel coating also presented cellular

attachment and spreading. Some differentiations into endoderm were spotted throughout

the scaffold. In both the Matrigel and gelatin coated scaffolds, cells were located on

multiple layers of the scaffold as observed in Figure 4-16.







day9
No Coat Gelatin Matrigel


hFo






Hoech





PI




AFP
/GFP


Bar 200um
Figure 4-14. Optical and fluorescent microscopy images on Day 9 of PCL phase
separated mES cell study.









day24
No Coat Gelatin Matrigel











Hoech







Pl





AFP
/GFP



Bar 200um

Figure 4-15. Optical and fluorescent microscopy images on Day 24 of PCL phase
separated mES cell study.























Figure 4-16. Fluorescent microscopy showing cells on multiple layers of the scaffold.
(Scale marker bar 200tm)

Flow cytometry

Figure 4-17 illustrates differences between the uncoated scaffold and the coated

scaffold. The uncoated scaffold had a lower individual cell size and few GFP positive

cells. Both Matrigel and gelatin coated scaffold had a higher population of individual

cells with larger cell sizes and coarser nuclear configurations then uncoated scaffolds.

No coat Gelatin Matrigel
S.%M 97av iDI3 flSt 9.6S

S'i .. \ Population Intensity
i High


** Low
00% OfP 0.0% 0% fP OJ ft O%.0%

Figure 4-17. Flow cytometry analysis at Day 24 of PCL phase separated mES cell study.

FESEM

Once the scaffolds had been fixed and dehydrated they were prepared for SEM

analysis as listed earlier. The Matrigel coated scaffolds had cellular attachment

throughout the samples and the cells were mainly found clustered together. It also

appears that the cytoplasm had collapsed in towards the nucleus as seen in Figure 4-









18(B). This effect is thought to be caused by how the cells adhered to the viscous

Matrigel. As the cells grasped the scaffold's structure, they clustered together (Figure 4-

18(A)) because of the hyrdogel properties of the coating.











A B

Figure 4-18. FESEM images of cellular attachment on Matrigel coated PCL scaffold. A)
2500x and B) 7000x. (Scale marker bar 10tm and 1 tm respectively).

Figure 4-19 displays a representative area of how the mES cells attached to the

gelatin coated PCL scaffolds. Once again the nucleus and cytoplasm are visibly seen

spread out on the surface of the scaffold, unlike the clusters formed with Matrigel (Figure

4-19 (A)). However, the cells did not collapse like the Matrigel coated scaffolds. The

gelatin coating was more of a thin sheet coating the scaffold and allowed the cells to

adhere and spread out instead of clustering.













Figure 4-19. FESEM images of cellular attachment on gelatin coated PCL scaffolds. A)
2500x and B) 7000x. (Scale marker bar 10tm and 1 tm respectively).









Reverse transcriptase-polymerase chain reaction (RT-PCR)

As seen in Figure 4-20, expression of various differentiation protein markers were

recorded after Day 24. These markers help determine the differentiation of the cells and

their possible final cell line. The "housekeeping" gene, P-actin, is used to prove that

cellular life exists and that the scaffolds did not create a toxic environment for the cells

[7,8]. Other forced expressions of transcription factors that were chosen to look for were

specific linage differentiation markers, such as GATA6, hepatocyte nuclear (HNF4), and

transthyretin (TTR) for primitive endoderm [7,8]. The expression of Albumin (ALB) can

imply the cells are becoming hepatic or visceral endoderm differentiated [7,8]. Also,

Nestin proteins were also examined as markers for neural differentiation.

After observing the scaffolds, differences were observed between protein markers

expressed in the RT-PCR data. P-actin was expressed for all the scaffolds proving that

cellular adhesion existed on all the scaffolds. The gelatin coated scaffolds proved to be

the most expressed scaffold of differentiation markers for hepatic/visceral endoderm

differentiation showing GATA6, HNF4, TTR, and ALB. Therefore gelatin coating

scaffolds seem to have the best possibility to further the ES cells to differentiate into a

hepatocyte cell linage. However, Matrigel coated scaffolds showed some evidence of

possible future neural differentiation with the expression of Nestin. The Matrigel coated

scaffolds also have a slight possibility to differentiate into an early hepatocyte/visceral

endoderm cell. The uncoated scaffolds showed no real assistance to help further the mES

cell populations into the desired cell lines according to this research (i.e. liver or nerve).







day24


p-actin


Cell
Viability


GATA6

HNF4

TTR

ALB

Nestin


Endoderm
Markers


EarlyHepatic
Marker
Neural Marker


Figure 4-20. RT-PCR analysis at Day 24 of PCL phase separated mES cell study.














CHAPTER 5
CONCLUSIONS

Scaffold Fabrication and Characterization

In this study, PCL was initially investigated for biodegradable embryonic stem cell

scaffold research. Two methods of fabrication were considered for this research: salt-

leaching and phase separation. When comparing PLGA to PCL, the PLGA scaffolds were

very brittle, had poor handling properties and were generally hard to manage. The PCL

scaffolds were more elastic and their structure was more durable while handling them.

When comparing the two methods of fabrication, many faults were found in the salt-

leaching procedure. Pockets seemed to form where the salt had leached out, instead of

the interconnected morphology desired for tissue engineering. The pocket morphology

was hypothesized to possibly limit the cells growth and proliferation throughout the

scaffold. However, the fibrous phase separated scaffolds had a porous, interconnected

morphology observed using FESEM. These scaffolds are believed to help contribute to

future ES cell studies.

In vitro Degradation Study

The in vitro degradation study confirmed what was already known, that PCL has a

slower degradation rate than PLGA. The PCL scaffolds withheld their morphology for

the entire nine week study, as where PLGA scaffolds had lost their scaffold functionality

by week 5. However, at the end of the study PCL scaffolds had become more porous,

offering the idea that preliminary PBS "bathing" may help the scaffold become a more

productive scaffold.









In vitro mES Cell Studies

Study 1

The Matrigel coated scaffold showed attachment, growth, endoderm differentiation,

and limited embryoid bodies when compared to the uncoated scaffold. The above

observations the Matrigel coating influences if the cells attach and spread on to the

scaffold. It was also found that the pocket morphology created by the salt leaching did

limit the cell's growth and proliferation throughout the scaffold. The cells stayed almost

entirely on the outside surface of the scaffolds and were unidentified in the core of the

scaffold.

Study 2

Study 2 confirmed that a coating is necessary for positive cellular attachment to

PCL scaffolds. At Day 9, the Matrigel coated scaffold seemed to have a slight advantage

over the gelatin coating in cellular attachment and had more endoderm differentiation

present. By Day 24, the Matrigel and gelatin coatings seemed comparable when

comparing representative fluorescent microscopy images and flow cytometry data.

However the expression of differentiation markers in the RT-PCR data displayed

dissimilarities in how coating materials affected the cell's differentiation. The Matrigel

coated scaffold had an expression of albumin and other visceral endoderm/early hepatic

markers (GATA6, HNF4, TTR). On the other hand the gelatin coated scaffold had an

expression of Nestin and a slight expression of albumin, along with other visceral

endoderm/early hepatic markers.

Final Thought

This study's goal was to fabricate a porous PCL scaffold for mES cell study. This

research demonstrates that scaffold morphology and various types of coatings can play a






47


vital role in how mES's adhere and differentiate with the coating/scaffold. It was found

that an open, interconnected biodegradable scaffold with an extracellular matrix dip

coating created an environment to promote cellular attachment, proliferation, and

differentiation throughout the scaffold. In addition, FESEM, flow cytometry, and RT-

PCR data illustrate that different coatings affect mES behavior and scaffold interaction.

Future research should be investigated on these topics to fully understand the role of

scaffold coating to steer mES toward a desired cell differentiation.















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BIOGRAPHICAL SKETCH

Michael Howell Tollon was born in Clearwater, FL, on March 2, 1980. He grew up

in Largo, FL, and attended Seminole High School. Michael completed his bachelor's in

materials science and engineering in August 2002 at the University of Florida. After

graduation he plans on seeking employment in the state of Florida.




