Potential applications of chitosan based hydrogels in regenerative medicine

POTENTIAL APPLICATIONS OF CHITOSAN-BASED HYDROGELS IN REGENERATIVE MEDICINE RUSDIANTO BUDIRAHARJO NATIONAL UNIVERSITY OF SINGAPORE 2014 POTENTIAL APPLICATIONS OF CHITOSAN-BASED HYDROGELS IN REGENERATIVE MEDICINE RUSDIANTO BUDIRAHARJO (M Eng.), Chulalongkorn University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information, which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Rusdianto Budiraharjo 23 June 2014 I ACKNOWLEDGEMENTS The completion of this PhD study would not be possible without the dedication, commitment, and assistance of numerous people I would like to thank my supervisor Prof Neoh Koon Gee for her guidance and insightful advices during my research in NUS Her hard working attitude, focus, and attention to details have never failed to impress me I would like to express my earnest gratitude to Ms Li Fengmei and Ms Li Xiang, for their assistance, both in their capacity as the Laboratory Officers as well as my personal friends, and their dedication to support me throughout my journey in NUS I am also gracious and humbled by the warm friendship and helping hands of my friends, who brighten my life during the hard time and make the joyful time so memorable To Chen Fei, Liu Gang, Poh Hui, Siew Lay, and Li Han, thanks for your friendship and those great times we spend for sports and great food To my friend Deny Hartono, who never fails to surprise me with his remarkable skills to provide me with great advices, which come full package with humors A special thank is given to Dicky Pranantyo, for his invaluable help during the revision of this thesis Finally, I would like to thank my greatest supporters of my study and life Their trust and faith in me have given me strength and allow me to endure even the most challenging time To my parents and family, thanks for the love and never ending support To my best friend Dr Wilaiwan Chouyyok, thanks for having an unconditional faith in me, even in the most trying of times II TABLE OF CONTENTS DECLARATION I   ACKNOWLEDGEMENTS II   TABLE OF CONTENTS III   SUMMARY .VII   LIST OF TABLES VIII   LIST OF FIGURES IX   LIST OF ABBREVIATIONS XIV   CHAPTER I INTRODUCTION   1.1 Background   1.2 Objectives and scope   1.3 Outline of the thesis CHAPTER LITERATURE REVIEW   2.1 Growth factor incorporation in a carrier   2.2 Chitosan as growth factor carrier for bone and wound healing 10   2.3 Role of HAP in dental physiology 11   2.4 Mineralized CMCS as a potential material for bone grafting 13   2.5 Overview of scaffold fabrication methods 15 CHAPTER ENHANCING BIOACTIVITY OF CHITOSAN FILM FOR OSTEOGENESIS AND WOUND HEALING USING COVALENTLY IMMOBILIZED BMP-2 OR FGF-2   3.1 Introduction 16   3.2 Experimental section 17   3.2.1 BMP-2 and FGF-2 loading on chitosan films 17   3.2.2 Quantification of loaded growth factor 21   3.2.3 SEM of the chitosan films 21   3.2.4 Degradation of films with covalently immobilized growth factor 21   III 3.2.5 Tensile properties of films with covalently immobilized growth factor 22   3.2.6 Growth factor release from the growth factor loaded films 22   3.2.7 Bacterial adhesion on the growth factor loaded films 22   3.2.8 Bioactivity assays of the growth factor loaded films 23   3.2.9 Statistical analysis 25   3.3 Results and discussion 26   3.3.1 BMP-2 and FGF-2 loading on the chitosan films 26   3.3.2 Degradation of films with covalently immobilized growth factor 28   3.3.3 Tensile properties of films with covalently immobilized growth factor 29   3.3.4 Retention of adsorbed and covalently immobilized growth factor 30   3.3.5 Bacterial adhesion on growth factor functionalized films 31   3.3.6 Bioactivity of the BMP-2 loaded films 32   3.3.7 Bioactivity of the FGF-2 loaded films 35   3.4 Summary 37 CHAPTER CHITOSAN FILMS WITH COVALENTLY CO-IMMOBILIZED BMP-2 AND VEGF FOR SIMULTANEOUS STIMULATION OF OSTEOGENESIS AND VASCULARIZATION   4.1 Introduction 38   4.2 Experimental section 39   4.2.1 Covalent co-immobilization of BMP-2 and VEGF on chitosan films 39   4.2.2 Quantification of immobilized growth factors and growth factor release 40   4.2.3 Cell attachment assay 40   4.2.4 Cell proliferation assay 41   4.2.5 ALP activity and calcium deposition assays 41   4.2.6 Gene expression of endothelial markers 43   4.2.7 Immunostaining of CD31 and vWF of ECFCs 44   4.2.8 Matrigel assay 44   4.2.9 Statistical analysis 44   4.3 Results and discussion 45   4.3.1 Amount and retention of growth factor on the functionalized films 45   4.3.2 Effects of immobilized BMP-2 and VEGF on osteoblasts and ECFCs 46   4.4 Summary 55 IV CHAPTER PROMOTING OSTEOGENIC DIFFERENTIATION OF OSTEOBLASTS AND BONE MARROW STEM CELLS USING HAPCOATED CMCS SCAFFOLDS   5.1 Introduction 