Development of chitin based materials for tissue engineering applications

DEVELOPMENT OF CHITIN BASED MATERIALS FOR TISSUE ENGINEERING APPLICATIONS CHOW KOK SUM NATIONAL UNIVERSITY OF SINGAPORE 2002... DEVELOPMENT OF CHITIN BASED MATERIALS FOR TISSUE ENGINE

DEVELOPMENT OF CHITIN BASED MATERIALS FOR

TISSUE ENGINEERING APPLICATIONS

CHOW KOK SUM

NATIONAL UNIVERSITY OF SINGAPORE

2002

DEVELOPMENT OF CHITIN BASED MATERIALS FOR

TISSUE ENGINEERING APPLICATIONS

CHOW KOK SUM

(B.Sc Hons, NUS)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE

2002

Acknowledgement

I would like to express my most heartfelt thanks to Professor Eugene Khor for his guidance and supervision throughout the course of this project Without his constant

support and patience, this project will not have been accomplished

My special appreciation to my friends and fellow lab mates for their invaluable friendship and encouragement Grateful to Irene, Mdm Loy, Mr Sim, Joanne and Ms Tan for their technical support Many thanks to Nelda, Sarah, Li Shan, Dawn, Ze Gang, Selina, Ling Ling, Xia Bing, Chin Teng, Ze Han and many others for their companionship and laughter during good and bad times

Last but not least, I would like to show my gratitude to National University of Singapore for granting me the research scholarship I am also grateful to the Dept of Chemistry for invaluable education and training during my undergraduate and postgraduate years

2.3) RESULTS AND DISCUSSION

2.3.a) Variation of Pre-Lyophilization Process and Drying Methods 52 2.3.b) Internal Bubbling Process (IBP) 66

3.3) RESULTS AND DISCUSSION

3.3.a) Synthesis of New Fluorinated Chitin Derivatives 110 3.3.b) Synthesis of Chitosan-Polypyrrole Hybrids 123 3.3.c) Synthesis of Reversible Water Swellable Chitin Gel 135

SUMMARY

As the field of tissue engineering emerged in the last decade, it has surfaced as a promising alternative approach in the treatment of malfunctioning or lost organs An important focus in this new approach is the search for suitable materials for development

of a variety of tissue engineering applications Chitin has been known to science for almost two centuries but the development of chitin chemistry and its application has lagged behind cellulose

Chitin is abundant in nature due to its compact intractable and inert structure resulted from strong hydrogen bonding network Chitin is known as one of the second most abundant polysaccharides in nature, after cellulose In crustaceans, chitin is present

in a complex structure with calcium carbonate, forming the rigid skeleton of carapace, shell and tail In insects, chitin is the main building block of the back plate This intractable characteristic of chitin is superior in the animal / plant kingdom as protective skeleton but is a major disadvantage for chemical / physical modification Therefore more efficient methods of reacting or modifying chitin (especially α-chitin as it is the most abundant of the 3 types of naturally occurring chitin) is necessary, in order to utilize this biomass as a major renewable raw materials

This dissertation presents new methodologies of developing chitin-based materials for tissue engineering applications In tissue engineering, a temporary matrix is required

to serve as an adhesive substrate for the implanted cells and a physical support to guide the formation of the new organs Investigations were conducted to develop a novel processing technique to fabricate highly porous chitin with a wider range of pore sizes

preparation, characterization and in vitro cytotoxic assessment of deoxy-fluorinated

chitin

Among the conducting polymers, polypyrrole has been one of the most widely studied because of its good chemical and thermal stability, ease of preparation and electroactivity6, &7 8 We have opted to introduce carboxylic acids substituents onto the C-3 position of the pyrrole monomer These in turn are made to react with the amino groups in chitosan creating covalent bonds between the polymers The result is a polypyrrole-chitosan hybrid material that is potentially biocompatible and electrical conducting

The incorporation of substantial amounts of carboxylic group into the intractable chitin backbone produced a highly swelled chitin hydrogel Swelling characteristics of the chitin hydrogel was determined by the hydrophilicity of the polymer By modifying the degree of hydrophilicity of the hydrogel, we could control its swellability and