Full Text

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FABRICATION OF COATED BIODEG RADABLE POLYMER SCAFFOLDS AND THEIR EFFECTS ON MURINE EMBRYONIC STEM CELLS By MICHAEL H. TOLLON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Michael H. Tollon

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ACKNOWLEDGMENTS I would like to thank Dr. Christopher Batich, Brad Willenberg, and Dr. Takashi Hamazaki for their help in my research. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 Significance..................................................................................................................1 Specific Aims................................................................................................................2 Specific Aim 1.......................................................................................................2 Specific Aim 2.......................................................................................................2 Study Design.................................................................................................................2 2 BACKGROUND..........................................................................................................4 Scaffolding in Tissue Engineering................................................................................4 Poly(DL-lactic-co-glycolic acid)..................................................................................5 Poly(-caprolactone).....................................................................................................9 Embryonic Stem cells.................................................................................................10 3 MATERIALS AND METHODS...............................................................................12 Scaffold Synthesis......................................................................................................12 Salt-leached Scaffold...........................................................................................12 Materials.......................................................................................................12 Molding........................................................................................................12 Bathing.........................................................................................................13 Drying and storage.......................................................................................14 Fibrous Phase Separated Scaffold.......................................................................14 Materials.......................................................................................................14 Molding........................................................................................................14 Layering.......................................................................................................15 Drying and Storage.......................................................................................15 iv

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Scaffold Characterization...........................................................................................15 General Observations..........................................................................................15 Scanning Electron Microscopy............................................................................15 In vitro Degradation Study.........................................................................................16 In vitro murine ES Cell Studies..................................................................................16 Sterilization..........................................................................................................17 Preparation of Coatings.......................................................................................17 Coating Procedure...............................................................................................18 Murine ES Cell Preparation and Seeding............................................................18 Preparation...................................................................................................18 Seeding.........................................................................................................19 mES Cell Study Analysis....................................................................................19 Fluorescent microscopy................................................................................19 Flow cytometry............................................................................................20 FESEM.........................................................................................................20 Reverse transcriptase-polymerase chain reaction (RT-PCR).......................21 4 RESULTS AND DISCUSSION.................................................................................22 Scaffold Characterization...........................................................................................22 Salt-leached Scaffold...........................................................................................22 General observations....................................................................................22 FESEM.........................................................................................................23 Fibrous Phase Separated Scaffold.......................................................................24 General observations....................................................................................24 FESEM.........................................................................................................24 Coatings...............................................................................................................25 In vitro Degradation Study.........................................................................................26 General Observations..........................................................................................26 Mass Analysis......................................................................................................28 Molecular Weight (MW) Analysis......................................................................30 pH Analysis.........................................................................................................31 FESEM (Week 1,3,5,7,9)....................................................................................33 PLGA...........................................................................................................33 PCL...............................................................................................................33 In vitro mES Cell Studies...........................................................................................35 PCL Salt-leached Scaffolds (Study 1).................................................................36 PCL Fibrous Phase Separated Scaffold (Study 2)...............................................37 Fluorescent microscopy................................................................................37 Flow cytometry............................................................................................41 FESEM.........................................................................................................41 Reverse transcriptase-polymerase chain reaction (RT-PCR).......................43 5 CONCLUSIONS........................................................................................................45 Scaffold Fabrication and Characterization.................................................................45 In vitro Degradation Study.........................................................................................45 v

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In vitro mES Cell Studies...........................................................................................46 Study 1.................................................................................................................46 Study 2.................................................................................................................46 Final Thought..............................................................................................................46 LIST OF REFERENCES...................................................................................................48 BIOGRAPHICAL SKETCH.............................................................................................51 vi

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LIST OF TABLES Table page 4-1 General observations of PLGA scaffolds in PBS.....................................................27 4-2 General observations of PCL scaffolds in PBS........................................................27 vii

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LIST OF FIGURES Figure page 3-1 Schematic drawing and picture of Teflon mold for salt-leached scaffold fabrication with mold release...................................................................................13 4-1 FESEM images illustrating the difference in washed and unwashed scaffolds.......23 4-2 FESEM images of scaffold......................................................................................25 4-3 FESEM images comparing the salt-leached and fibrous phase separated scaffold morphologies............................................................................................................25 4-4 FESEM images of Matrigel and gelatin coated PCL scaffolds................................26 4-5 PLGA average percent mass loss during degradation study....................................29 4-6 PCL average percent mass loss during degradation study.......................................29 4-7 PLGA molecular weight change during degradation study.....................................30 4-8 PCL molecular weight change during degradation study........................................31 4-9 Graph presenting pH change of PBS for PLGA scaffolds during degradation study.........................................................................................................................32 4-10 Graph presenting pH change of PBS for PCL scaffolds during degradation study.32 4-11 FESEM images of degrading PLGA scaffolds at. A) Week 1, B) Week 3, C) Week 5, D) Week 7 and E) Week 9.........................................................................34 4-12 FESEM images of degrading PCL scaffolds at. A) Week 1, B) Week 3, C) Week 5, D) Week 7 and E) Week 9...................................................................................35 4-13 Optical and fluorescent microscopy images of PCL salt-leached scaffold on day 6................................................................................................................................37 4-14 Optical and fluorescent microscopy images on Day 9 of PCL phase separated mES cell study..........................................................................................................39 4-15 Optical and fluorescent microscopy images on Day 24 of PCL phase separated mES cell study..........................................................................................................40 viii

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4-16 Fluorescent microscopy showing cells on multiple layers of the scaffold..............41 4-17 Flow cytometry analysis at Day 24 of PCL phase separated mES cell study..........41 4-18 FESEM images of cellular attachment on Matrigel coated PCL scaffold...............42 4-19 FESEM images of cellular attachment on gelatin coated PCL scaffolds.................42 4-20 RT-PCR analysis at Day 24 of PCL phase separated mES cell study.....................44 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science FABRICATION OF COATED BIODEGRADABLE POLYMER SCAFFOLDS AND THEIR EFFECTS ON MURINE EMBRYONIC STEM CELLS By Michael H. Tollon May 2005 Chair: Christopher Batich Major Department: Materials Science and Engineering In the past decade, tissue engineering has become a great interest in materials science research. Embryonic stem (ES) cell transplantation has become one of the most researched therapies for restoring tissue and organ function. Many studies have investigated the use of porous biodegradable scaffolds to promote cell adhesion, growth, proliferation, differentiation, and to help steer the course of tissue development. Research has shown that extracellular matrices and the basement membranes affect various cell types and cellular behaviors. However, the effects of these materials on ES cell behavior are currently understudied and poorly understood. In this study, the synthetic biodegradable polymers polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) were chosen to create an interconnected foam structure. A phase separated scaffold method was developed and compared to previously designed salt-leached scaffolds. In vitro degradation studies were conducted on salt-leached scaffolds at 37oC, while submerged in a phosphate buffered saline solution. x

PAGE 11

These scaffolds were analyzed for mass loss, pH, molecular weight, and morphology. The PCL scaffolds maintained their structure and functionality during the 9 week degradation study when compared to PLGA. Multiple coating techniques were explored to modify cellular results using these scaffolds. When the phase separated PCL scaffolds were coated with Matrigel and gelatin solutions murine ES (mES) cells attached, spread, and differentiated within the scaffolds. Comparisons of the coatings effect on the mES cells were analyzed using fluorescent microscopy, flow cytometry, scanning electron microscopy, and reverse-transcriptase polymerase chain reaction. In conclusion it was found that a scaffolds coating/extracellular matrix affects a mES cells morphology and differentiation. xi

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CHAPTER 1 INTRODUCTION Significance Tissue engineering has become of great interest to materials science research in the past decade. It has become one of the most researched therapies for tissue and organ revival [1,2]. The use of tissue engineered devices has the potential to reduce the annual cost of tissue loss and end-stage organ failure, which is estimated to reach over $10 billion in the U.S. alone [3]. Liver regeneration, in recent years, has become one of the most sought after goals in tissue engineering [4]. Liver diseases, such as viral hepatitis (A, B, and C), cirrhosis, and cancer, have created this major necessity for success in tissue engineering. It has been shown that acute liver failure has a mortality rate of more than 80% and it is estimated that one in 10 Americans, or 25 million people in this country suffer from a form of liver disease [4,5]. Approximately 30,000 individuals in the U.S. die each year due to the lack of a cure for this disease and a shortage of donor organs [4-6]. One of the challenges in tissue engineering concerns introducing and controlling the stem cells into a specific lineage and to fit them into the desired tissue architecture. Embryonic stem cells (ES cells) can be cultivated in an undifferentiated state indefinitely in vitro and have the potential to differentiate into multiple cell lineages. The most widely used method to differentiate ES cells in vitro begins with cell aggregation in a suspension culture [7,8]. However these cell aggregates, also known as embryoid bodies, have poor ability to generate the target cellular phenotypes, such as hepatocytes. 1

PAGE 13

2 Therefore, bioengineered scaffolds have been fabricated to help cellular attachment, proliferation and to avoid embryoid body formation. Porous, biodegradable polymers have been found to play a vital role by creating a scaffold structure for tissue regeneration [9-11]. The scaffolds structural integrity is a key attribute during the course of tissue formation [2,12]. Scaffolds supply the necessary foundation for cell attachment, proliferation and maintaining differentiated functions, while limiting embryoid body formation [7,9,10]. Other important factors for cell scaffolding success are the addition of growth factors and/or other protein coatings [2,12,13]. Presently there is no ideal scaffold developed to achieve the ultimate goal of ES cell differentiation towards a hepatocyte lineage. Specific Aims Specific Aim 1 Fabricate biodegradable poly(-caprolactone) scaffolds with porous morphologies for murine embryonic stem cell study. Specific Aim 2 Investigate differentiation of murine embryonic stem cells on the constructed biodegradable polymer scaffolds coated with Matrigel or gelatin. Study Design In this study, poly(-caprolactone) (PCL) was investigated for biodegradable embryonic stem cell scaffold research. Two methods of fabrication: salt-leaching and phase separation, were considered to create an open, interconnected foam structure for murine embryonic stem (mES) cell evaluation. The PCL salt-leached method was compared to previously constructed poly(lactic-co-glycolic acid) scaffolds described in the literature [2,12,14]. In vitro degradation studies were conducted on the salt-leached

