INVESTIGATING THE COMPATIBILITY OF a PROTOTYPE SCAFFOLD IN a NOVEL NEAR CELLSHEET APPROACH

LIST OF FIGURES Figure 1 UNOS organ transplant statistics for 1990 to 1999 documenting the wait-listed patients O and transplants ● [Drury JL, 2003] 18 Figure 2 Biaxially stretched PCL f

ENGINEERING PCL-BASED NANO-MATERIAL

FOR TISSUE REPAIR

ALEX FOO HSIEN LOONG B.Eng (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

NUS FACULTY OF MEDICINE GRADUATE PROGRAM IN BIOENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2009

ACKNOWLEDGEMENTS

I am greatly indebted to the following people in the course of this project

I will like to thank my thesis supervisors Prof Teoh Swee Hin and Prof Hanry Yu for their guidance in this project

I will also like to thank my project collaborator Dr Lou Yan-Ru for her generous support in hMSC culture, in particularly her patience and clear advice with various molecular biotechnologies such as RT-PCR and Western Blotting

I will like to thank Dr Jeremy Teo for passing down his extensive knowledge in microCT imaging and his gallant help to analyze the results I will also like to specially acknowledge my most under-appreciated cinematographer Ms Ong Siew Min for her assistance during video filming

I will like to thank the following people for their friendship during my stay: Mr Talha Arooz, Dr Suzanne Ng San San, Dr Khong Yuet Mei, Mr Lim Teck Chuan, Dr Edwin Chow, Dr Kan Shyi-Herng, Dr Amy Chou and Dr Toh Xue Li

I will like to thank Prof Jackie Ying, Ms Noreena AbuBakar, IBN administrative, safety and facilities department for their support in providing me a pleasant environment to work in

I will like to acknowledge the research funding from IBN, BMRC, A-Star, as well as the generous scholarship from NUS

I will like to thank my family for being understanding, patient and supportive of my own selfish pursuits I will also like to humbly dedicate this thesis to my niece Sydney Leong and hope that this will inspire her in her future endeavors when she grows up

Last but not least, I will like to thank God for His unfailing love during this journey of faith

TABLE OF CONTENTS

ACKNOWLEDGEMENTS - 2

SUMMARY - 6

LIST OF PUBLICATIONS - 7

LIST OF TABLES - 8

LIST OF FIGURES - 9

LIST OF VIDEOS - 12

LIST OF SYMBOLS AND ABBREVIATIONS - 13

I INTRODUCTION - 17

II LITERATURE REVIEW - 22

2.1 The use of scaffolds in TE - 22

2.2 Nanofibers - 24

2.2.1 Non-conventional fabrication approach - 25

2.2.2 Polyelectrolyte complexation - 26

2.2.3 Self-assembly - 28

2.2.4 Electrospinning - 32

III OBJECTIVES - 40

IV MATERIALS AND METHODS - 43

4.1 Fabrication of FSS - 43

4.2 Characterization of FSS - 44

4.3 Non-invasive surface modification of FSS - 47

4.4 Cell culture - 49

4.5 Biological characterizations - 50

4.6 Specific tests for primary hepatocytes - 51

4.7 Specific tests for osteoblastic hMSC - 52

4.8 Statistical methods - 55

V RESULTS - 56

5.1 Specific Aim 1 - 56

5.2 Specific Aim 2 - 76

5.3 Specific Aim 3 - 89

VI DISCUSSION - 110

VII CONCLUSIONS - 117

VIII REFERENCES - 118

SUMMARY

Conventional approach to TE commonly involves the use of synthetic scaffoldings that often lack the nano-scale features typically found in the native environment Such dissimilarity has been speculated to be one of the instrumental factors contributing to the poor maintenance of the cells under in vitro conditions An alternative solution is

to culture them on fibrillar surfaces generated rapidly from emerging technologies such as electrospinning Our group has come up with a new PCL-based nano-material design (FSS) that can ensure the structural stability of the otherwise fragile substrate irregardless of the pore dimensions A highly permeable material with ease of handling can therefore be fabricated and its potential use as a sandwiching or stacking membrane was examined Morphological and functionality studies on overlaid primary hepatocytes revealed a 3D-like cytoskeletal structure and improved urea secretion A more extensive bone differentiation profile was also observed in hMSC when individual pre-seeded low density FSS were stacked together prior to their exposure to osteogenic medium Preliminary results also identified physical communications to play a significant role in priming the cells to be more receptive to external signaling cues Such benefits in biological responses were however not extended to hepatic layers probably due to an inadequate cell density used during the test We propose here an alternative material that can be stacked up to better mimic the stratified architecture of the innate surrounding

LIST OF PUBLICATIONS

(1) Foo HL, Taniguchi A, Yu H, Okano T, Teoh SH, Catalytic Surface

Modification of Rolled-Milled Poly (ε-Caprolactone) Biaxially Stretched to Ultra-Thin Dimension Materials Science & Engineering C – Biomimetic and Supramolecular Systems, 2007 27 (2): p 299 – 303

(2) Ong SM, Zhang C, Toh YC, Kim SH, Foo HL, Tan CH, van Noort D, Park S,

Yu H, A Gel-Free 3D Microfluidic Cell Culture System Biomaterials, 29 (22):

p 3237 – 3244

LIST OF PATENTS

(1) Foo A, Yu H, Bioactive Scaffold With Controllable Mass Transfer Properties

Independent of Mechanical Strength Provisional Patent (25 June 2008)