N Kasuya., K Iiyama, G Meshitsuka, A Ishizu, Preparation of

6-O-deoxy-6-fluorocellulose, Carbohydrate Research, 260, 251, 1994

2

N Kasuya, K Iiyama, A Ishizu, Synthesis and characterization of highly substituted

deoxyfluorocellulose acetate, Carbohydrate Research, 229, 131-139, 1992

H Tseng, K Takechi, K I Furuhata, Chlorination of chitin with sulfuryl chloride under

homogeneous conditions, Carbohydrate Polymers, 33 13-18, 1997

6

M.T Nguyen, A F Diaz, A novel method for the preparation of magnnetic

nanoparticles in a polypyrrole powder, Adv Materials, 858, 1994

SUMMARY

7

S Machida, S Miyata, A Techagumpuch, Chemical synthesis of highly electrically

conductive polypyrrole, Synthetic Met, 31(3), 311, 1989

8

S Machida, S Miyata, A Techagumpuch, Chemical synthesis of highly electrically

conductive polypyrrole, Synthetic Met., 1989, 31, 311

CHAPTER ONE: INTRODUCTION

CHAPTER ONE: INTRODUCTION

PREFACE

Polysaccharides are naturally occurring macromolecules, built by condensation of high molecular weight polymers with simple monosaccharide units In contrast to functional roles of nucleic acids and proteins, they have been considered to be substances

of less importance and regarded primarily as structural materials and suppliers of water and energy However, in recent years, polysaccharides inherent properties in biological systems are better understood and interest in polysaccharides chemistry is unabated The development of new delicate methods of extraction, separation, modification and analysis has resulted in the discoveries of novel polysaccharides and realization of their potential applications1

Among the polysaccharides, cellulose and chitin are the two most abundant biopolymers Although cellulose has been studied extensively, only limited attention has been paid to chitin In 1811, Professor Braconnot, director of botanical garden at the Academy of Sciences, Nancy, France, discovered a substance in fungi and named it

“fungine” This discovery occurred 30 years before Payen’s isolation of cellulose2 In

1823, Odier isolated an insoluble residue from the elytrum of the cockchafer beetle with hot potassium hydroxide solution and called it chitin The name chitin is derived from the Greek word “chiton”, meaning “coat of mail” since it functions as protective coat for invertebrates Despite the early discovery and an annual production of at least 10 gigatons/year in the biosphere, chitin remains an almost unused biomass resource3

CHAPTER ONE: INTRODUCTION

The main driving force for the development of new applications for chitin and its derivatives lies with the fact that these polysaccharides represent a renewable source of natural biodegradable polymers Since chitin is the second most abundant biomass after cellulose, academic and industrial scientists are faced with a great challenge to find new and practical applications for this material Among the potential explorations identified for chitin based materials is in the emerging field of tissue engineering

Tissue engineering provides an interesting glimpse into the future of medicine Using this technology, it will be possible for doctors to routinely repair or replace failing

or aging body parts tissues with laboratory-grown parts such as bone, cartilage, blood vessels, and skin4 There has also been an increasing public and media interest in tissue engineering, from the first tissue engineered skins to be approved by the US Food and Drug Administration (FDA) to the controversial use of human embryonic stem cells, which are taken from aborted fetuses or discarded embryos5, & 6 7 Although there is growing excitement in the field of tissue engineering, it is still in its infancy Success will depend largely on the ability to understand complex cellular interactions and application

of appropriate scaffolding materials, growth factors and cell populations

This dissertation is divided into two parts, namely the fabrication of chitin matrixes and chemical modifications of chitin The first part of the thesis will present the research focused on developing new methods to fabricate chitin matrixes These matrixes have the potential to provide a structural framework for cells and facilitate formation of new tissues in tissue engineering The second part of the thesis will investigate the chemical

CHAPTER ONE: INTRODUCTION

modifications of chitin that can impart various desirable characteristics in tissue engineering