PAGE 14

3 scaffolds at 37oC while submerged in a phosphate buffered solution (PBS) with a pH of 7.4. The scaffolds were analyzed and compared for mass loss, molecular weight, morphology, and pH change of the PBS throughout the nine week study. The salt-leached and phase separated PCL scaffolds were then analyzed for biological assessment with the help of Dr. Naohiro Terada and Dr. Takashi Hamazaki, from the Department of Pathology, Immunology and Laboratory Medicine at the University of Florida. Murine ES cells were seeded on both scaffold designs with various coating materials applied. Next, the coatings were investigated for the possible significant differences in cellular results. The biological assessments were analyzed using fluorescent microscopy, flow cytometry, field emission scanning electron microscopy (FESEM), and reverse transcriptase-polymerase chain reaction (RT-PCR),

PAGE 15

CHAPTER 2 BACKGROUND Scaffolding in Tissue Engineering Tissue engineering using cell transplantation has become one of the most researched therapies for restoring tissue and organ function [1,2]. Already, research groups are investigating possible tissue engineered products such as cartilage, bone, heart valves, nerves and muscle [15]. In this technique, the scaffold plays the fundamental role of promoting cell: adhesion, growth, proliferation, differentiation, and will help steer the course for tissue development [2,12]. The 3D architecture of the scaffold will ultimately define the newly formed soft or hard tissue [15]. The crucial function of the scaffold design is to produce an ideal structure that can act as the natural extracellular matrix (ECM) until host cells can repopulate and resynthesize a new natural matrix. The scaffolding material and design must be carefully selected to assure that the seeded cells are biocompatible with the fabricated scaffold [16]. Porous scaffolds with an open pore system are often desired for maximizing cell seeding, attachment, growth, and ECM production [9,10]. A scaffolds pore size, porosity, and the global continuity of the pores are used to characterize the pore structure [2]. Several successful methods to fabricate highly porous polymer scaffolds include particulate-leaching, gas foaming, phase separation, emulsion freeze drying, and 3D printing methods. These techniques can be used to fabricate sponge-like scaffolds or combined to develop more complex superstructures [2,12,14]. 4

PAGE 16

5 Biodegradable polymers have also shown their importance in tissue engineering scaffolds, since the scaffold material should not remain in the final engineered tissue. The scaffolds degradation rate may affect many cellular processes including cell growth, tissue regeneration, and host response. The degradation of the scaffold should either be similar to or slower than the rate of tissue formation for optimum results [9-11]. Scaffold coatings have also been experimented with lately in tissue engineering research. It is believed that with a scaffolds physical support, the coating can provide chemical cues which could support an environment for ES cells to organize and differentiate [13]. Coatings can range from biodegradable polymers to growth factors that may help promote cell adhesion, growth, proliferation, differentiation, and steer the course for tissue development [2,12,13]. According to Ma et al., the understanding of the principles of scaffolding is far from satisfactory, and the ideal scaffolds are yet to be developed [12]. Poly(DL-lactic-co-glycolic acid) For the last two decades, synthetic biodegradable polymers have been increasingly used in the medical field, compared to natural polymers [17-19]. Amongst them, the thermoplastic poly(esters) like poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and especially poly(lactic-co-glycolic acid) (PLGA) have generated a tremendous interest due to their excellent biocompatibility and biodegradability [17-20]. The discovery and the synthetic work on low molecular weight oligomeric forms of lactide and/or glycolide polymers was first carried out several decades ago [18,19]. The methods to synthesize high molecular weights of these polymers were first reported during the late 1960s and early 1970s. A number of groups have published pioneering work on the utility of these polymers to make sutures/fibers [18,19]. These fibers had

PAGE 17

6 several advantages like good mechanical properties, low immunogenicity and toxicity, excellent biocompatibility, and predictable biodegradation kinetics [18,19]. The wide acceptance of the lactide/glycolide polymers as suture materials, made them attractive candidates for biomedical applications like ligament reconstruction, tracheal replacement, surgical dressings, vascular grafts, nerve, dental, and fracture repairs [18-20]. Many researchers have investigated and documented the biodegradation, biocompatibility, and tissue reaction of PLGA [19]. Various polymeric devices like microspheres, microcapsules, nanoparticles, pellets, implants, and films have been fabricated using these polymers for the delivery of a variety of drug classes. They are also easy to formulate into drug carrying devices for various applications which have been approved by the FDA for drug delivery use [17-20]. Understanding the chemical, physical and biological properties of a polymer is helpful, before formulating a tissue scaffold device. The various properties of the PLGA co-polymer are better understood by considering PLA and PGA as individual polymers. The polymer PLA can exist in an optically active stereoregular form (L-PLA) and in an optically inactive racemic form (D,L-PLA) [17,19,20]. L-PLA is found to be semi-crystalline in nature due to high regularity of its polymer chain while D,L-PLA is an amorphous polymer because of irregularities in its polymer chain structure [18,20]. Hence the use of D,L-PLA is usually preferred over L-PLA as it enables more homogeneous dispersion of the drug in the polymer matrix [20]. PGA is highly crystalline because it lacks the methyl side groups of the PLA [18,20]. Lactic acid is more hydrophobic than glycolic acid and therefore lactide-rich PLGA copolymers are less hydrophilic, absorb less water, and subsequently degrade more slowly [17,18].

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7 The physical properties such as the molecular weight and the polydispersity index affect the mechanical strength of the polymer and its ability to be formulated as a drug delivery device [18,19]. Also these properties may control the polymer biodegradation rate and hydrolysis [18]. The commercially available PLGA polymers are usually characterized in terms of intrinsic viscosity, which is directly related to their molecular weights [18]. The mechanical strength, swelling behavior, capacity to undergo hydrolysis, and subsequently the biodegradation rate are directly influenced by the crystallinity of the PLGA polymer [18]. The resultant crystallinity of the PLGA copolymer is dependent on the type and the molar ratio of the individual monomer components (lactide and glycolide) in the copolymer chain [17]. PLGA polymers containing 50:50 ratio of lactic and glycolic acids are hydrolyzed much faster than those containing higher proportion of either of the two monomers [19]. PLGAs prepared from L-PLA and PGA are crystalline copolymers while those from D,L-PLA and PGA are amorphous in nature [18,19]. It has been found that PLGAs containing less than 70% glycolide are amorphous in nature [21]. The degree of crystallinity and the melting point of the polymers are directly related to the molecular weight of the polymer [18,19]. The glass transition temperatures (Tg) of the PLGA copolymers are above the physiological temperature of 37C and hence they are normally glassy in nature [18,19]. Thus, they have a fairly rigid chain structure, which gives them significant mechanical strength to be formulated as a degradable device [18,19]. It has been reported that the Tgs of PLGA decrease with the decrease of lactide content in the copolymer composition and with decreasing molecular weight [22].

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8 The PLGA polymers chosen should also have considerable mechanical strength, since tissue scaffolds are subjected to significant physical stress. Different factors like the molecular weight, copolymer composition (lactide/glycolide ratio), crystallinity, and geometric regularity of individual chains significantly affect the mechanical strength of the particular polymer [17-19]. For both in vitro and in vivo the PLGA copolymer undergoes degradation in an aqueous environment (hydrolytic degradation or biodegradation) through cleavage of its backbone ester linkages [17-19]. The polymer chains undergo bulk degradation and the degradation generally occurs at a uniform rate throughout the PLGA matrix [18]. It has been recorded that the PLGA biodegradation occurs through random hydrolytic chain scissions of the swollen polymer [18,19]. The carboxylic end groups present in the PLGA chains increase in number during the biodegradation process as the individual polymer chains are cleaved; these are known to catalyze the biodegradation process [18,19]. It has also been reported that large fragments are degraded faster internally and amorphous regions degrade faster than crystalline. The biodegradation rate of the PLGA copolymers are dependent on the molar ratio of the lactic and glycolic acids in the polymer chain, molecular weight of the polymer, the degree of crystallinity, and the Tg of the polymer [18,19]. A three-phase mechanism for the PLGA biodegradation has been proposed [23]: 1. Random chain scission process. The molecular weight of the polymer decreases significantly, but no appreciable weight loss and no soluble monomer products are formed. 2. In the middle phase a decrease in molecular weight accompanied by rapid loss of mass and soluble oligomeric and monomer products are formed. 3. Soluble monomer products formed from soluble oligomeric fragments. This phase is that of complete polymer solubilization.