LIST OF TABLES

Table 2 Desirable features in an ideal scaffold [Murugan R, 2007] 24

Table 3 Mechanical properties of thin films from various processes [Tiaw

KS, 2007]

58

Table 5 Comparing the fluorescence density between the top and bottom

layer

64

Table 6 Comparing XPS stoichometric ratios between various surfaces 71

Table 7 Comparing XPS stoichometric ratios between surfaces anchored or

grafted with collagen

72

Table 8 Comparing the water permeability between different substrates 80

Table 9 Urea response towards change in fiber density and overlay time 87

Table 10 Expression of bone-specific genes with respect to stacking 96

Table 11 Gene and protein expression with respect to overlay time 99

LIST OF FIGURES

Figure 1 UNOS organ transplant statistics for 1990 to 1999 documenting

the wait-listed patients (O) and transplants (●) [Drury JL, 2003]

18

Figure 2 Biaxially stretched PCL film and its properties 58

Figure 6 Non-invasive surface modification of FSS via collagen gelation

Figure 10 Comparing between surfaces previously anchored or grafted

with collagen

72

Figure 11 Investigating cell behavior on modified high density FSS 73

Figure 12 Investigating cell behavior on modified low density FSS 75

Figure 13 Benefits of membrane-based sandwich model in liver TE 78

Figure 15 Effect of FSS on viability of primary hepatocytes during overlay 81

Figure 16 Investigating the benefits of sandwiching primary hepatocytes

with FSS

83

Figure 17 Optimization of sandwiched culture with FSS 85

Figure 18 Increasing the transplanted cell number per unit area 91

Figure 19 Improvements in osteogenic responses of hMSC during stacking 94

Figure 20 Examining the effect of fiber density on stacking consequences

Figure 23 Comparing the gene expression of hMSC post-exposed to

vitamin D3-supplemented osteogenic medium in dense and

dispersed culture

103

Figure 24 Total and normalized functions of primary hepatocytes in a

non-stacked and non-stacked configuration

106

Figure 25 Investigating the effect of fiber density on primary hepatocyte

during stacking

107

Figure 26 Investigating the presence and importance of physical contacts

in stacking model of primary hepatocytes

108

Figure 27 Investigating the reason for stacked primary hepatocytes to

exhibit inferior functions on low density FSS in spite of the

presence of physical contacts

108

Figure 28 Schematic illustration of Wnt pathway and the associated

molecules (Fretz JA, 2007)

116

LIST OF VIDEOS

Video 1 Ease of handling a physiologically wetted fibrous mesh with FSS

Video 2 Ease of harvesting electrospun fibers from collector with FSS

Video 3 Two stacked layers remained strongly intact in spite of vigorous

swirling, indicating the tight interactions between them

LIST OF SYMBOLS AND ABBREVIATIONS

“So the Lord God caused the man to fall into a deep sleep; and while he was sleeping,

He took one of the man’s ribs and closed up the place with flesh Then the Lord God made a woman from the rib He had taken out of the man”

– Genesis 2: 21 – 22 (NIV) [The first written account of TE]

I INTRODUCTION

Organ failure is a major health problem in the United States that accounts for about 8 million surgical procedures (Murugan R, 2007) and $400 billion dollars or 50 % of the total annual health care expenditure (Nature Biotechnology Review, 2000) Conventional treatment involving the use of medical devices was often found to be inadequate for complete tissue recovery as a result of several shortcomings These include a shortened shelf-life due to mechanical wear or secondary inflammatory responses (Hutmacher DW, 2006) Capsular contracture of breast implants, insulating barriers around electrodes and scarring of heart valve sewing rings are just some of the activities that had reportedly led to implant failure As a result, not even durable items like hip or knee prostheses are able to last beyond 15 years There are also methods such as radical cystectomy that attempt to substitute the malfunctioning organ (bladder) with a similar analog (intestinal tissue) in the host However, post-surgery side effects were often encountered due to slight functional differences in the replacement (McCook A, 2007) Not only were the patients at risk of developing stones, as many as 50 % of them were inflicted with metabolic acidosis due to fluid absorption by the graft They were further burdened with the daunting cost of $70,

000 even without factoring the cost of the complications as described above

While there are more effective options in allogeneic transplants, severe shortage of donor organs has greatly limited the number of lives that can be saved For example, only 23, 407 out of 103, 309 people received transplants from 2000 – 2001 (http://ustransplant.org) A mere 14 % heart transplants, each allegedly costing $500,

000, were performed on Americans aged 55 or younger in 2001 (Moreno-Borchart A, 2004; McCook A, 2007) Approximately 15 % of the remaining candidates did not

survive the wait (Stock UA, 2001) whereas those who did receive the new lease of life are dependent on immunosuppressive agents for the rest of their lives A worrying trend can further be detected in the following figure (Drury JL, 2003) Despite a tremendous expansion in the waiting list from 1990 to 2000, the number of transplants had however remained more or less constant This gap, in light of an ageing population and rising medical costs, is only expected to widen in the future It is therefore imperative to propose another strategy in order to address the shortfall in time