1.1) CHITIN

1.1.a) INTRODUCTION

Chitin8 is a linear amino polysaccharide consisting of -1, 4-linked

N-acetyl-D-glucosamine Chitin is analogous in chemical structure to cellulose (Figure 1), where substitution of the C2 hydroxyl group (OH) for the acetamide group (NHCOCH3) as the only structural difference Chitosan is known as the deacetylated form of chitin The main difference between chitin and chitosan lies in the degree of deacetylation A fully acetylated or deacetylated polymer is not naturally occurring The only means to differentiate chitin from chitosan is by considering their respective acetyl contents or degree of acetylation It is generally accepted that chitosan is the one with degree of acetylation below 50% Hence, chitin was termed to those having degree of acetylation over 50%9 Chitin and chitosan are among the polysaccharides found in nature to have nitrogen attached to the biopolymer backbone Other nitrogen-containing polysaccharides include polygalactosamine and mucopolysaccahrides

CHAPTER ONE: INTRODUCTION

OOH

O

N H A cOH

OOH

O

O HOH

OOH

O H

n

OOH

O

OH

OOH

Figure 1: Chemical structures of cellulose, chitin and chitosan

* where Ac = COCH3 group

CHAPTER ONE: INTRODUCTION

1.1.b) SOURCES

Chitin is one of the most plentiful polysaccharides produced in nature by biosynthesis It only ranks second to cellulose as the most abundant organic compound on earth10 Cellulose is the key carbohydrate that plants use to build cell walls Likewise to these compounds, chitin also contributes strength and protection to the organism Chitin

is widely found in animals particularly in the shells of crustaceans, such as shrimp, crab

as well as in the exoskeleton of marine zoo-plankton It is present in a complex structure with calcium carbonate, forming the rigid skeleton of carapace, shell or tail of the organism

Chitin is also found in the insect kingdom, it forms the main building block of the back plate The chitinous shell or exoskeleton, does not grow, and is periodically molted After the old shell is shed, a new, larger shell is secreted by the epidermis, providing room for future growth of the insect

Apart from animals, chitin is also found in plants It is present in vast majority as the principle protective layer in the cell walls of yeast, mushrooms and most classes of fungi (Figure 2) Chitin’s occurrence as main structural component of the cell wall provided strong and intractable material for protection

CHAPTER ONE: INTRODUCTION

In fungi, chitin is present in cell walls in the form of micro-fibrils1,11 Chitin is also the major organic component in crustaceans12 Every year, almost 100 billion tons of discarded crustacean shells sink through the world’s oceans This has attracted much attention to crustaceans as a source of raw material for chitin production13 Therefore, most of the chitin produced commercially is derived from crab, shrimp, and crayfish exoskeletons obtained as waste from the seafood processing industry14

Figure 2: Adapted schematic diagram showing location of chitin fibers in an arthropod

cuticle15

CHAPTER ONE: INTRODUCTION

1.1.c) STRUCTURAL CLASSIFICATION

Chitin and chitosan tend to adopt a helix structure The helical strands can be classified into three crystalline forms namely , and (Figure 3) These crystalline networks are aligned by chains in bonded “layers” connected by N-H…O=C hydrogen bonds through the C2 amide linkages16, 17 The most abundant form is -chitin, usually found in crabs In -chitin, the molecules are aligned in an anti-parallel manner This molecular arrangement is favorable for the formation of strong hydrogen bonds resulting

in the most stable and abundant of the three crystalline forms In order to utilize the abundance of -chitin, it is used as the raw material in this project

Figure 3: Representation of molecular arrangement for and -chitins indicating

hydrogen bonding interactions3

CHAPTER ONE: INTRODUCTION

-chitin is usually found in squids and cuttlefish11 Molecules in -chitin are packed in a parallel fashion, producing weaker intermolecular interactions -chitin is easily transformed into -chitin -chitin is a mixture of and -chitin with both parallel and anti-parallel arrangements