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9 The role of enzymes in any PLGA biodegradation is unclear [18,19]. Most of the literature indicates that the PLGA biodegradation does not involve any enzymatic activity and is purely through hydrolysis [18]. However, some findings have suggested an enzymatic role in PLGA breakdown based upon the difference in the in vitro and in vivo degradation rates. It has also been found that motion and buffers may affect their rate differences [19]. However, it is known that PLGA biodegrades into lactic and glycolic acids [17-19]. Lactic acid enters the tricarboxylic acid cycle and is metabolized and subsequently eliminated from the body as carbon dioxide and water [17-20]. Glycolic acid is either excreted unchanged in the kidney or it enters the tricarboxylic acid cycle and eventually eliminated as carbon dioxide and water [18]. Poly(-caprolactone) Synthetic biodegradable polymers over the years have gradually taken over the medical field, compared to natural polymers [17-19]. Polycaprolactone (PCL), a polyester biodegradable polymer, is becoming one of these biomedical materials of interest [24]. Polycaprolactone is synthesized by a ring opening polymerization of the -caprolactone monomer. PCL is a semi-crystalline polymer which exhibits a low melting point (57oC) and a low glass transition temperature (-62oC) [24-26]. It is considered a soft and hard-tissue biocompatible, non-toxic polymer [24-26]. The rubbery characteristics of PCL have been utilized in low molecular weight drug delivery, resorbable sutures, and bone graft substitutes. It has been found that PCL demonstrates a lower tensile modulus and strength than PLA, but higher extensibility, which is important in tissue scaffolding [27].

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10 Like other polyesters, PCL will undergo auto-catalyzed bulk hydrolysis degradation because of the susceptibility of its aliphatic ester linkage. However, the hydrophobic, semi-crystalline polymer retards degradation and resorption kinetics when compared to other aliphatic polyesters such as PLGA, which makes it more suitable for long term implantable devices [24-26]. Bulk hydrolysis breaks the ester linkage, which creates fragmentation and the release of oligomeric species. Low molecular-weight fragments are eventually engrossed by giant cells and macrophages. The byproduct e-hydroxycaproic acid, is either metabolized via the tricarboxylic acid (TCA) cycle or removed by direct renal secretion [24,25,28]. It is also possible for PCL to enzymatically degrade (enzymatic surface erosion) by lipases and esterases, though this is rare [24,29]. Embryonic Stem cells Embryonic stem (ES) cells have shown potential success for cell transplantation in tissue scaffolding because of their ability to differentiate into multiple somatic cell linkages [13]. The most widely used method to differentiate ES cells in vitro initiates with cell aggregation in suspension culture [7]. The cell aggregates, called embryoid bodies, are made up of multicellular, multi-differentiated structures replicating an in vivo developmental program [7]. These embryoid bodies are complicated to study and to determine their differentiation state within the embryoid bodies. Once embryoid bodies form, their reproducibility and function towards creating useful tissue structures diminish [7]. However, it has also been found that porous biodegradable polymer scaffolds support ES cells during the formation of 3D tissues. These scaffolds have the opportunity to prevent embryoid bodies from forming. The scaffolds porosity and biodegradability provides space for cell adhesion, growth, proliferation, and differentiation [2,9,11,13].

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11 Hence, these dimensional scaffolds are expected to be useful to carry stem cells and to allow them to be more useful (than without scaffolds) for regenerative medicine. This has been demonstrated for PLGA sponges and for collagen gels for instance. However these structures are weak or brittle, and there is a need for a longer lasting, tougher scaffolds.

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CHAPTER 3 MATERIALS AND METHODS Scaffold Synthesis Salt-leached Scaffold Materials The polymers used for this scaffold fabrication were polycaprolactone (PCL) (Mw ~ 120,000) provided by Sigma Aldrich and poly (DL-lactic-co-glycolic acid) (PLGA) in a 50:50 mole ratio (Mw ~ 85,000) provided by Birmingham Polymers. Polymer samples were first dissolved with pesticide grade methylene chloride (Fisher) to get the desired weight/volume of 5% (PCL) and 10% (PLGA). Molding A Teflon mold was fabricated by Analytical Research Systems (Gainesville, FL) for this scaffold fabrication method. It has spaces for 8 casting cavities at 1cm x 1cm per cavity. An L-shaped mold release was placed in each cavity to help free the scaffold from the cavity (Figure 3-1). Recipes for polymer solution and NaCl (250-500m) particles were established to fill all 8 cavities to maximize results per mold. For the 10% PLGA solution a mixture of 10g NaCl and 3mL polymer solution was used. As for the 5% PCL, a blend of 10g NaCl and 4mL of polymer solution was used. Once NaCl particles are combined and thoroughly mixed in with the appropriate amount of polymer solution, the mixture is dispensed and packed into each cavity. The Teflon mold was then placed into a vacuum oven for 45 minutes to evaporate off the solvent. Once removed from the vacuum oven, each scaffold was detached from its cavity by freeing the edges of the 12

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13 scaffold and pulling up on the mold release. Deionized (DI) water may also help free the scaffold from the mold. The stainless steel mold release was removed from the scaffold and the bathing process begins. Figure 3-1. Schematic drawing and picture of Teflon mold for salt-leached scaffold fabrication with mold release. Bathing Once the scaffolds are extracted from the mold, they were placed in a 90mm diameter Petri dish. The dish was then filled with 45mL of DI water and rotated at 50rpm on an orbital shaker (Bellco Biotechnology) for 7 days. For the first 2 hours, DI water was exchanged every 30 minutes to help extract the NaCl from the scaffold. The following exchanges occur daily. Every time the DI water is exchanged, each scaffold was removed from the DI water bath and squeezed while excess water was siphoned off using a transfer pipette. The scaffolds are then placed back into a fresh DI water bath. The bathing process leached out the salt particles to create voids in the scaffolds morphology.

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14 Drying and storage After the total bathing process, the scaffolds are removed and are squeezed and siphoned off using a transfer pipette. Next, the scaffolds were air dried over night and then dried in a vacuum oven for 45 minutes. After being dried, they were stored under vacuum until needed for future experiments. Fibrous Phase Separated Scaffold Materials The polymers used for this scaffold fabrication were polycaprolactone (PCL) (Mw ~ 120,000) provided by Sigma Aldrich. The polymer was first dissolved using histological grade acetone (Fisher) to get the desired 5% weight/volume. The solutions were then refrigerated until needed. Molding First, pipette 1mL of 5% (wt/vol) PCL onto a 75mm diameter watch glass. While holding the glass in one hand, rotate it in a slow circular pattern. Next, spray 2mL of DI water at the glass approximately 4 to 6 inches away with a sterile syringe and needle (22 gauge) in a left to right fashion covering the entire watch glass. This generates a phase separation forcing the polymer out of the solution creating fibrous webbing. Once the phase separation starts to occur, pour the excess cloudy solution into the waste storage. Once the webbing has formed, thoroughly rinse the fibrous web with DI water. Rotate the watch glass 90o and repeat the above steps on top of the newly formed fibrous web to create another polymer deposit. Once the second application has been applied, the web is soaked with methanol to help free it from the glass and then dried in a vacuum oven for 30 minutes. After dried, this web is now considered one layer of the final scaffold.

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15 Multiple layers are attached together to create the full scaffold. Layers are stored under vacuum until needed for the layering process. Layering Four layers are first placed on top of each other in a 75mm diameter watch glass. Next, the layers are united together by spraying 1mL of the polymer solution with a sterile syringe and needle (22 gauge) in a left to right fashion covering the entire layer. Quickly spraying DI water over the layers to cause a phase separation once again, which fastens the four layers together. Once the phase separation is visible, pour the cloudy solution into waste storage which was discarded at a later date. Thoroughly rinse the layers with DI water to clean out any excess solution. Next, flip over the newly formed scaffold and repeat the above steps to bond the layers again from the other side. Drying and Storage After the layers have been connected, the scaffold is soaked in methanol, removed and then dried in a vacuum oven for 30 minutes. The scaffolds are subsequently stored under vacuum until needed for future experiments. Scaffold Characterization General Observations After fabrication, the scaffolds were characterized. The scaffolds were weighed and basic handling properties were explored. Comparisons were made for each polymer and scaffold fabrication method used, and with the literature as far as possible. Scanning Electron Microscopy The scaffolds morphology was examined by field emission scanning electron microscopy (FESEM) (JEOL 6335F). After the scaffolds were dried, they were mounted on an aluminum stub and a AuPd sputter coating was applied. The samples were imaged