56   5.2 Experimental section 58   5.2.1 Preparation of HAP-coated CMCS scaffolds 58   5.2.2 SEM and energy dispersive X-ray (EDX) analysis 59   5.2.3 X-ray diffraction (XRD) 60   5.2.4 Fourier transform infra red (FTIR) spectroscopy 60   5.2.5 Ca/P ratio determination 60   5.2.6 Thermogravimetric analysis (TGA) 61   5.2.7 Evaluation of osteoblast functions 61   5.2.8 Evaluation of gene expression from stem cells 63   5.2.9 Statistical analysis 65   5.3 Results and discussion 65   5.3.1 Properties of the coated scaffolds 65   5.3.2 Effects of the coated scaffolds on osteoblasts 69   5.3.3 Effects of the coated scaffolds on bone marrow stem cells 74   5.4 Summary 75 CHAPTER BIOACTIVITY STUDY OF MTA-COATED CMCS SCAFFOLDS AS DENTIN REMINERALIZATION PATCH IN A TOOTH MODEL   6.1 Introduction 77   6.2 Experimental section 78   6.2.1 Preparation of CMCS scaffolds 78   6.2.2 Scaffold mineralization in the tooth model and bulk solution 78   6.2.3 Scaffold characterization 80   6.2.4 Statistical analysis 81   6.3 Results and discussion 82   6.3.1 Properties of the CMCS scaffolds 82   6.3.2 Scaffold mineralization in the tooth model and bulk solution 84   6.4 Summary 89 V CHAPTER CONCLUSIONS   7.1 Summary of major achievements 90   7.2 Suggestions for future work 92 REFERENCES 95 APPENDIX I LIST OF PUBLICATIONS 114 VI SUMMARY Chitosan is a highly promising biomaterial for regenerative medicine due to the combination of its advantageous biological properties and malleability In this thesis, four potential applications of chitosan-based hydrogels are highlighted The first study shows that chitosan films with covalently immobilized bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promoted osteoblast and fibroblast functions to a greater extent than corresponding films with adsorbed BMP-2 or FGF2, due to the higher amount of growth factor retained by covalent immobilization than by adsorption In the second study, BMP-2 and vascular endothelial growth factor (VEGF) were covalently co-immobilized on chitosan films, resulting in simultaneous stimulation of osteoblast and endothelial colony forming cell functions in an additive fashion In the third study, hydroxyapatite (HAP) of different morphologies was coated on carboxymethyl chitosan (CMCS) scaffolds Regardless of the different coating morphology, the HAP-coated scaffolds promoted osteoblast functions and osteogenic differentiation of bone marrow stem cells to a larger extent than noncoated CMCS scaffold Finally, mineral trioxide aggregate (MTA)-coated CMCS scaffolds were evaluated as dentin remineralization patch in a tooth model, and they induced significantly more HAP formation than non-coated CMCS scaffold Overall, these studies demonstrate the feasibility and efficacy of covalent immobilization of growth factor for expanding the potential applications of chitosan hydrogel in bone and wound healing They also highlight the benefits of using the mineralization ability of CMCS hydrogels to improve bone and tooth regeneration VII LIST OF TABLES Table 3.1 List of BMP-2 and FGF-2 loaded chitosan films 20 Table 4.1 Growth factor loading and release from BMP-2 and VEGF functionalized films 42 Table 4.2 Primers for quantitative PCR analysis of CD31 and vWF expression by ECFCs 43 Table 5.1 Ionic composition of mineralizing solutions used in the preparation of HAP-coated CMCS scaffolds 60 Table 5.2 Primers for PCR analysis of osteogenic marker expression by stem cells 64 Table 5.3 Ca/P ratio and amount of HAP on the coated CMCS scaffolds 69 Table 6.1 Ionic composition of SBF 80 Table 6.2 Mercury porosimetry results of CaC scaffold 84 Table 6.3 Ca/P ratio of CaP crystals formed on CaC and CaMT scaffolds that were mineralized in the tooth model over 14 days 86 VIII Gümüşderelioğlu M.; Aday S Heparin-functionalized chitosan scaffolds for bone tissue engineering Carbohydr Res 2011, 346, 606-613 Habibovic P.; de Groot K Osteoinductive 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Neoh K G.; Kang E T Enhancing bioactivity of chitosan film for osteogenesis and wound healing by covalent immobilization of BMP-2 and FGF-2 J Biomater Sci Polym Ed 2013, 24, 645-662 114 ... listed in Table 3.1 Adsorption of growth factor was carried out by immersing a pristine disc in 100 µl of loading solution containing µg/ml of BMP-2 or FGF-2 in PBS for h, followed by a rinsing... efficacy of covalent immobilization of growth factor for expanding the potential applications of chitosan hydrogel in bone and wound healing They also highlight the benefits of using the mineralization... thesis was to explore the potential applications of chitosan- based hydrogels in regenerative medicine, with the focus on two main strategies: (1) covalent immobilization of growth factors to enhance