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 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 TABLE OF CONTENTS CHAPTER ONE: INTRODUCTION PREFACE 1.1) CHITIN 1.1.a) Introduction 1.1.b) Sources 1.1.c) Structural Classification 1.1.d) Solubility 1.1.e) Biodegradability 1.1.f) Health Issues, Benefits and Risks 1.1.g) Limitations 10 11 13 1.2) TISSUE ENGINEERING 1.2.a) Introduction 1.2.b) Cell-Polymer Matrix Construct 1.2.c) Polymer Matrix 1.2.d) Cell-Polymer Matrix Generation 14 18 19 20 1.3) AIM OF PROJECT 22 1.4) REFERENCES 24 CHAPTER TWO: FABRICATION OF CHITIN MATRIXES 2.1) INTRODUCTION 2.1.a) Variation of Pre-Lyophilization Process and Drying Methods 2.1.b) Internal Bubbling Process (IBP) 34 36 37 2.2) EXPERIMENTAL 2.2.a) General 2.2.b) Variation of Pre-Lyophilization Process and Drying Methods 2.2.c) Internal Bubbling Process (IBP) 38 44 48 2.3) RESULTS AND DISCUSSION 2.3.a) Variation of Pre-Lyophilization Process and Drying Methods 2.3.b) Internal Bubbling Process (IBP) 52 66 2.4) SUMMARY 81 2.5) REFERENCES 83 ii TABLE OF CONTENTS CHAPTER THREE: CHEMICAL MODIFICATION OF CHITIN 3.1) INTRODUCTION 3.1.a) Synthesis of New Fluorinated Chitin Derivatives 3.1.b) Synthesis of Chitosan-Polypyrrole Hybrids 3.1.c) Synthesis of Reversible Water Swellable Chitin Gel 90 93 94 96 3.2) EXPERIMENTAL 3.2.a) General 3.2.b) Synthesis of New Fluorinated Chitin Derivatives 3.2.c) Synthesis of Chitosan-Polypyrrole Hybrids 3.2.d) Synthesis of Reversible Water Swellable Chitin Gel 98 99 102 108 3.3) RESULTS AND DISCUSSION 3.3.a) Synthesis of New Fluorinated Chitin Derivatives 3.3.b) Synthesis of Chitosan-Polypyrrole Hybrids 3.3.c) Synthesis of Reversible Water Swellable Chitin Gel 110 123 135 3.4) SUMMARY 141 3.5) REFERENCES 143 CONCLUSION 154 APPENDIX 158 PUBLICATIONS 163 iii 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 -chitin as it is the most abundant of the 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 iv SUMMARY that can accommodate the ever-increasing number of potential tissue engineering applications In this study, we also explore important parameters that affect the morphology, pore size, water uptake ability and in vitro cytotoxicity of these porous matrixes To date, only examples of deoxyfluorocellulose have been reported1,2&3 Fluorinated chitin derivatives were not known although the bromination, chlorination and iodination of chitin have been investigated4,5 In this dissertation, we present the first 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, & 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 v SUMMARY intractability Depending on the application, the degree of swelling could be customized accordingly N Kasuya., K Iiyama, G Meshitsuka, A Ishizu, Preparation of 6-O-deoxy-6fluorocellulose, Carbohydrate Research, 260, 251, 1994 N Kasuya, K Iiyama, A Ishizu, Synthesis and characterization of highly substituted deoxyfluorocellulose acetate, Carbohydrate Research, 229, 131-139, 1992 N Kasuya, K Iiyama, G Meshitsuka, T Okana, A structural study of fluorinated cellulose: crystallization of fluorinated cellulose by conversion treatments used for cellulose, Carbohydrate Polymers, 34, 229-234, 1997 M Sakamoto, H Tseng, K I Furuhata, Regioselective chlorination of chitin with Nchlorosuccinimide triphenylphosphine under homogenous conditions in lithium chloride N,N-dimethylacetamide, Carbohydrate Research, 265, 271-280, 1994 H Tseng, K Takechi, K I Furuhata, Chlorination of chitin with sulfuryl chloride under homogeneous conditions, Carbohydrate Polymers, 33 13-18, 1997 M.T Nguyen, A F Diaz, A novel method for the preparation of magnnetic nanoparticles in a polypyrrole powder, Adv Materials, 858, 1994 vi