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16 at 10kV with a working distance of 15mm. Images were taken at low magnifications to identify the scaffolds structure. In vitro Degradation Study A nine week in vitro degradation study was conducted on both the PCL and PLGA salt-leached scaffolds to study their molecular weight (MW) loss, mass loss and morphology changes. For both polymers, 12 groups were divided up with 6 scaffolds per group (144 total samples). Each group of 6 scaffolds was placed in 21x70mm, 4DR vial (Fisher) with 12mL of phosphate buffer saline (PBS) (1X) (Sigma) with a pH of 7.4. Each group was then placed into a rotating incubator (Robbins Scientific) at 37oC rotating at 10rpm. The PBS was exchanged at 12 hours, 24 hours, 36 hours, day 2, day 3, day 4, day 5, day 6, week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, and week 9 for each group. Groups were extracted from the study at the following designated times: 12 hours, day 2, week 1, day 10, week 2, week 3, week 4, week 5, week 6, week 7, week 8, and week 9. After each group was extracted, it was rotated at 25 rpm in a bath of DI water for 15 minutes to clean the scaffolds of PBS. At this point the scaffolds were air dried overnight and then vacuum dried for 2 hours the following day. For each group, all six samples were weighed dry, five samples were used for gel permeation chromatography (GPC), and one sample was used for FESEM analysis. In vitro murine ES Cell Studies Once scaffold fabrication was complete, the scaffolds were evaluated for their possible significance in tissue engineering. Dr. Naohiro Terada and Dr. Takashi Hamazaki, from the Department of Pathology, Immunology and Laboratory Medicine at the University of Florida, greatly assisted in the cellular studies. Both PCL salt-leached and fibrous phase separated scaffolds were used, with main emphasis on the fibrous

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17 phase separated scaffold. The interest of the studies was to compare the salt-leached method to the novel, fibrous phase separation procedure, while also judging the difference in coating materials. The first study involved Matrigel coated and uncoated salt-leached PCL scaffolds which were analyzed by fluorescent microscopy. The second study involved Matrigel coated, gelatin coated, and uncoated fibrous phase separated scaffolds that were analyzed using fluorescent microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry and FESEM. Sterilization The scaffolds were sectioned into 1in x 1in samples (thickness was dependent on original scaffold) and placed in a bath overnight of 70/30 solution of ethanol and DI water respectively. The scaffolds were dried, washed with PBS twice and then dried once again. The scaffolds were kept sterile until coated. Preparation of Coatings The two coating solutions were prepared as listed below (700L): 10% (vol/vol) Matrigel Matrix (BD Bioscience) mixed with D-MEM/F-12 serum free media 0.1% gelatin solution (Specialty Media) in ultra pure water The Matrigel Matrix is a solubilized basement membrane preparation extracted from the Englbreth-Holm-Swarm (EHS) mouse sarcoma, which is a tumor rich in extracellular matrix proteins [30]. Its main components are laminin, collagen IV, entactin, and heparan sulfate proteoglycan. The matrix also contains TGFfibroblast growth factor (FGF), tissue plasminogen activator (TPA), and other growth factors which are found naturally in EHS tumors [31]. At room temperature, the Matrigel Matrix polymerizes to produce a biologically active matrix material that resembles the

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18 mammalian basement membrane. Matrigel is a viscous gel and must be kept frozen for storage purposes. Gelatin is a pure protein obtained by the partial hydrolysis of collagen. This collagneous material has been used from food substitutes to scientific studies. It also provides a basement membrane in cellular studies and can increase a surfaces hydrophilicity [4]. The gelatin solution has a viscosity close to water and differs greatly from the Matrigels hydrogel properties. Coating Procedure After the preparations have been completed, the coating procedure is started immediately. For each coating material, the same procedure is followed. The scaffold is first submerged in the coating solution for 2 minutes. Next, the scaffold is removed with excess coating solution being siphoned off. The scaffold was then incubated for 5 minutes at 37oC. The process is repeated until 3 coatings have been applied and incubated, respectively. Murine ES Cell Preparation and Seeding Preparation The cell line used for the studies was murine Alpha fetoprotein/green fluorescent protein (Afp/GFP) ES stem cells. This cell line can be used to monitor primitive endoderm differentiation [7,8]. The mES cells were maintained prior to the study in an undifferentiated state on gelatin-coated dishes in Knock-out Dulbeccos Modified Eagle Medium (DMEM) (Gibco) containing 10% knockout serum replacement (Gibco), 1% fetal bovine serum (FBS) (Atlanta biologicals), 2mM L-glutamine, 100units/mL penicillin, 100g/mL streptomycin, 25mM HEPES (Gibco), 300M monothioglyercol (Sigma), and 1000unit/mL recombinant mouse LIF media (Chemicon).

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19 The cells were suspended before seeding them on the scaffold. The media was siphoned out of the culture dish trying not to disturb the cells. A wash of PBS was applied to the Petri dish and cells, and then siphoned off. While rotating the dish in a circular pattern, 1mL of 0.25% Trypsin/EDTA (Gibco) was added to dissociate the cells from the dish. The mES cells were then suspended in the mES Differentiated Media: 500mL of IMDM (Gibco), 5mL of penicillin/streptomycin (Gibco), 16L of monothiglycerol (Sigma). Next, 100mL were removed from the prepared solution and stored for later use. Finally, 100mL of 20% FBS is added to the remaining solution. The mES Differentiated Media was then stored for future cell experiments. Seeding The sectioned and coated scaffolds were first placed in its individual 12 well culture dish. Next100L of mES cell suspension (2.17x106 cells/mL) was added in each well. The suspension was placed on top of the scaffold to help cellular adhesion. The culture wells were then incubated for 30 minutes at 37oC. Once removed from the incubator, 1mL of mES Differentiated Media was added to each culture dishes. The scaffolds were incubated at 37oC again until needed for observation. The scaffolds were observed with an optical fluorescent microscope on day 0, 2, 4, 7, 9, 21, 24 for cellular analysis. mES Cell Study Analysis Fluorescent microscopy The cells were observed under a fluorescence microscope (Olympus IX70) and three fluorescent labels were used. The Afp/GFP cells will express a green fluorescent protein after they have differentiated to visceral endoderm. Hoechst 33342 is a blue

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20 fluorescent stain used to mark the nucleus of cells, both dead and alive. Propidium iodide (PI), a red fluorescent stain, was also used because its only permeable to non-living membranes, and is used for detecting dead cells. By comparing GFP, Hoechst and PI presence, one can distinguish live cells from dead ones and identify if the embryonic stem cells have differentiated into the more mature endodermal cells [7]. Flow cytometry Cells were dissociated with 0.25% trypsin/EDTA after day 24 of the experiment. The flow cytometry was performed on FACS Sort (BD Biosciences) on a data set of 30,000 cells, which was then recorded using CellQuest Acquisition software (BD Biosciences). Flow cytometry helps illustrate the separation of GFP-positive and negative cells and the cells size [7]. FESEM The scaffolds morphology and cellular attachment was examined by FESEM (JEOL-6335F). Once the cell study was completed, the cells were fixed in a 2.5% glutaraldehyde/PBS fixation made from 50% glutaraldehyde (Electron Microscopy Sciences) and PBS (1X) with a pH of 7.4 (Gibco). The scaffolds were rinsed with DI water and then dehydrated with ten minute baths of 70%, 80%, 95%, and 100% (three times) of ethanol/DI water solutions [32]. After the scaffolds were fixed and dehydrated, they were mounted on an aluminum stub and a C evaporation coating was applied. The samples were imaged at 10kV with a working distance of 15mm. Low acceleration voltages were used initially to ensure no beam damage. Images were taken at high and low magnifications to identify the scaffolds structure and any possible cellular attachment.

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21 Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was extracted by using RNA aqueous kit (Ambion). Then cDNA was synthesized by using SuperScript II first-strand synthesis system with oligo(dT) (Gibco). PCR was performed by using Taq DNA polymerase (Eppendorf). For each gene, the DNA primers were originated from different exons to ensure that the PCR product represents the specific mRNA species and not geomic DNA [7]. Gene markers are then compared to determine possible cells lineages.

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CHAPTER 4 RESULTS AND DISCUSSION Scaffold Characterization Salt-leached Scaffold General observations PLGA. During fabrication, the PLGA scaffolds were extracted from the mold fairly easy using the L-shaped mold release mentioned earlier. However, there was a slight indentation from the mold release but this did not cause any significant effects on the overall scaffold. After fabrication, the PLGA salt-leached scaffolds were weighed and observed for general handling properties. The weights of the final scaffolds were 12.0 2.51mg and exemplified a rigid character after drying. Their dimensions were 9 1mm in diameter with a thickness of 6 2mm. The scaffolds were very brittle and difficult to work with. The PLGA scaffolds compared well with similar particle leaching methods used in prior work [10,12,14]. PCL. During fabrication, the PCL scaffolds were extracted from the mold effortlessly from the mold. Again, there was a slight indentation from the mold release, but no affects to the overall scaffolds structure. After fabrication, the PCL salt-leached scaffolds were weighed and observed for general handling properties. The weights of the scaffolds were 13.6 .85mg and illustrated an elastic quality when compared to the PLGA scaffolds. Their dimensions were 9 1mm in diameter with a thickness of 4 2mm. During literature searches, nothing was found on salt-leached scaffolds using PCL, but the scaffolds did compare morphologically to other salt-leached scaffolds. 22