1.1.d) SOLUBILITY

The strong inter / intra molecular hydrogen bonding network of -chitin is the key basis for its intractability and insolubility in the common solvents used for dissolution of cellulose It swells slightly in basic solvents and does not swell at all in common organic solvents It is found to be soluble only in special solvents such as 5% lithium chloride (LiCl) in N, N-dimethylacetamide (DMAc) or a mixture of DMAc with N-methyl-2-pyrrolidone (NMP) containing 5-8% LiCl Chitin was also reported to dissolve in hydrochloric acid or sulfuric acid but observed to experience hydrolysis under heating and prolonged stirring3 Therefore, under mild conditions in dilute acids, chitin is not soluble In fact, chitin is purified by stirring in 50% HCl at 25oC for 1 day In sharp contrast to the intractable nature of -chitin, -chitin swells highly in water because of the weak intermolecular hydrogen bonding Chitosan, the deacetylated form with a basic amino group is soluble in aqueous acidic medium with pH < 6.5; such as in dilute acetic, lactic and hydrochloric acid solutions Thus, chitosan precipitates at pH above 6.5 and its applications was limited owing to the insolubility at neutral or high pH region18

CHAPTER ONE: INTRODUCTION

1.1.e) BIODEGRADABILITY

The earliest report of enzymatic degradation of chitin is that of Beneke in 19052 Both chitin and chitosan are reported to be biodegradable19 in nature The biodegradation process is carried out by many varieties of microorganisms20 In most instances, the

complete enzymatic hydrolysis of chitin to N-acetyl-D-glucosamine requires a system of

chitinolytic enzymes The chitinolytic enzymes comprise of chitinase acetylglucosaminidase (EC 3.2.1.14), β-D-acetylglucosaminidase (EC 3.2.1.30) and β-N-acetylhexosaminidase (EC 3.21.52) Chitinase 1,4-β-poly-N-acetylglucosaminidase (EC 3.2.1.14) performs random hydrolysis of the chain, β-D-acetylglucosaminidase (EC 3.2.1.30) hydrolyzes the terminal non-reducing sugar moiety and β-N-acetylhexosaminidase (EC 3.21.52) removes the successive sugar units from the non-reducing end21

1,4-β-poly-N-Chitinolytic enzymes (CE) are also present in higher plants, even though plants do not have chitin as a structural component This characteristic may be attributed to the self-defense mechanism of plants against pathogenic microbes and insects that have chitin as their exoskeleton22 CE have also been found in the digestive tracts of some vertebrate species such as rodents and more recently discovered in humans23 The existence of chitinolytic enzymes in the gastrointestinal tract and lung may suggest their possible role in digestion and/or defense

CHAPTER ONE: INTRODUCTION

Chitin in humans is also degraded by lysozyme24 The degree of deacetylation of chitin affects its biodegradability and chitin with a degree of deacetylation of 0.7 is most susceptible to lysozyme25,26 This is attributed to the strong interaction between the acetyl group of chitin with subsite-C in the active cleft of lysozyme27 The susceptibility to lysozyme degradation has formed the basis of the medical applications of chitin28 Among the examples is the gradual re-sorption of chitin dressing as artificial skins with simultaneous replacement of natural tissue29 Due to the lysozymic degradation process, sustained release of an active ingredient from chitin films and micro spheres can also occur30

1.1.f) HEALTH ISSUES BENEFITS AND RISKS

For years, chitin and its derivatives have been used for nutritional and medicinal purposes in the Far East Many people do take dietary supplements made from chitin and chitosan to improve their health They also cited improvements in skin, hair and nail health The low toxicity of chitin was demonstrated to be mostly due to its biodegradability and the fast metabolization of hydrolysate from the system31 Studies showed that chitin and chitosan have a low degree of toxicity with LD50 in laboratory mice is > 10g/kg body weight, which is close to that of salt and sugar32 Chitin was reported to show good biocompatibility as a wound-healing accelerator33

CHAPTER ONE: INTRODUCTION

Muzzarelli reported that oral administration was recognized as safe, non-toxic and deprived of any activity on certain drugs34