Figure 1: UNOS organ transplant statistics for 1990 to 1999 documenting the listed patients (O) and transplants (●) [Drury JL, 2003]

wait-An alternative answer may very well be provided in a multidisciplinary field also known as TE where various aspects of medicine, materials science, engineering and biology are combined to help persuade the body to regenerate itself through the use of cells, supporting structure and biomolecules Ironically, its humble beginning was traced back not to any scientific journals, but rather a famous “Healing of Justinian”

portrait by Italian renaissance painter Fra Angelica (1400 – 1455) where two brothers Saints Damien and Cosmos were drawn in the process of transplanting a homograft limb onto a wounded soldier (Vacanti CA, 2006) It was not until centuries later in the

1970 - 80s before a landmark study by John Burke and Iannas Yannos was carried out

to orchestrate regeneration of a full-thickness dermal wound via the use of dermal fibroblasts in collagen matrix Their work, representing one of the earliest attempts to illustrate the influence of exogenous materials on tissue healing, further went on to generate currently the most successful tissue engineered product: the Integra dermal regeneration template (Shastri VP, 2006) They further inspired many other similar projects involving mostly the use of naturally occurring and therefore easily available polymers as containing vessels for the cells While the results were encouraging, unpredictable outcomes due to poor control of physical and chemical properties of the scaffolds were often the bottleneck, and it was only in the mid-1980s before another broad definitive step was taken, this time by Joseph Vacanti and Robert Langer who spearheaded the vision to use intelligently designed synthetic materials instead for cell delivery (Vacanti CA, 2006) This in turn paved the way for the emerging field to be formally recognized at the National Science Foundation in 1987 where its name was first coined (Nerem RM, 2006) TE was however only catapulted into public awareness with the broadcast of the infamous “mouse with the human ear” model by Charles Vacanti at University of Massachusetts Medical Center (Stock UA, 2001) In fact, this imagery had became such an icon that it was even picked up by a television network to be incorporated into one episode of popular Crime Scene Investigation series Despite several criticisms from animal welfare organizations, the vast potential

of TE was finally presented to the world In 1991, a young patient with Poland’s syndrome became the first human to receive a tissue engineered implant (Vacanti CA,

2006) This was followed closely by another major milestone in 1997 when the first manufactured living human skin equivalent complete with a keratinized epidermis and dermis was made commercially available by Novartis Pharmaceuticals under the name Apligraf (Nature biotechnology review, 2000) Research and development expenditure was even expected to reach approximately 22 % annual growth rate, with that year alone sky-rocketing to half a billion dollars (Nature biotechnology review, 2000)

Despite the hype, successful commercial products have so far been limited The following table gives a grim overview of current status in these engineered organs (Stock UA, 2001) Few have progressed beyond in-vivo studies, and those that are in clinical trials are mostly cartilage or skin substitutes where the demands for oxygen and nutrients are less stringent, the functions they are required to perform less complicated and critical, and the number of cell types involved in their regeneration fewer (Moreno-Borchart A, 2004; Bock AK, 2003) Instead of the $100 billion industry speculated by Barron (Palmer J, 2000), the total estimated sales in 2004 amounted to no more than 100 million with less than 100 companies and many more that went out of business or file for bankruptcy (Nerem RM, 2006) The industry had apparently rapidly declined to its pre-hype 90’s level with only approximately 2500 employees In short, TE has yet to live up to the potentials it communicated to the world 10 years ago It has remained for most part a laboratory science that merely holds an empty promise to revolutionize medicine There is an urgent need for tissue engineers and even policy-makers to re-examine their strategies

Table 1: Current status in TE [Stock UA, 2001]

II LITERATURE REVIEW

2.1 The use of scaffolds in TE

Despite the emergence of cell-based methodologies, scaffold remains an integral component in TE that should not be neglected.There is probably no universal scaffold that works equally well in all applications Despite the exhausting number of factors

to consider in scaffold design and the impossibility to realize all of them in one, it is still imperative to satisfy a minimum set of biochemical and physical requirements (Yeong WY, 2004; Hutmacher DW, 2004; Zhang H, 2005; Hutmacher DW, 2001) Since the primary goal of a scaffold is to temporary mimic the functions of natural ECM until the transplanted cells generate adequate amount of their own, it should therefore have suitable architecture with sufficient area as well as biocompatible surface chemistry for the cells to adhere, proliferate and secrete In addition, it should also have high porosity with interconnected network that not only can facilitate mass transfer of nutrients and excreted wastes, but also provide sufficient channels for infiltration to take place either during seeding or in vivo tissue remodeling events such as neural / vasculature anastomosis The scaffold should also exhibit appropriate mechanical properties: a substrate too rigid will result in the inability of the cells to recognize and recruit the receptors into focal adhesion plaques for signal propagations;

on the other hand, a material too compliant will bring about poor cell attachment due

to its poor resistance against the tractional forces generated by the assembling cytoskeletons They should also have appropriate roughness as a substrate too smooth will result in poor attachment whereas a surface too rough will cause the adhering cells to either fail to bridge the irregularities or be simply physically damaged by the presence of sharp edges