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23 FESEM The scaffolds were characterized using FESEM to compare morphologies. Images were taken before and after washing to show the salt-leaching process. The results are shown in Figure 4-1. The figure also displays the porous structure of both PLGA and PCL scaffolds. It should also be noted that the salt-leaching can create pockets in the scaffolds instead of the desired interconnected porous microstructure. As noted before, an interconnected porous structure helps provide for maximizing cell seeding, attachment, and growth [9,10]. Therefore this method may not be the most desirable for mES cell studies. A B C D Figure 4-1. FESEM images illustrating the difference in washed and unwashed scaffolds. A) PCL unwashed, B) PCL unwashed, C) PLGA washed and D) PCL washed. (Scale bar 100m)

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24 Fibrous Phase Separated Scaffold General observations After fabrication, the PCL salt-leached scaffolds were weighed and observed for general handling properties. The weights of the scaffolds layers were 136 8.56mg and demonstrated an elastic quality when handled in the lab. Once the layers were bonded together, the final scaffolds weight was 661 8.55mg. Also the scaffold became more rigid after the final bonding of the layers was complete, but still had elastic handling qualities. The scaffolds final dimensions were 50 3mm in diameter with a thickness of 4 2mm. During literature searches, nothing was found on using a PCL/acetone phase separation system to create multi-layered cellular scaffolds. FESEM The scaffolds were characterized using FESEM to compare morphologies of the phase separation scaffolds. Images were taken of both layers and completed scaffolds to present the microstructure. The results show an interconnected porous scaffold as seen in Figure 4-2. As stated earlier, it should be noted that the salt-leaching can create pockets in the scaffolds, as where this novel procedure has a more open morphology. A comparison image is shown in Figure 4-3 of the morphologies for both methods of fabrication. It is important that the scaffold has an interconnected porous structure, which helps provide for maximizing cell seeding, attachment, and growth [9,10]. Therefore this method may be more desirable for mES cell studies when compared to the salt-leaching method.

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25 A B Figure 4-2. FESEM images of scaffold. A) 100x and B) 2500x. (Scale marker bar 100m and 10m respectively) A B Figure 4-3. FESEM images comparing the salt-leached and fibrous phase separated scaffold morphologies. (Scale marker bar 100m) Coatings Figure 4-4 displays the effect of coatings on the fibrous phase separated PCL coated scaffolds. The Matrigel coated scaffold had a much rougher, bumpy surface after it had been dried and prepared for FESEM analysis. This is probably a result of fact that the Matrigel coating is much more viscous and globular then the gelatin coating. The gelatin coating seen in Figure 4-4 (B) looks more like thin sheet coating over the scaffold. It was hypothesized that the morphologies of these coatings may affect the cells morphology on the coating surface. However, the true morphologies of these coatings

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26 cannot be represented using FESEM imagining and is believed that the coatings would be hydrated and swollen in solution. A B Figure 4-4. FESEM images of Matrigel and gelatin coated PCL scaffolds. A) Matrigel and B) gelatin. In vitro Degradation Study During the nine week in vitro degradation study, general visible observations were conducted on both the PCL and PLGA salt-leached scaffolds. For both of the polymers studied, their respective groups were extracted from the study at the following designated times: 12 hours, day 2, week 1, day 10, week 2, week 3, week 4, week 5, week 6, week 7, week 8, and week 9. Upcoming sections will discuss mass, molecular weight, pH, and morphology analysis for both PLGA and PCL scaffolds for each extraction date. General Observations General, visible observations were recorded on the scaffolds degradation, which are displayed in Table 4-1 and Table 4-2. Each week the samples were inspected for any obvious changes in the scaffolds in the PBS (pH 7.4) solution during the experiment.

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27 Table 4-1. General observations of PLGA scaffolds in PBS. General Observations during degradation Week 1 No visible change Week 2 Light particulates Week 3 Light particulates Week 4 Medium/light particulates Scaffolds have become softer and more delicate to handle Week 5 Medium/light particulates Extracted samples stuck to drying plate Week 6 Light particulates Scaffolds are starting to conglomerate Week 7 Light/no particulates Scaffolds are conglomerating Week 8 Light/no particulates Scaffolds are conglomerated After being dry, scaffolds turned brown Week 9 Scaffolds have conglomerated into a shiny, grey clump Table 4-2. General observations of PCL scaffolds in PBS. General Observations during degradation Week 1 No visible change Week 2 No visible change Week 3 No visible change Week 4 Very fine particulates Scaffolds seem to be flattening out Week 5 Light particulates Porosity is becoming to increase Week 6 Light particulates Porosity slightly increases Week 7 Fine, light particulates Week 8 Fine, light particulates Week 9 Fine, light particulates

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28 To the visible eye, the PCL scaffolds preserved their shape and morphology when compared to the PLGA scaffolds. The PLGA scaffolds seemed to lose its shape by week 3 and by week 5 had lost its scaffold function. The PLGA scaffolds also seemed to give off particulates at an earlier stage of the degradation study. The PCL scaffolds also did not conglomerate in later stages of the study like the PLGA scaffolds. Finally, no visible color change occurred in the PCL scaffolds during the study, unlike the grey/brown color the observed for the PLGA scaffolds. Mass Analysis Throughout the nine week in vitro degradation study, the average percent mass loss was recorded for both the PLGA and PCL salt-leached scaffolds. Each group of scaffolds were extracted and evaluated from the study at their designated point in time. Figures 4-5 and 4-6 display the results of the average percent mass remaining at extraction, during the in vitro degradation study for both PLGA and PCL respectively. Trendlines were applied to help evaluate the data. Discussion. As noted above, the PLGA scaffolds loss a significant amount of mass during the study when compared to their PCL counterpart. The PLGA scaffolds mass started to drop drastically after day 28 and continued to plummet after day 49. However, the PCL scaffolds kept a slightly consistent linear slope throughout the nine week study. As noted earlier in Table 4-1, PLGA scaffolds on week 5 stuck to the drying plate and broke into pieces when trying to remove for weighing, causing the discrepancy in Figure 4-5. After this incident, scaffolds were dried on a Teflon plate to avoid the mishap again. The large error bars after day 49 on Figure 4-5, are because the PLGA scaffolds had conglomerated together distorting the scaffolds weights.

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29 0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00100.00010203040506070Degradation time (days)Average % Mass remaining Figure 4-5. PLGA average percent mass loss during degradation study. (Polynomial trendline) 70.0075.0080.0085.0090.0095.00100.00010203040506070Degradation time (days)Average % Mass remaining Figure 4-6. PCL average percent mass loss during degradation study. (Polynomial trendline)

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30 Molecular Weight (MW) Analysis For the duration of the nine week in vitro degradation study, the molecular weight was analyzed for both the PLGA and PCL salt-leached scaffolds. Each group of scaffolds were extracted and evaluated from the study at their designated point in time. Figures 4-7 and 4-8 display the results of the molecular weight change during the in vitro degradation study for both PLGA and PCL respectively. Trendlines were applied to help evaluate the data. Discussion. Figures 4-7 and 4-8 display the obvious dissimilarities in the two polymers MW loss. The PLGA scaffolds exponentially decreased in MW during the in vitro degradation study, where again the PCL scaffolds had a slight linear slope. By day 28, the PLGA scaffolds had lost nearly 87% of their original MW; where as the PCL scaffolds had retained almost 98% of their original MW. However, from day 28 till the end of the study, the MW loss of the PGLA scaffolds leveled out. y = 77301e-0.0565x0100002000030000400005000060000700008000090000010203040506070Degradation time (days)Molecular Weight (Mw) Figure 4-7. PLGA molecular weight change during degradation study. (Exponential trendline)

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31 100000105000110000115000120000125000010203040506070Degradation time (days)Molecular Weight (Mw) Figure 4-8. PCL molecular weight change during degradation study. (Polynomial trendline) pH Analysis During the in vitro degradation study, the buffers pH was evaluated from both the PLGA and PCL salt-leached scaffolds. At each designated PBS exchange time point, each vial was analyzed using a pH meter. Figures 4-9 and 4-10 display the results of the PBS solutions pH change during the in vitro degradation study for both PLGA and PCL respectively. Trendlines were applied to help evaluate the data. Discussion. As Figure 4-9 notes, the pH trendline severely dives to very acidic values after day 14, implying that the PLGA scaffolds are beginning to degrade. By day 63, the pH had risen slightly, indicating that the polymers degradation rate had tapered off [33]. Figure 4-10 illustrates that the PCL scaffolds buffer solution hardly shifted in pH during the nine week study, with no significant drop off.