A beneficial effect of chitin-chitosan as a food supplement is the reduction of plasma cholesterol and triglycerides due to its ability to bind dietary lipids, thereby reducing intestinal lipid absorption It was also reported to improve the HDL-cholesterol/total cholesterol ratio 35, 36 Plasma cholesterol in animals on cholesterol-free diet, however, is not affected; indicating that endogenous biosynthesis of cholesterol remains intact 37 Chitosan acts by forming gels in the intestinal tract which entrap lipids Unfortunately, other nutrients, including fat-soluble vitamins and minerals also have the tendency to be trapped, thus interfering with their absorption 38

Dietary chitin-chitosan may influence calcium metabolism by accelerating its urinary excretion The reported undesirable effects are a marked decrease in plasma vitamin E level, reduction in bone mineral content and growth retardation27 Ascorbic acid or vitamin C, enhances gel formation of chitosan, thereby increasing the reduction of plasma cholesterol Bile acid composition and short-chained fatty acid content in the cecum are altered by chitosan which impedes lipid emulsification and absorption35

Chitin-chitosan was also reported to inhibit in vitro growth of microorganisms including Candida and in vivo has a protective effect on Candida infection The

antibacterial and anti-yeast activities of chitin-chitosan are desirable properties and may

be useful in preventing infection of wounds by direct application35

CHAPTER ONE: INTRODUCTION

On prolonged ingestion, however, it may alter the normal flora of the intestinal tract that may result in the growth of resistant pathogens Although studies with cells, on tissues and animals indicate that chitin-chitosan promotes wound healing, increases immune response, and possesses antitumor activity 39, these claims need to be further validated in human subjects by clinical trials Therefore, certain medical precautions should be observed with long-term ingestion of high doses of chitin-chitosan to avoid potential adverse metabolic consequences 40

1.1.g) LIMITATIONS

The acetamido groups in chitin lead to strong hydrogen bonding producing compact structures The strong inter / intra molecular hydrogen bonding in the 3D crystalline structures of chitin as well as its high molecular weights (usually in the order

of 106) results in its insolubility in most organic solvents This intractable characteristic

of chitin is superior in the animal / plant kingdom as protective skeleton but is a disadvantage for chemical / physical modification Chitin also exhibits poor accessibility / reactivity to reactants when compared to cellulose In chitosan, the presence of less acetamido groups has produced a weaker hydrogen-bonding network The disruptions of hydrogen bonding in chitosan enable it to be soluble in acidic medium and be manipulated for various applications Therefore, utilization of chitin as a raw material has been limited to a handful of applications

CHAPTER ONE: INTRODUCTION

1.2) TISSUE ENGINEERING

1.2.a) INTRODUCTION

Tissue Engineering is a potential new area for development that brings together various disciplines such as bioengineering, material science, chemistry, cellular biology and medicine Fundamentally, tissue engineering aims to develop biological replacements that restore, sustain or improve tissue function It also aims to apply the biological replacements to medical situations where tissue has been lost through trauma

or disease41 Tissue engineering is defined as the application of engineering disciplines to either maintain existing tissue structures or to enable tissue growth42 (Figure 4)

Surgical approaches to overcome tissue loss include organ transplantation from one individual to another, tissue transfer from a healthy individual to an affected site in the patient and replacement of tissue function with mechanical devices (such as prosthetic valves and kidney dialysis machines) Other medical strategies may include pharmacology supplement of the metabolic products of missing or non-functional tissue

CHAPTER ONE: INTRODUCTION

Figure 4 Schematic adaptation of the tissue engineering approach42

While these approaches incorporate enormous advancements in the field of medicine, they also have some intrinsic limitations Organ transplantation is constrained

by the number of available donors More than 50,000 people are on the transplant waiting lists for various organs in the United States alone Furthermore, immuno-suppression in organ transplant beneficiary carries a lifetime risk of potential morbidity and mortality