The role of a good scaffold does not lapse even after implantation There is still the need to control their degradation profiles so that they can transfer stress at an appropriate rate to the healing tissues and continue to offer, prior to neotissue formation, some form of mechanical strength and stiffness that can protect the relatively fragile tissue implant against excessive deformation from any forms of stresses associated in a native environment particularly if it is constantly experiencing high loading The latter include forces brought about by wound contraction or other types of remodeling processes The modulus for hard and soft tissues should essentially always remain in the respective range of 10 – 1500 and 0.4 – 350 MPa despite the dynamic changes occurring at the scaffold (Hollister SJ, 2005) The breakdown will therefore need to be either in sync or slower than the rate of tissue regeneration (Murugan R, 2007) In addition, there should not be any severe adverse host responses triggered by the presence of the synthetic implant and its disintegrated components The scaffold should therefore be made from materials whose degraded by-products can be easily removed through the body natural excretory system to avoid undesirable accumulation that may in turn evoke the immune system

Scaffold should be manufactured via a controllable, reproducible and cost-effective high throughput process in order to facilitate its translation towards clinical applications It should also be able to adopt a more pro-active role in tissue remodeling by offering the flexibility to accommodate biological components such as growth factors without denaturing them It should further be sterilizable by an accessible technique without risking changes to its original structural or other related properties Size and shape of the final construct should also be contemplated,

particularly if it is to be customized for individuals These desirable features of a scaffold are summarized in the following table (Murugan R, 2007)

Table 2: Desirable features in an ideal scaffold [Murugan R, 2007]

2.2 Nanofibers

In vivo, cells are immobilized within tissue and bound to a diverse array of scaffoldings known as the ECM The latter consists predominantly of interwoven protein fibers such as collagen or elastin 10 – 300 nm in diameter functioning to provide tensile strength to the surrounding (Sniadecki NJ, 2006) Unfortunately, conventional scaffold manufacturing technologies are unable to replicate these fibril ropes and meshes, even though it has became evidently clear the profound effects their spatial presentation will have on the cells The following section will thus describe in greater details some of the emerging approaches to better mimic the physical 3D environment via nanofibers production

2.2.1 Non-conventional fabrication approach

One such procedure comprises of thermally induced spinodal liquid-liquid phase separation and crystallization in the polymer-rich component (Ma PX, 1999) 3D matrices with continuous fibrous network on the walls were successfully retrieved upon freeze-drying These scaffolds were determined to have considerable mechanical strength that could be further improved by modulating the polymer concentration and varying the fiber density (Chen VJ, 2004) Reproducible architecture had also been enforced by carrying out the process in a pre-defined mold previously manufactured via reverse Solid Freeform Fabrication (Chen VJ, 2006) In combination with a suitable porogen, interconnected macropores with varying shapes (spherical) (Chen

VJ, 2004) and arrangements (orthogonal or helicoidal tubular) (Zhang RY, 2000) were created Not only did the amount of adsorbed serum proteins increase by 4.2 fold, macromolecules such as fibronectin and vitronectin were detected exclusively on these nanofibrous surfaces rather than solid walls control (Woo KM, 2003) Cell attachment was reported to be 1.7 fold higher as a result Sustained expression of α2 integrins on osteoblasts had also been observed even in the presence of collagen inhibitor like 3,5-dehydroproline In addition, degradation kinetics of these phase-separated scaffolds was also found to be significantly faster in spite of the hydrophobicity as a result of more sites being made available for hydrolysis due to a 100-fold increase in surface area (Chen VJ, 2006) In fact, loss in mass for the first 12 months was determined to be as high as 50 %

Parallel fabrication of a multitude of nanofibers with regular morphology had also been reported elsewhere This was achieved by rapid rotation of a standard spin coater

previously loaded with polymer solution (Weitz RT, 2008) Fibers with diameter ranging from 25 nm - 5 µm and length up to 0.5 mm were collected probably due to

an instability triggered by the competition between centrifugal and curvature-induced Laplace force Thin liquid jets, stretched during rotation, emerged from the outwardly driven polymer to yield solidified nanofibers upon solvent evaporation A minimum rotating speed was found to be necessary as an increase in velocity would result in a corresponding decrease in viscosity Product morphology was also determined to be sensitively dependent on solvent vapor pressure Not only would slow drying be caused by a value too low, it would also facilitate the relaxation of polymer chains to their non-stretched conformation, thereby resulting in thick fibers with high frequency

of coalescence as well as susceptibility to breakage

2.2.2 Polyelectrolyte complexation

Unlike polycondensation, complex coacervation involves the formation of insoluble nano-materials due to charge neutralization at the interface of two electrolyte solutions with opposite polarity Though more commonly used for generating micro- (Zhu JH, 2005) and nanoparticles (Hartig SM, 2007), continuous fiber collection (at least until either one of the starting materials is depleted) can also be accomplished with the use of a roll-up apparatus An extensive description of the resulting morphology had been reported (Wan ACA, 2004) Typically, it consists of a primary fiber with elongated beadings occurring at regular interval along its axis The former itself comprises of another group of thinner fibers bundling together in a parallel fashion (Yamamoto H, 2001)

A more detailed mechanism for fiber fabrication had been suggested by Leong KW and his team (Wan ACA, 2004) They proposed an initiating (and most decisive) phase where oppositely charged polyions would interact to generate a local increase in viscosity Not only would it prevent bulk mixing of the two solutions, it could also block the formation of precipitates that would otherwise cause undesirable perturbations to the interface when dragged along a convective current created by the upward shear motion of fiber drawing Subsequent fiber drawing would however disrupt this equilibrated layer into numerous individual nucleating sites where more complexations into “nuclear” fibers would take place until they eventually condensed into a bead along the primary fiber axis Such defect could be removed by reducing the drawing speed to a rate lower than “bead point” where the droplet would have sufficient time to coalesce at the solution-air boundary Fiber production would however fail should the system have charge density or concentration lower than the requisite amount to form the semi-permeable barrier in the first place