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32 4.404.705.005.305.605.906.206.506.807.107.40010203040506070Degradation time (days)pH Figure 4-9. Graph presenting pH change of PBS for PLGA scaffolds during degradation study. (Polynomial trendline) 6.406.606.807.007.207.40010203040506070Degradation time (days)pH Figure 4-10. Graph presenting pH change of PBS for PCL scaffolds during degradation study. (Polynomial trendline)

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33 FESEM (Week 1,3,5,7,9) Throughout the study, the scaffolds morphology was analyzed by SEM for both the PLGA and PCL salt-leached scaffolds. Each group of scaffolds were extracted from solution and evaluated from the study at their designated point in time. Figures 4-11 and 4-12 display the morphology change of the scaffolds during the in vitro degradation study for both PLGA and PCL respectively. PLGA During the degradation study, the PLGA scaffolds underwent vast morphological changes as seen in Figure 4-11. From week 1 to week 3, the porosity of the scaffold seemed to increase slightly during the study. However, by week 5 the scaffolds had lost all of their functional scaffolding properties needed for tissue engineering, as described earlier in the background. The scaffolds were no longer porous and had developed into a rough surface mass. By week 9, the scaffolds had greatly degraded and all that was left was a smooth mass. PCL The PCL scaffolds morphology varied little during the in vitro degradation study. However it was noted that the visible porosity of the scaffolds did increase after week 6. After the nine week study, the PCL scaffolds still withheld their vital scaffold properties of porosity and interconnected pores, needed for tissue engineering purposes. In fact, the PCL scaffolds could be considered a more likely candidate after the study than before, since the scaffolds seemed to become more porous after some degradation. These results can be seen below in Figure 4-12.

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34 A B C D E Figure 4-11. FESEM images of degrading PLGA scaffolds at. A) Week 1, B) Week 3, C) Week 5, D) Week 7 and E) Week 9. (Scale marker bar 100m)

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35 A B C D E Figure 4-12. FESEM images of degrading PCL scaffolds at. A) Week 1, B) Week 3, C) Week 5, D) Week 7 and E) Week 9. (Scale marker bar 100m) In vitro mES Cell Studies Once scaffold fabrication was complete, the scaffolds were evaluated for in vitro mES cell differentiation. Dr. Naohiro Terada and Dr. Takashi Hamazaki, from the Department of Pathology, Immunology and Laboratory Medicine at the University of Florida, greatly assisted in the cellular studies. Both coated and uncoated PCL salt-leached and fibrous phase separated scaffolds were used in the studies. The interests of these studies were to

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36 evaluate the difference between the salt-leached and the novel, fibrous phase-separation procedures, while also considering the various coating materials. PCL Salt-leached Scaffolds (Study 1) The scaffolds were prepared and examined to determine the cells growth, survival and differentiation state. Under fluorescent microscopy, Afp-GFP/mES cells express a green fluorescent protein once the cells have differentiated toward endodermal cells. Figure 4-13 illustrates the differences in how the Afp/GFP mES cells interacted with the uncoated and coated scaffolds. Day 6. Figures 4-13 [A-B] are representative areas showing that some of the mES cells had aggregated forming embryoid bodies. The outer layer of the embryoid bodies had also differentiated into endodermal cells shown by the GFP expression marker. After looking at the uncoated scaffolds, it seems the mES had not attached to the scaffold and are aggregating without interacting with the scaffold. Figures 4-13 [C-D] are representative areas demonstrating how some of the cells have attached to the Matrigel coated scaffold and differentiated into endodermal cells. However, it was noted that the cellular attachment was limited by the scaffolds morphology. The cells seemed to exist only in pockets of the scaffold and were not found through the entire structure. A few embryoid bodies were also found with the Matrigel coated scaffolds. These embryoid bodies are believed not to have attached to the scaffold.

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37 A B C D Figure 4-13. Optical and fluorescent microscopy images of PCL salt-leached scaffold on day 6. A) Optical image of uncoated PCL, B) GFP image of uncoated PCL, C) optical image of Matrigel coated PCL and D) GFP image of Matrigel coated PCL. (Scale marker bar 200m) PCL Fibrous Phase Separated Scaffold (Study 2) Fluorescent microscopy The scaffolds were prepared and examined to determine cellular growth, survival and differentiation fate. As noted before, Afp-GFP mES cells express green fluorescent protein when the cells differentiate toward endodermal cells. Hoechst 33342, a blue fluorescent dye specifically staining cell nuclei (both dead and alive), and PI, a red fluorescent dye staining only dead cell nuclei, where used to help illuminate the cellular results. Figures 4-14 and 4-15 illustrate representative areas of the mES cell-scaffold interactions with the various scaffold and coating types. Day 9. After observing the scaffolds with an optical/fluorescent microscope, cells were mainly located on corners of the scaffolds. Figure 4-14 displays representative

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38 areas of were cells were found on each specific scaffold coating. The uncoated scaffolds exhibited unattached embryoid bodies with the outer layers differentiating to endodermal cells. Some cells attached and had also differentiated into endoderm. By comparing the nuclei stains, most cells seemed dead on the scaffold. The gelatin coated scaffolds formed did not seem to form embryoid bodies or differentiated into endodermal cells. However, the cells that did attach seemed alive and spreading onto the scaffold. The Matrigel coated scaffolds showed little evidence of embryoid bodies forming in the fluorescent microscopy. The mES cells seemed to have attached, spread and differentiated endodermally in locations in the scaffolds interconnected structure. After monitoring the Matrigel coated scaffolds, cells seemed to be alive rather than dead. Day 24. At Day 24, mES cells were discovered throughout the scaffolds and representative areas were selected for fluorescent microscopy seen in Figure 4-15. The uncoated PCL scaffolds had some cellular attachment and differentiation into endoderm. Some embryoid bodies were also visible in the culture well. The gelatin coated scaffold displayed both cellular attachment and spreading. Some differentiations into endodermal cells were found, along with some embryoid bodies. Also, few dead cells were spotted using PI and fluorescent microscopy. The Matrigel coating also presented cellular attachment and spreading. Some differentiations into endoderm were spotted throughout the scaffold. In both the Matrigel and gelatin coated scaffolds, cells were located on multiple layers of the scaffold as observed in Figure 4-16.

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39 Figure 4-14. Optical and fluorescent microscopy images on Day 9 of PCL phase separated mES cell study.

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40 Figure 4-15. Optical and fluorescent microscopy images on Day 24 of PCL phase separated mES cell study.

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41 A B Figure 4-16. Fluorescent microscopy showing cells on multiple layers of the scaffold. (Scale marker bar 200m) Flow cytometry Figure 4-17 illustrates differences between the uncoated scaffold and the coated scaffold. The uncoated scaffold had a lower individual cell size and few GFP positive cells. Both Matrigel and gelatin coated scaffold had a higher population of individual cells with larger cell sizes and coarser nuclear configurations then uncoated scaffolds. Figure 4-17. Flow cytometry analysis at Day 24 of PCL phase separated mES cell study. FESEM Once the scaffolds had been fixed and dehydrated they were prepared for SEM analysis as listed earlier. The Matrigel coated scaffolds had cellular attachment throughout the samples and the cells were mainly found clustered together. It also appears that the cytoplasm had collapsed in towards the nucleus as seen in Figure 4

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42 18(B). This effect is thought to be caused by how the cells adhered to the viscous Matrigel. As the cells grasped the scaffolds structure, they clustered together (Figure 4-18(A)) because of the hyrdogel properties of the coating. A B Figure 4-18. FESEM images of cellular attachment on Matrigel coated PCL scaffold. A) 2500x and B) 7000x. (Scale marker bar 10m and 1m respectively). Figure 4-19 displays a representative area of how the mES cells attached to the gelatin coated PCL scaffolds. Once again the nucleus and cytoplasm are visibly seen spread out on the surface of the scaffold, unlike the clusters formed with Matrigel (Figure 4-19 (A)). However, the cells did not collapse like the Matrigel coated scaffolds. The gelatin coating was more of a thin sheet coating the scaffold and allowed the cells to adhere and spread out instead of clustering. A B Figure 4-19. FESEM images of cellular attachment on gelatin coated PCL scaffolds. A) 2500x and B) 7000x. (Scale marker bar 10m and 1m respectively).

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43 Reverse transcriptase-polymerase chain reaction (RT-PCR) As seen in Figure 4-20, expression of various differentiation protein markers were recorded after Day 24. These markers help determine the differentiation of the cells and their possible final cell line. The housekeeping gene, -actin, is used to prove that cellular life exists and that the scaffolds did not create a toxic environment for the cells [7,8]. Other forced expressions of transcription factors that were chosen to look for were specific linage differentiation markers, such as GATA6, hepatocyte nuclear (HNF4), and transthyretin (TTR) for primitive endoderm [7,8]. The expression of Albumin (ALB) can imply the cells are becoming hepatic or visceral endoderm differentiated [7,8]. Also, Nestin proteins were also examined as markers for neural differentiation. After observing the scaffolds, differences were observed between protein markers expressed in the RT-PCR data. -actin was expressed for all the scaffolds proving that cellular adhesion existed on all the scaffolds. The gelatin coated scaffolds proved to be the most expressed scaffold of differentiation markers for hepatic/visceral endoderm differentiation showing GATA6, HNF4, TTR, and ALB. Therefore gelatin coating scaffolds seem to have the best possibility to further the ES cells to differentiate into a hepatocyte cell linage. However, Matrigel coated scaffolds showed some evidence of possible future neural differentiation with the expression of Nestin. The Matrigel coated scaffolds also have a slight possibility to differentiate into an early hepatocyte/visceral endoderm cell. The uncoated scaffolds showed no real assistance to help further the mES cell populations into the desired cell lines according to this research (i.e. liver or nerve).