SUMMARY S Machida, S Miyata, A Techagumpuch, Chemical synthesis of highly electrically conductive polypyrrole, Synthetic Met, 31(3), 311, 1989 S Machida, S Miyata, A Techagumpuch, Chemical synthesis of highly electrically conductive polypyrrole, Synthetic Met., 1989, 31, 311 vii CHAPTER ONE: INTRODUCTION CHAPTER THREE: CHEMICAL MODIFICATIONS OF CHITIN 44 B Garner, A Geogevich, A.J Hodgson, L Liu, G.G Wallace, Polypyrrole-heparin composites as stimulus responsive substrates for endothelial cell growth., J Biomed Mater Res., 44, 121-129, 1999 45 H C Li, E Khor, Interaction of collagen with polypyrrole in the production of hybrid materials, Polym Int., 35, 53, 1994 46 E Khor, H C Li, A Wee, In situ polymerization of pyrrole in animal tissue in the formation of hybrid, Biomaterials, 16, 657, 1995 47 H C Li, E Khor, A collagen polypyrrole hybrid: influence of 3-butanesulfonate substitution, Macromol Chem Phys., 196, 1801, 1995 48 E Khor, J L H Whey, Interaction of chitosan with polypyrrole in the formation of hybrid biomaterials, Carbohyd Polym., 26, 183, 1995 49 K Nakamae, T Nizuka, T Miyata, M Furukawa, T Nishino, K Kato, T Inoue, A.S Hoffman, Y Kanzaki, Lysozyme loading and release from hydrogels carrying pendent phosphate groups, J Biomater Sci Polymer Edn, 9(1), 43, 1997 50 M Kanke, H Katayama, S Tsuzuki, H Kuramoto, Application of chitin & Chitosan to Pharmaceutical Preparations I: Films Preparation and In Vitro Evaluation, Chem Pharm Bull., 37(2), 523, 1989 Page 150 CHAPTER THREE: CHEMICAL MODIFICATIONS OF CHITIN 51 J.L West, S.M Chowdhury, M.J Slepian, J.A Hubbell, Inhibition of thrombosis and intimal thickening by in situ photopolymerization of thin hydrogel barrier, Proc Natl Acad Sci USA, 91, 5967, 1994 52 A.S Swahney, C.P Pathak, J.J Rensburg, R.C Dunn, J.A Hubbell, Optimization of photopolymerised bioerodible hydrogel properties for adhesion prevention, J Biomed Mater Res, 28, 581, 1994 53 S Hirano, K Horiuchi, Chitin gels, Int J Biol Macromol, 11, 253, 1989 54 S Tokura, N Nishi, Novel drug delivery system by chitin derivatives, Macomol Symp., 99, 201, 1995 55 E Khor, A.C.A Wan, C.F Tee, G.W Hastings, Reversible Water-Swellable Chitin Gel, J Polym Sci A: Polym Chem., 35, 2049, 1997 56 A.C.A Wan, E Khor, Surface Carboxylation of a Chitin Hydrogel, J Bioactive and Compatible Polymers, 12, 208, 1997 57 A Kimbaris, G Varvounis, Reduction of 2- and 3- acrylpyrroles: A new synthesis of the pyrrolo[1,2-b] cinnolin-10-one ring system from 1-(4-methylphenyl)sulfonyl-1Hpyrrole, Tetrahedron, 56(49), 9675, 2000 Page 151 CHAPTER THREE: CHEMICAL MODIFICATIONS OF CHITIN 58 M Kakushima, P Hamel, R Frenette, J Rokach, Regioselective synthesis of acylpyrroles, J Org Chem, 48, 3214, 1983 59 K Kurita, S Inoue, S Nishimura., Preparation of soluble chitin derivatives as reactive precursors for controlled modifications: tosyl chitins and iodo chitin, J Polym Sci., Part A: Polym Chem., 29, 937, 1991 60 M Zhang, H Inui, S Hirano, A facile method for the preparation of 6deoxyderivatives of chitin., J Carbohy Chem., 16(4-5), 673-679, 1997 61 M Zhang, A Haga, H Sekiguchi., S Hirano, Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx Mori) pupa exuvia, Int J Biol Macromol., 27, 99-105, 2000 62 N Kasuya, K Iiyama, G Meshitsuka, A Ishizu, Preparation of 6-deoxy-6- fluorocellulose, Carbohydrate Research, 260, 251-257, 1994 63 T Oishi, T Kawamoto, M Fujimoto, Synthesis and polymerization of maleimides containing perfluoroalkyl groups, Polym J., 26(5), 613-622, 1994 64 A Domard, C Gey, M Rinaudo, C Terrassin, 13C and 1H NMR spectroscopy of chitosan and N-trimethyl chloride derivatives, Int J Biol Macromol., 9, 233-237, 1987 Page 152 CONCLUSION CONCLUSION