CHAPTER ONE: INTRODUCTION

Transferring tissue from the donor to another site in the same individual often entails various shortcomings such as imperfect match for the reconstructive need or potential for donor site morbidity and complication in the transferred tissue Mechanical devices on the other hand, may have limited resilience in a biological system, lack of mechanisms for self-repair It also may sustain inflammation or infection at the transplant site, impose anti-coagulation and will not develop as the recipient grows These shortcomings have been recognized and studied Therefore, current researchers have look beyond transplantation for solutions43 (Table 1)

Tissue engineering is one of a new generation of treatment strategies that provide alternative solution to the replacement or restoration of tissue or organ function with constructs that contain specific growing populations of living cells The transplantation of cells on matrixes is distinguished from the introduction of isolated cells by the presence

of a biological scaffold It differs from the encapsulated systems in that cells have direct contact with the host site rather than is separated by a semi-permeable membrane

Three avenues have been explored in creating new tissues namely, a) introduction

of living cells; b) development of the encapsulated systems and c) transplantation of cells into the matrixes44

CHAPTER ONE: INTRODUCTION

Table 1: Shortcomings associated with interventions for tissue deficit or dysfunction45

Organ transplantation Costly, operative risk, donor shortage and

immunosuppression

Surgical reconstruction Insufficient graft tissue, inappropriate graft

tissue and poor graft function at ectopic location

Extracorporeal devices Temporary support, limited spectrum of

replacement functions, thromboembolic complications with vascular manipulations

Implantable devices Poor vascularization, immunosuppression,

fibrous tissue ingrowth and insufficient nutrient /waste exchange

CHAPTER ONE: INTRODUCTION

1.2.b) CELL-POLYMER MATRIX CONSTRUCT

The novel concept of growing cells on polymer matrixes that could generate tissues originated from various biological observations41 (a) Tissue go through continuous regeneration, adaptation and replacement (b) fully grown cells can reorganize into their natural histological state when in appropriate cell culture environments, (c) due

to a lack of scaffold to guide restructuring, isolated cells suspensions have limited growth46 and (d) the amount of tissue that can be implanted is also restricted by diffusion limit for gas and nutrient exchange47

Cell-matrix construct is employed as a scaffold to guide tissue regeneration involving isolating appropriate cell populations and transferring to polymer matrixes for

in vivo implantation The matrix is required to consists of several functions namely, (a) be

biocompatible to the host; (b) ease of remolding into a variety of shapes and structures during fabrication; (c) able to retain their shape when implanted; (d) offer sustainable mechanical supports; (e) and provide sufficient spaces between cells to facilitate gas and nutrients exchange Eventually, the biodegradable matrix will degrade, adsorb and gradually be replaced by regenerated tissue thus avoiding any kind of inflammatory response from the host48

Employing cell-matrix construct for tissue engineering is an approach that enable experimental control at three stages of assembly to achieve optimal system namely the cells, the polymer matrix and the method used for the construct assembly

CHAPTER ONE: INTRODUCTION

1.2.c) POLYMER MATRIX

Various development and advances in the field of polymer chemistry have contributed to the engineering of customized synthetic polymer-matrixes Various polymer-matrixes were also constructed by utilizing natural biomaterials as raw materials 49 Variables such as polymer porosity and degradation rate can be systematically regulated Defined shapes and sizes of polymers with specific intrinsic architectures can be readily and reliably manufactured

A wide variety of synthetic biodegradable polymers have been investigated as matrixes, among which are vascular grafts made from endothelial cells and expanded polytetrafluoroethylene (ePTFE, DacronTM)50, liver equivalents from hepatocytes and PGA or poly(vinyl-alcohol) (PVA, IvalonTM)51 and intestinal tubes made from enterocytes and polylactic-co-glycolic acid (PLGA VicrylTM)52