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44 Figure 4-20. RT-PCR analysis at Day 24 of PCL phase separated mES cell study.

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CHAPTER 5 CONCLUSIONS Scaffold Fabrication and Characterization In this study, PCL was initially investigated for biodegradable embryonic stem cell scaffold research. Two methods of fabrication were considered for this research: salt-leaching and phase separation. When comparing PLGA to PCL, the PLGA scaffolds were very brittle, had poor handling properties and were generally hard to manage. The PCL scaffolds were more elastic and their structure was more durable while handling them. When comparing the two methods of fabrication, many faults were found in the salt-leaching procedure. Pockets seemed to form where the salt had leached out, instead of the interconnected morphology desired for tissue engineering. The pocket morphology was hypothesized to possibly limit the cells growth and proliferation throughout the scaffold. However, the fibrous phase separated scaffolds had a porous, interconnected morphology observed using FESEM. These scaffolds are believed to help contribute to future ES cell studies. In vitro Degradation Study The in vitro degradation study confirmed what was already known, that PCL has a slower degradation rate than PLGA. The PCL scaffolds withheld their morphology for the entire nine week study, as where PLGA scaffolds had lost their scaffold functionality by week 5. However, at the end of the study PCL scaffolds had become more porous, offering the idea that preliminary PBS bathing may help the scaffold become a more productive scaffold. 45

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46 In vitro mES Cell Studies Study 1 The Matrigel coated scaffold showed attachment, growth, endoderm differentiation, and limited embryoid bodies when compared to the uncoated scaffold. The above observations the Matrigel coating influences if the cells attach and spread on to the scaffold. It was also found that the pocket morphology created by the salt leaching did limit the cells growth and proliferation throughout the scaffold. The cells stayed almost entirely on the outside surface of the scaffolds and were unidentified in the core of the scaffold. Study 2 Study 2 confirmed that a coating is necessary for positive cellular attachment to PCL scaffolds. At Day 9, the Matrigel coated scaffold seemed to have a slight advantage over the gelatin coating in cellular attachment and had more endoderm differentiation present. By Day 24, the Matrigel and gelatin coatings seemed comparable when comparing representative fluorescent microscopy images and flow cytometry data. However the expression of differentiation markers in the RT-PCR data displayed dissimilarities in how coating materials affected the cells differentiation. The Matrigel coated scaffold had an expression of albumin and other visceral endoderm/early hepatic markers (GATA6, HNF4, TTR). On the other hand the gelatin coated scaffold had an expression of Nestin and a slight expression of albumin, along with other visceral endoderm/early hepatic markers. Final Thought This studys goal was to fabricate a porous PCL scaffold for mES cell study. This research demonstrates that scaffold morphology and various types of coatings can play a

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47 vital role in how mESs adhere and differentiate with the coating/scaffold. It was found that an open, interconnected biodegradable scaffold with an extracellular matrix dip coating created an environment to promote cellular attachment, proliferation, and differentiation throughout the scaffold. In addition, FESEM, flow cytometry, and RT-PCR data illustrate that different coatings affect mES behavior and scaffold interaction. Future research should be investigated on these topics to fully understand the role of scaffold coating to steer mES toward a desired cell differentiation.

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49 10. Lu L, Peter S, Lyman M, Lai H, Leite S, Tamada J, Uyama S, Vacanti J, Langer R, Mikos A, In Vitro and In Vivo Degradation of Porous Poly(DL-Lactic-co-Glycolic Acid) Foams Biomaterials, 2000. 21 (18): p. 1837-1845. 11. Holy C, Dang S, Davies J, Shoichet M, In Vitro Degradation of a Novel Poly(Lactide-co-Glycolide) 75/25 Foam Biomaterials, 1999. 20 (13): p. 1177-1185. 12. Ma P, Choi J, Biodegradable Polymer Scaffolds w ith Well-Defined Interconnected Spherical Pore Network Tissue Engineering, 2001. 7 (1): p. 23-33. 13. Levenberg S, Huang N, Lavik E, Roge rs A, Itskovitz-Eldor J, Langer R, Differentiation of Human Embryonic Stem Cells on Three-dimensional Polymer Scaffolds Proceedings of the National Academy of Sciences of the USA, 2003. 100 (22): p. 12741-12746. 14. Liao C, Chen C, Chen J, Chiang S, Lin Y, Chang K, Fabrication of Porous Biodegradable Polymer Scaffolds Using a Solvent Merging/Particulate Leaching Method Journal of Biomedical Materials Research, 2002. 59 (4): p. 676-681. 15. Hutmacher D, Scaffold Design and Fabrication Technologies for Engineering Tissues-state of the Art and Future Perspectives Journal of Biomaterials Science Polymer Edition, 2001. 12 (1): p. 107-124. 16. Li W, Laurencin C, Caterson E, Tuan R, Ko F, Electrospun Nanofibrous Structure: A Novel Scaffold for Tissue Engineering Journal of Biomedical Materials Research, 2002. 60 (4): p. 613-621. 17. Jalil R, Nixon JR, Biodegradable Poly(Lactic Acid) and Poly(Lactide-coGlycolide) Microcapsules: Problems Asso ciated with Prepar ative Techniques and Release Properties Journal of Microencapsulation, 1990. 7 (3): p. 297-325. 18. Wu X, Synthesis and Properties of Biodegradabl e Lactic/Glycolic Acid Polymers In: Wise et al., editors. Encycl opedic Handbook of Biomaterials and Bioengineering. New York: Marcel Dekker, 1995. p. 1015-54. 19. Lewis D, Controlled Release of Bioactive Ag ents from Lactide/Glycolide Polymers In: Chasin M, Langer R, editors. Biodegradable Polymers as Drug Delivery Systems. New York: Marcel Dekker, 1990. p. 1-41. 20. Tice T, Biodegradable Controlled-rel ease Parenteral Systems Pharmaceutical Technology, 1984. 11 : p. 26-35. 21. Gilding D, Reed A, Biodegradable Polymers for use in SurgeryPoly(Glycolic)/Poly(Lactic Acid) Homo and Copolymers Polymer, 1979. 20 (12): p. 1459-1464. 22. Jamshidi K, Hyon S, Ikada Y, Thermal Characterization of Polylactides Polymer, 1988. 29 (12): p. 2229-2234.

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50 23. Raghuvanshi R, Singh M, Talwar G, Biodegradable Delivery System for Single Step Immunization with Tetanus Toxoid. International Journal of Pharmaceutics, 1993. 93(1-3): p. R1-R5. 24. Armani D, Liu C, Microfabrication Technology for Polycaprolactone, Biodegradable Polymer. Journal of Micromechanics and Microengineering, 2000. 10(1): p. 80-84. 25. Kweon H, Yoo M, Park I, Kim T, Lee H, Lee H, Oh J, Akaike T, Cho C, A Novel Degradable Polycaprolactone Networks for Tissue Engineering. Biomaterials, 2003. 24(5): p. 801-808. 26. Coombes A, Rizzi S, Williamson M, Barralet J, Downes S, Wallace W, Precipitation Casting of Polycaprolactone for Applications in Tissue Engineering and Drug Delivery. Biomaterials, 2004. 25(2): p. 315-325. 27. Williamson M, Coombes A, Gravity Spinning of Polycaprolactone Fibres for Applications in Tissue Engineering. Biomaterials, 2004. 25(3): p. 459-465. 28. University of Minnesota, Bioabsorbable Polymers, 1998, http://www.courses.ahc. umn.edu/medical-school/BMEn/5001/notes/bioabs.html, 3/1/2005. 29. Shimao M, Biodegradation of Plastics. Current Opinion in Biotechnology, 2001. 12(3): p. 242-247. 30. Kleinman H, McGarvey M, Liotta L, Robey P, Tryggvason K, Martin G, Isolation and Characterization of Type IV Procollagen, Laminin, and Heparan Sulfate Proteoglycan from the EHS Sarcoma. Biochemistry, 1982. 21(24): p. 6188-6193. 31. McGuire P, Seeds N, The Interaction of Plasminogen Activator with a Reconstituted Basement Membrane Matrix and Extracellular Macromolecules Produced by Cultured Epithelial Cells. Journal of Cellular Biochemistry, 1989. 40(2): p. 215-227. 32. Universiteit Utrecht/Cell Biology, Standard Glutaraldehyde Fixation for SEM of Tissue Fixative, 2000, http://www.bio.uu.nl/mcb/EMSA/protocols/glutafix.htm, 3/1/2005. 33. Lu L, Garcia C, Mikos A, In Vitro Degradation of Thin Poly(DL-Lactic-co-Glycolic Acid) Films. Journal of Biomedical Materials Research, 1999. 46(2): p. 236-244.

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BIOGRAPHICAL SKETCH Michael Howell Tollon was born in Clearwater, FL, on March 2, 1980. He grew up in Largo, FL, and attended Seminole High School. Michael completed his bachelors in materials science and engineering in August 2002 at the University of Florida. After graduation he plans on seeking employment in the state of Florida. 51