Collected fibers can further be easily processed into a nonwoven 3D fibrous scaffold either via needle-punching (Lim SH, 2006) or hydro-entanglement (Wan ACA, 2006) The latter was able to hold firm the structure by forming a cross-linking bridge between two adjacent fibers, without which swelling would occur and the resulting high water pressure would force ionic groups on one fiber to complex with oppositely charged species on another strand Cell adhesive peptides such as RGD were also incorporated to enhance the compatibility (Wan ACA, 2006) hMSC enclosed in an alginate-chitin based structure were shown to remain viable and proliferate for more than a month without losing its osteogenic / chondrogenic lineage (Yim EKF, 2006)

In addition, as a result of its water compatibility, biological substrates can also be

incorporated during manufacturing without any denaturation risk Multiple nucleating sites aggregated over the particle while retaining the original physical properties of the fiber (Wan ACA, 2004) High encapsulation efficiency and uniformity were also reported The release profiles of a variety of substances in a typical complexing system were further found to be largely dependent on the molecular weight and ionic character of the species (Liao IC, 2005) While neutral agents like dexamethsone were completely discharged within 3 hours, a more sustained profile was achieved in polar molecules such as recombinant human platelet derived growth factor, bovine serum albumin or avidin due to their partial interactions with charged residues on the polyelectrolytes Successful examples so far include human β-nerve growth factors (Wan ACA, 2004) and DNA analogs (Lim SH, 2006) that had been integrated without sacrificing their bioactivity to either differentiate PC12 cells in a dose-dependent manner or stimulate them in synthesizing desired proteins for extended period

2.2.3 Self-assembly

Alternatively, similar nanofibrous structure can be manufactured through a process that makes use of the natural amphiphilic property in proteins to self-assemble non-covalently among themselves without any human intervention under thermodynamic equilibrium Despite being mediated by relatively weaker forces such as ionic and hydrogen bonding, hydrophobic interactions, simple van der Waals as well as aromatic stacking, the combined effects are strong enough to yield a rather geometrically stable supramolecular architecture Short oligomeric peptides constitute one of the most common form of starting materials for such system since they can be easily synthesized from de novo design via conventional F-moc mature solid-phase synthesis chemistry (Gelain F, 2007) Not only is such approach available at an

affordable cost, resulting construct can also be custom-made for specific application due to the ease of manipulating the physico-chemical properties from a library of 20 natural amino acids and a growing number of artificial derivatives (Leon EJ, 1998)

Three theoretical models are currently in use to enable better understanding of the assembly process (Zhang SG et al, 2002) The first makes use of molecular dynamics simulation to explore the packing order between the peptides and the energy landscape associated with structural deformation A second semi-continuum model helps to simplify thermodynamical analysis of higher ordered structures such as bundling by assuming each monomer as a communicating brick while the last fully continuum model exploits order parameters to develop a field-theoretic expression of free energy for structural features larger than the size of individual peptide

Three types of SAPs had therefore been identified (Zhang SG, 2002) Type I comprise

of synthetic molecular velcros 8 – 16 amino acids long with negative apartite or glutamate in the hydrophilic domains as well as a regular alternating pattern between hydrophobic alanine or leucine and positively charged lysine or arginine Complementary ionic bonds that subsequently develop between two such entities thus provide the driving force for them to adopt a particular conformation specific to the participating molecules Type II however are those that will undergo drastic structural changes upon variation in external stimuli or reaction time and are therefore known as molecular switches These transitions were believed to be due to the co-existence of two sequence motifs in one molecule For instance, DAR16-IV, despite adopting a beta sheet structure under ambient condition, was observed to transform into alpha helical at high temperature (Altman M, 2000) while LKL16 oligopeptides progressed

from beta sheet tape to fiber morphology with prolonged assembling time (Koga T, 2006) Similar effects may also be induced by altering the pH or even crystal lattice packing (Zhang SG, 2002) Type III on the other hand contain specific domains that allow them to self assemble onto a surface instead of among themselves The first component is usually a terminal segment of functional groups that other bioactive agents recognize; the second is a flexible spacer (usually a string of hydrophobic amino acids such as alanine or valine) to minimize non-specific binding; the third is a cysteine, asparatic acid or lysine residue that can covalently interact respectively with any gold-, amine- or carboxylic-functionalized surface (Zhao XJ, 2004)

Other types of SAPs are also presently being developed into new materials Vauthey S

et al designed a 7-8 residues peptide comprising of > 4 hydrophobic amino acids at its lipophilic tail and only 1 – 2 hydrophilic residues at its carboxyl side chain and C terminus (Vauthey S, 2002) Open-ended surfactant-like nanotubes and vesicles 30 –