The most widely used polymers in tissue engineering have been the poly( -hydroxyl acids) of the aliphatic polyester, eg: polyglycolic acid (PGA), polylactic acid (PLA) and copolymers (PLGA) of these materials The broad applications of the PLGA family to tissue engineering are attributed to a long-standing established safety record in humans where PGA was first developed as DexonTM, a synthetic absorbable suture53 These

polymers were approved for in vivo use by the FDA for certain applications and were

readily processed into a variety of shapes and foams using melt and dissolution

CHAPTER ONE: INTRODUCTION

techniques54 Properties of synthetic polymers can be altered by modifications to the functional groups (backbone and side-chain), polymer architecture (linear, branched, comb or star) and polymer combinations of either physically mixed polymer blends/interpenetrating networks or chemically bonded co-polymers 55

1.2.d) CELL-POLYMER MATRIX GENERATION

With the existence of fabricated polymer matrix and cultivated cell populations, construct generation methods can proceed The method involves (a) introduction of cells

to the polymer matrix usually refer to as “seeding”; (b) optimization of parameters for cells attachment, (c) initiation of tissue formation and growth

Seeding may be statically applied directly between the cell suspension and polymer matrix or performed under agitated dynamic conditions It was reported that successful seeding of thin polymer matrixes (<2mm) can be achieved with static methods, a higher yield and level spatial cell distribution with thicker (>2mm) matrixes require dynamic systems56,57

In the context of tissue engineering, “bioreactor” presents a similarity to a dynamic tissue culture environment where tissue-specific mechanical forces such as stretch, pressure and shear forces are applied Gas and nutrient exchange is amplified by a continuous exchange of fresh culture medium Among the examples include stretch delivery to

CHAPTER ONE: INTRODUCTION

cultured myoblasts58, chrondrocytes in vitro application of pressure or simulated

micro-gravity59 or preservation of hepatocyte cultures under flow conditions60

After the “bioreactor” phase, a conditioning period will follow; (a) formation of tissue from the individual cells, (b) development of tissue micro-structure by subjecting specific mechanical forces, (c) enhancement of construct’s mechanical strength by the deposition

of connective tissues, (d) polymer degradation prior to in vivo transfer and possibility of

foreign body response and (e) provision of a temporary window for incorporation of a micro-vascular system into the tissues

As the field of tissue engineering transpires in the last decade, it is now recognized that cells seeded on a three-dimensional polymer matrix can, in fact, be substituted by native

tissues under appropriate in vivo and in vitro conditions Future achievements will hinge

upon formulating strategies to manufacture specific matrixes that will promote in-growth

of the host vasculature or to form a basic micro-vasculature system in donor engineered structures With this in mind, this project was aimed at the fabrication of chitin-based materials as potential polymer-matrix construct material in tissue engineering applications

tissue-CHAPTER ONE: INTRODUCTION

1.3) AIM OF PROJECT

This project aims to address the following questions:

1) Can chitin (the 2nd most abundant biomass in nature) be used as a raw material in

tissue engineering? In this instance, we have investigated into the fabrication of chitin

matrixes This involves the study of new preparation methodologies to overcome the

present limitation, characterization and cytotoxicity assessment of the scaffold materials

Present limitations:

a) Due to the intrinsic properties of chitin that is not soluble in common organic solvents and unable to form a stable suspension with salts, all the reported methods of porous matrix fabrication methods such as salt leaching, emulsion, thermal phase separation, etc are not useable Therefore, until now no chitin porous matrixes have been reported

b) Most of the current methods produce porous matrices that are limited to pore sizes below 500 µm Thus preventing development of porous matrices of larger surface areas for expanding the current pool of applications

CHAPTER ONE: INTRODUCTION

Goals:

a) To develop novel methods for the fabrication of porous chitin matrices to act

as scaffoldings materials in tissue engineering applications

b) To develop novel methods for expanding the current pore size limit of 500 µm c) To develop fabrication procedure that is cost effective and simple

2) Can we overcome the intractability and inertness of chitin by new chemical

modifications methods? By doing so, we can impart new properties desirable for tissue

CHAPTER ONE: INTRODUCTION

1.4) REFERENCES

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CHAPTER ONE: INTRODUCTION

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CHAPTER ONE: INTRODUCTION

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