In this dissertation, new methodologies have been developed to utilize chitinbased materials for potential applications in tissue engineering Physical and chemical modifications of chitin were adopted as a systematic attempt to address the current limitations associated with the utilization of chitin as a raw material In the aspect of physical modifications, chitin was subjected to various treatments in order to create matrixes with various pore sizes The intrinsic properties of chitin that is intractable, inert and insoluble in common organic solvents presented new challenges in the fabrication of matrixes Therefore, until now no chitin porous matrixes have been reported Lyophilization (L) and internal bubbling process (IBP) were developed to address this limitation The key in matrix fabrication by these two methods is simplicity and better control of pore sizes in the procedures With lyophilization (L), chitin matrixes obtained were between 10-500 m and internal bubbling process (IBP) produced pores with dimensions of between 100-1000 m The utility of these procedures enable careful tailored pore size matrixes for tissue-engineering applications with specific pore size requirement The wide range of pore sizes can also accommodate the expanding lists of tissue regeneration systems The simplicity and cost effectiveness of the two procedures further substantiate their efficacy for matrix fabrication Page 154 CONCLUSION Current methodologies of chemical modifications of chitin are developed nearly decade ago With increasing need to expand the number of chitin derivatives for new applications, methodologies were extended to prepare products previously not available New products were obtained from direct fluorination of chitin with diethylamino sulfur trifluoride (DAST) Controlled fluorination with DAST is feasible, giving a range of degree of substitution between 50-98% depending on reaction time MTT assay shows that fluorinated chitin materials are non-cytotoxic and this property appears to be a function of the degree of fluorine present in the derivative Further investigations of this new fluorinated chitin as well as comparison with deoxybromo chitin, deoxychloro chitin, deoxyiodo chitin as well as deoxyfluoro cellulose will provide better insights into potential cells adhesion and vascular prostheses systems in tissue engineering The successful preparation of chitosan-pyrrole substituted derivatives demonstrated that biopolymer-polypyrrole hybrids can be covalently bonded With the conductive characteristics of polypyrrole, this work will pave the way into the preparation of conductive biopolymer Further work for optimization of hybrids for higher conductivity levels present potential utilizations in the area of nerve or brain cells generation Page 155 CONCLUSION Chitin as one of the most important biomass in the world; currently the usage of chitin is limited by its inherent intractability and insolubility Due to its abundance, this biomass plays a very important role in providing us with sustainable resource Tissue engineering is an area of enormous interest, high cost and will have a tremendous impact on medicine in coming decades With the increasing cost of medical treatments and research, it is pivotal to search for an alternative cheaper renewable source of materials This work presented the integration of chitin and tissue engineering The availability of new chitin methodologies will provide a pool of potential materials for tissue engineering This will also promote better understanding of chitin as a raw material and a renewable resource for the future Page 156 APPENDIX APPENDIX MATERIALS AND METHODS Chitin Chitin was obtained