50 nm in diameter were obtained under aqueous condition Due to their structural similarity with membrane proteins, these self-assembled macrostructures are presently being considered as either molecular devices for biomedical sensing or lab models for studying accurately cellular responses to environmental cues (Zhao XJ, 2004) In another study, a SAP system was designed to promote mineralizations whose crystallographic c axes were aligned along the long axes of fibers like those at native bone tissues (Hartgerink JD, 2001) Apart from facilitating crystal growth via the incorporation of phosphoserine and aspartic acid, these nucleations were also initiated

by concentrating the inorganic cations to local supersaturation on surfaces where repeating anionic groups were exposed Interfacing ability with inorganic materials like nickel were also improved by fusing histidine repeats in the amino acid sequences

(Zhang SG et al, 2002) Alternatively, a platform provided by combinatorial biology protocols such as phage- or cell-surface display can be used to help generating massive libraries of peptides for downstream screening of their binding efficiency on inorganic surfaces (Sarikaya M, 2003)

In particularly, 3D peptide scaffolds comprising of isotropic lattice with interweaving fibers 10 nm in diameter may also be formed spontaneously via exposing type I SAP

to monovalent salts-containing physiological solutions Overbeek theory had identified such self-assembling to be regulated by the superposition of van der Waals / hydrophobic attraction and electrical double-layer repulsion (Caplan MR, 2002) The latter was determined to constitute a kinetic barrier that must be minimized to near kT by charge screening from the salts in order to allow the approach of the oligopeptides Not only were the resulting hydrogels highly hydrated, structural characterizations also revealed matching porosity with their native analogs In addition to the lack of severe immune responses from the host (probably due to their ionic nature), they were also found to be biodegradable in an in-vivo environment despite their in-vitro stability towards heat, chemicals and proteolytic enzymes (Leon EJ, 1998)

Derjaguin-Landau-Verwey-SAPs are also amenable to further modification via a level as small as one amino acid Biologically active domains can therefore be left flagging via a glycine spacer on the C-termini of one filament without being accidentally deleted during peptide synthesis (Gelain F, 2007) Zhang SG and his coworkers functionalized their SAP scaffold through direct solid phase peptide extension at the amino terminal with three short sequence motifs from laminin I as well as collagen IV, and reported not only

cobblestone phenotype in human aortic endothelial cells, but also improved low density lipoprotein uptake, nitric oxide release as well as basement membrane deposition (Genove E, 2005) Similarly, Silva GA et al incorporated a peptide epitope comprising of isoleucine, lysine, valine and alanine that selectively directed neural progenitor cells rapidly into beta-tubulin-positive neurons (Silva GA, 2004) Lengthy axons projecting into the permissive scaffold were observed to form active synapses with one another (Zhang SG, 2003) These in vitro experiments were further translated into successful in vivo repatching of severed brain sections (Zhang SG, 2003) To date, these customized designer scaffolds had been shown to support a variety of mammalian cells, including chondrocytes (Kisiday JD, 2005), liver progenitor (Semino CE, 2003) and embryonic stem cells (Garreta E, 2006), without interfering intercellular communication

The non-invasive nature of SAP can also enable them to accept fragile biological specimens and deliver them via a mechanism similar to those described for polyelectrolyte complexation (Nagai Y, 2006) Alternatively, some agents can be further stabilized by fusing their ends to the self-assembling sequence that can only be liberated in the presence of locally expressed proteases Insulin-like growth factor-1 tethered to the peptide scaffold via a biotin / streptavidin sandwich approach remained capable of inducing in vivo Akt phosphorylation and cardiomyocytes maturation even after two weeks (Davis ME, 2006)

2.2.4 Electrospinning

Among all the techniques available, electrospinning offers one of the simplest and cost-effective ways to manufacture nano-sized features rapidly Despite the relatively

modest setup comprising of only a few portable components such as syringe pump, high voltage source as well as a collector,the mesh-like product had served ever since

1934 in diverse areas ranging from optics, chemo-sensing and filtration to biomedical applications such as drug delivery, wound dressing and scaffolding for TE Presently, they can even offer, in place of animal-derived biological coatings, 3D nanofibrous topographical cues to cells via commercially available 96 well templates (Corning® labware)

Often described as an electrohydrodynamic problem, various theoretical models had been developed in order to better appreciate electrospinning, one of which had identified three different forms of physical instability experienced by a charged jet in

an external electric field: Rayleigh, axisymmetric and non-axisymmetric conducting mode (Shin YM, 2001) The importance of last event, also known as whipping, was further implicated in experimental observations where its onset would always precede the production of fibers with submicron diameter Such spiraling was subsequently determined to occur through a force either resulting from dipoles previously induced

by the dynamic interactions between the external electric field and the fluctuating centerline or the mutual repulsions among the surface charges on the accelerating jet (Shin YM, 2001) The dominant instability was found to be strongly dependent on both the fluid parameters as well as the static charge density on the jet and only the bending mode is preferred for defect-free smooth fibers with minimal beadings

Mit-uppatham C et al had identified a total of 6 different types of forces participating

in a seemingly straightforward process: gravity, surface tension, electrostatic forces for transporting the charged jet onto collector plate, coulombic repulsion between

similar charged species within the fluid, viscoelastic forces from polymer chains entanglement as well as friction between the jet and its surrounding air (Mit-uppatham

C, 2004) These can in turn be manipulated to achieve downstream spatial, morphological and dimensional effects on the collected nanofibers, thereby making electrospinning a process very much susceptible to endo- and exogenous environment The latter are classified into three main categories: ambient conditions (temperature, humidity and air velocity), solution properties (viscosity, conductivity, surface tension, polymer molecular weight, dipole moment and dielectric constant) as well as processing parameters (flow rate, electric field strength, distance between tip and collector, needle tip design, and collector composition and geometry) (Pham QP, 2006)

Various models including one involving an orthogonal experimental design had been proposed to determine the importance of each contributing factor by considering them either as an individual (Thompson CJ, 2007) or correlated entities (Cui WG, 2007) A trend in which solution concentration and polymer molecular weight interacted to reign as the more influential variables on both fiber diameter and morphology had also been predicted (Cui WG, 2007) A low value in the former was reported to yield beadings as a result of the primary jet breaking up due to their inability to offset a stronger coulombic repulsion (Pham QP, 2006) Fibers may also remain wet enough

to retain sufficient mobility to contract into beadings in response to surface tension at the collector (Mituppatham C, 2004) Presence of junctions and fiber bundlings are further commonly encountered under such circumstances (Deitzel JM, 2001) Fortunately, these defects can be easily minimized via increasing the concentration to

an optimal range unique for individual polymer / solvent type Anything beyond this

window will however fail to maintain a consistent polymer supply at the spinneret due

to a sharp increase in zero shear viscosity A thick oscillating filament was found to accumulate till it gathered sufficient mass to fall under gravity instead (Deitzel JM, 2001) Attempts made to determine the minimum polymer concentration for a defect-free electrospun fabric identified critical chain overlap C* and entanglement concentration Ce as the two important variables demarcating respectively 3 regimes that include dilute, semidilute unentangled and semidilute entangled (Gupta P, 2005) C* was defined to be the point where the concentration inside a single macromolecular chain matches that of the bulk solution It had also been approximated to be 3M / (4πNAR3g) (NA = Avogadrio number; Rg = radius of gyration;

M = molecular weight of polymer) (Yu JH, 2006) On the other hand, the expression for Ce in a good solvent was estimated to be pMe / Mw (p = polymer density; Me = entanglement molecular weight of the undiluted polymer) (Eda G, 2007) SEM revealed that fiber formation would still be initiated at concentration higher than C*, though the presence of beading could only be removed when the concentration became twice of Ce (or 5 – 10x of C*) and entanglements within the solution were so extensive that contraction of jet radius was prevented by providing sufficient resistance against surface tension In another study, Shenoy SL et al developed a model based on a semiempirical approach and established the minimum requirement for fiber formation to be one entanglement per polymer chain but 2.5 for them to be defect-free (Shenoy SL, 2005) Strong viscoelastic force arising from high polymer concentration was also found to result in larger diameter due to its antagonistic response towards fiber stretching In fact, a generic power law had been observed between these two variables (Ki CS, 2005; Demir MM, 2002; Deitzel JM, 2001; McKee MG, 2004) For a polyethylene terephthalate-co-polyethylenimine system, the

exponent was revealed to be ~ 3.0 (McKee MG, 2004) On the other hand, there was also a bimodal distribution in some systems comprising of smaller secondary strands dispersing among larger primary filaments when concentration rose above a certain threshold (Deitzel JM, 2001) It is however currently unknown if they had been caused by an increased frequency of splaying events, or a greater stability in auxilliary fibers due to the more concentrated source

The influence of polymer molecular weight can be derived from the following rationale For chain entanglements to be present, the [η]c component ([η] = intrinsic viscosity; c = polymer concentration), otherwise known as Berry number Be from the Flory-Huggins theory, should have a value greater than 1 (Gupta P, 2005) The visco-term is in turn correlated to molecular weight M via Mark-Houwink-Sakurada equation where [η] = KMa (McKee MG, 2004; Gupta P, 2005) A higher concentration of low molecular weight chains must therefore be present before they can overcome the small hydrodynamic volume they occupy to topologically constrain one another (Eda G, 2007) Grafahrend D et al were unable to achieve fiber formation with molecular weights below 25 kDa even at high concentration of their block copolymer solution (Grafahrend D, 2007) Conversely, large beading defects and fragile mechanical strength were removed simply via more than 3-fold increase in molecular weight of the chitosan solution from 30kDa to 106 kDa (Geng XY, 2005)

In fact, beadings in some cases were not present in spite of an extremely low concentration as a result of pre-existing undissolved entangled domains in the solvent (Eda G, 2007) Similar observations where C* was reduced with increasing molecular weight of poly (methyl methacrylate) were also reported elsewhere (Gupta P, 2005)

In addition, a power law relationship analogous to the one reported in polymer

concentration was also determined between fiber diameter and molecular weight irregardless of the solvent types (Eda G, 2007) At a concentration where bead-free morphology was achieved, the exponent was found to be an unexpected -0.5 for linear atactic polystyrene (i.e.: increased molecular weight, smaller fiber diameter) This could be due to the presence of a large hydrodynamic sphere with extensive coiling at higher molecular weight and [η] These substantial entanglements even at low concentration could in turn minimize the requisite polymer amount to stabilize one unit fiber length, thereby enabling a smaller diameter to be achieved without losing its geometrical stability