from Polyscience, USA, and purified by stirring in 1M NaOH at room temperature for days and in 1M HCl for 1h The degree of acetylation (DA) was determined by FT-IR1 spectroscopy (Figure 34) The DA by the improved method proposed was obtained from the relationship: DA = (A1655 / A3450) x 115 IR transmission of at least 10% at 3450cm-1 was used to ensure the validity of the method reported From the FT IR spectrum, the values of (A1655 / A3450) was determined to be (6.8 / 8.5) and the DA of chitin was estimated by: DA = (6.8 / 8.5) x 115 ~ 92% Page 158 APPENDIX Figure 34: FTIR spectrum of chitin Page 159 APPENDIX The DA value for chitin obtained from FTIR method mentioned previously was confirmed by elemental analysis Anal Calc for (Chitin: C8H13NO5)0.9 (Chitosan: C6H11NO4)0.1 : C: 47.75, H: 6.40, N: 6.96 Found: C: 47.55, H: 6.35, N: 6.75 From the elemental analysis values, the DA for chitin was estimated at ~90% Therefore, the DA obtained from FTIR method is in agreement with results obtained from elemental analysis Solvents and reagents All other solvents and reagents were analytical grade, obtained from commercial sources and used without further purification unless stated otherwise Preparation of 0.5% Chitin Solution in 5% Lithium Chloride (LiCl) / Dimethyl Acetamide (DMAc) Anhydrous LiCl (5.0g) was dried at 130oC for about ½h, cooled inside a desiccator and dissolved in 100ml of DMAc by magnetic stirring Chitin flakes (0.05g) was suspended in this solution and shaken overnight at 150rpm, 4oC in a refrigerated shaking incubator to give 100ml of 0.5% (w/w) of chitin solution in 5% DMAc/LiCl solvent system The viscous clear solution was filtered through glass wool and stored in glass containers at room temperature Page 160 APPENDIX REFERENCES A Baxter, M Dillon, K.D.A Taylor, G.A.F Roberts, Improved method for IR determination of the degree of N-acetylation of chitosan Int J Biol Macromol, 14, 166, 1992 Page 161 PUBLICATIONS PUBLICATIONS 1) K.S Chow, E Khor; Synthesis of soluble Chitin Derivatives; ADVANCES IN CHITIN SCIENCE, Vol 3, 147, Proceedings of the 3rd Asia Pacific Chitin / Chitosan Symposium, Keelung, Taiwan, ROC; 1998 2) K.S Chow; E Khor; Syntheses and Characterization of New Chitin Derivatives; Proceedings of the 1st Singapore Chemical Conference, Singapore; 1998 3) K.S Chow, E Khor; Reversible Water-Swellable Chitin Gel; Modulation of Swellability; ADVANCES IN CHITIN SCIENCE, Vol 4, 334, Proceedings of the 3rd International Conference of the European Chitin Society, Potsdam, Germany; 1999 4) K.S Chow, E Khor; Fabrication of Porous Chitin Matrixes; ADVANCES IN CHITIN SCIENCE, Vol 4, 355, Proceedings of the 3rd International Conference of the European Chitin Society, Potsdam, Germany; 1999 5) Y.L Lam, K.S Chow, E Khor; Preparation and Characterization of Covalently Bonded Biopolymer-Polypyyrole Hybrid Materials; J POLYM RES; 6(4): 203210; OCT 1999 6) W.K Loke, S.K Lau, L.Y Lim, E Khor, K.S Chow; Wound Dressing with Sustained Anti-Microbial Capability; JOURNAL OF BIOMEDICAL MATERIALS RES; Vol 53, 8-17; 2000 Page 163 PUBLICATIONS 7) K.S Chow, E Khor; Novel Fabrication of Open-Pore Chitin Matrixes; BIOMACROMOLECULES; 1(1): 61-67; SEP 2000 8) K.S Chow, E Khor, A.C.A Wan; Porous Chitin Matrixes for Tissue Engineering: Fabrication and In Vitro Cytotoxic Assessment; J POLYM RES; 8(1): 2-35; MAR 2001 9) K.S Chow, E Khor; New fluorinated chitin derivatives: synthesis, characterization and cytotoxicity assessment; CARBOHYD POLYM; 47(4): 357363; MAR 2002 Page 164 .. .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... 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... 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