Early works in electrospinning dealt mainly with organic polymers or natural derivatives like collagen, gelatin, chitosan, chitin, cellulose and starch (Murugan R, 2006) The range of raw materials had however in recent years became more diverse

as unconventional substrates, including those with electronic, magnetic, optical and biological properties, can now also be easily processed For instance, the gelling tendency of alginate even at low concentration was overcame via the combined actions from poly(ethylene oxide) copolymer, Triton X-100 surfactant and dimethyl sulfoxide cosolvent (Bhattarai N, 2006) They could therefore be manufactured into a nanofibrous scaffold with not only structural resemblances to native glycosaminoglycan proteins, but also satisfactory elasticity and tensile strength when used in concomitantly with collagen Hyaluronic acid is another example of a polysaccharide molecule that had been electrospun despite experiencing similar challenges (Li JX, 2006) Recent new members include even fibrinogen (Ye Q, 2000) and calf thymus sodium-DNA (Fang X, 1999) Hidden regions such as β 15-42 epitope on the parent molecule had also been exposed by the spinning to improve not

only cell proliferation and migration, but also the mechanical stability of the scaffold and its resistance to enzymatic degradation via an increase in internal crosslinkings (McManus MC, 2007)

Apart from the typical organic-based biological materials, inorganic elements such as ceramics were also co-electrospun with proteins to result in a composite structure Key features in their primary strategy comprised of increasing the viscoelastic property of the reaction mixture with a polymer, as well as controlling the hydrolysis rate of the resulting sol-gel precursor via either pH or ageing conditions previously initiated by the air moisture (Li D, 2004) A continuous gel network was generated, and the organic phase was subsequently removed with calcinations at elevated temperatures to yield a thinner yet mechanically superior pure inorganic nanofibrous structure A more uniform nano-crystallites due to the quick diffusion of water vapor into the thin fibers had also been reported Such approach had been adopted for the production of bioactive glass nanofibers (84 mm in diameter) in bone TE (Kim HW, 2006) Depositions of poorly crystallized apatite were observed along the surface upon contact with simulated body fluid, and their growth were attributed to local supersaturation of calcium and phosphate ions at exposed Si-O- centers previously formed by cationic exchange between the glass network and their surroundings (Xia

W, 2007; Kim HW, 2006)

Electrical conducting polymers such as polypyrrole, polyaniline and poly (vinylidene fluoride) further constitute another unique class that were recently manufactured in nanofibrous form rather than a conventional film Charge-initiated changes could therefore be stimulated on ions-sensitive tissues like nerves or cardiac muscles (Li

MY, 2006) by either electrophoretically redistributing their cell surface receptors or improving proteins adsorption with alterations in local electrical fields of the ECM (Kotwal A, 2001) Other interesting materials include stimuli-responsive polymers Aligned fibrous 3D scaffold fabricated from thermal sensitive hydroxybutyl chitosan was dissolved at 40C to yield a directionally intact hMSC sheet (Dang JM, 2007) The group observed a transient overexpression of myogenic genes even in non-differentiating medium and attributed them to alterations in the intranuclear environment as a result of stretching induced specifically by the topographical features In another study, Wang LG et al reported the synthesis of a nanofibrous mat with the ability to experience pH-induced volume transitions (Wang LG, 2007) This was achieved in a triblock polymer with an ABA configuration, where B was the pH sensitive domain poly[2-(diethylamino)ethyl methacrylate] and A a hydrophobic glassy polymer such as poly(methyl methacrylate) that allowed the structure to undergo continuous volumetric oscillations without fracture by forming an even distribution of crosslinks Expansion in such system was enabled when the polymer chains began to polarize and start repelling one another while contraction was evoked when they became electrically neutral instead at the coil-globule transition point of

pH 5 Unlike hydrogel, electrospinning of these materials resulted in superior surface area to volume ratio that in turn minimized the response time previously severely constrained by diffusion limitations Maximum dimensional variations were further accomplished through improving the microdomains arrangement with annealing in the presence of tetrahydrofuran

III OBJECTIVES

Conventional scaffold designs often result in constructs that do not possess the nanoscale complexity of the native environment Cells in such a surrounding are however embedded in a plethora of ECM proteins like collagen or elastin fibers, each

10 – 300 nm in diameter with extensive entanglements among themselves to give rise

to a nonwoven mesh of considerable tensile strength and elasticity (Sniadecki NJ, 2006) Extracted basement membrane revealed an architecture consisting of 70 nm wide nanopores as well as a felt-like landscape with peaks and valleys approximately

100 nm in height and depth Various aspects of these nano-topographical features, such as scale, shape and even symmetry, had been shown to result in downstream events independent of those typically caused by biochemical and mechanical cues (Stevens MM, 2005) For instance, the extent of orientation in smooth muscle cells was inversely proportional to feature size (i.e.: the smaller they are, the better the alignment) (Hu W, 2005) Rat cardiomyocytes also self assembled more readily into aggregates with stronger action potential when cultured on nanopillars that were determined to be functioning individually as a guiding post to these migrating cells (Kim D-H, 2006) Recent evidences had further shown their osteogenic effects on bone progenitor cells even in the absence of appropriate supplements (Dalby MJ, 2007) In fact, one of the most noticeable early responses in cells had been found to be

a heightened perception resulting from an increase in the number of sensing organelles such as filopodia and microspikes (Stevens MM, 2005)

Among all the techniques available, electrospinning offers one of the simplest and cost-effective ways to manufacture nano-sized features rapidly Briefly, the process involves subjecting a polymer solution at a needle tip to charge induction The