謝國煌臺灣大學：化學工程學研究所張根育Chang, Ken-YuKen-YuChang2007-11-262018-06-282007-11-262018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/52298骨創傷的修復在臨床醫學上是個很重要的課題，藉由組織工程的發展，我們得以發展出能夠引導骨缺陷修復的人工植入材料。本論文的主軸乃是設計及開發以幾丁聚醣天然高分子為主之生醫材料，藉由不同形式之基材設計與生物活性分子之導入，欲發展出能夠取代現今骨修復之填充材料，使其具有臨床應用性之價值。本論文主要包括了以下之幾部分: (1) 骨引導及骨誘導性之幾丁聚醣薄膜材料之開發:乃是利用化學固定骨形態生長因子於幾丁聚醣薄膜上，進行體外骨母細胞培養及大型骨缺陷之動物體內測試。研究結果發現經由化學固定能夠使生長因子發揮長效且專一作用於受傷部位之功能，且表面固定有生長因子之薄膜顯著提高細胞活性及分化，證實經過化學固定後骨生長因子仍保有生物活性。在植入兔子橈骨之大型骨缺陷後，本研究開發之表面固定生長因子之幾丁聚醣薄膜能不同於對照組與純幾丁聚醣薄膜，在短時間內(六週)即引導骨缺陷之修復癒合。 (2) 三維立體之多巨孔性幾丁聚醣支架之開發:乃是以溫度相轉換法為基礎，加以修正，以改良過之方式製造具有專一方向性之巨大孔洞分佈的多孔性基材。 (3) 幾丁聚醣支架於不同骨生長動物模型比較:前法製作出之幾丁聚醣支架分別進行兔子橈骨大型骨缺陷與老鼠門牙拔除後齒槽骨再生之體內測試。研究結果發現，純幾丁聚醣支架於植入橈骨缺陷中12週後仍未出現骨癒合的徵兆，只有在骨斷面新生出些許的骨痂；然而在植入牙齒拔牙後之傷口後一個月，不僅齒槽骨的再吸收明顯被抑制，且齒槽骨大量地再生而填充滿整個拔牙傷口。此研究結果代表幾丁聚醣支架的骨引導性明顯會隨不同的植入部位而有所變化，而此研究開發出之純幾丁聚醣支架在齒槽骨引導再生上有非常突出之表現。 (4) 骨引導及骨誘導性之多巨孔幾丁聚醣支架之開發:乃是沿用化學固定骨生長因子之方式，將骨型態生長因子固定於多孔性支架表面，製作出具長效型且易植入之骨填充材料。動物實驗中經由與純幾丁聚醣、表面物理吸附骨生長因子之幾丁聚醣支架之比較發現，化學固定骨生長因子之支架大大的提升了骨組織再生及癒合的速度與機率。 (5) 幾丁聚醣與氫氧基磷灰石複合多孔性支架之開發:藉由與陶瓷材料製作成複合基材，能夠提升支架之機械性質與骨引導性。研究結果發現複合支架仍保有專一方向性、多孔性及巨大孔洞之特徵。隨氫氧基磷灰石之導入，材料之機械性質上升而裂解速率與程度隨之下降，且於兔子橈骨模型中發現骨缺陷能在短時間內修復且重整為緻密骨之組織結構。Tissue engineering has shown great promise as a viable alternative to current bone graft solutions due to its use of biocompatible, biodegradable scaffolds as support systems for cellular attachment, proliferation, migration, and maintenance of normal phenotypic expression. The overall goal of this thesis was to design and develop biodegradable, biocompatible chitosan-based scaffolds that will be practical alternatives to current bone repair materials. The first specific aim was to develop a new method to prepare an osteoinductive-osteoconductive bioimplant based on chitosan and recombinant human bone morphogenetic protein-2 (rhBMP-2). BMP-2 was chemically immobilized on the chitosan membrane in order to provide a bioactive surface that can enhance bone-regeneration capacity. Cellular evaluation demonstrated this novel rhBMP-2-immbilized membrane to be biocompatible and osteoionductive, with evidence of enhanced cellular proliferation and early alkaline phosphatase expression. Accelerated bone healing observed histologically and radiographically in the rabbit radius critical-sized defect indicated that it would seen to be applicable for inducing significant and localized bone formation in future guided tissue regeneration. The next objective was to develop 3-dimensional chitosan scaffolds. The macroporous chitosan sponges were fabricated by the modified method based on thermally induced phase separation. A combination of solid-liquid phase separation/solvent-extraction/neutralization/freeze drying paths were successfully developed to fabricate highly anisotropic chitosan scaffolds with high porosities (>90%) and macropores (>100µm) which may favor cell growth, migrate, nutrient transportation and further bone tissue regeneration. When implanted in a segmental long bone defect, however, macroporous chitosan sponges did not show sufficient osteoconductivity for complete bone defect healing. It was therefore another aim of this thesis to chemically immobilize rhBMP-2 on macroporous chitosan scaffolds in order to provide osteoinductivity for quick and promoted bone regeneration. Bone defect bridging and union was achieved within 4 weeks in 8 of 10 specimens. Such a high bone healing efficiency of rhBMP-2-immobilized chitosan sponges revealed that they are good candidate bioimplants for guided tissue regeneration application. A trial related to implantation of pure chitosan sponge in the tooth extraction socket was also made in this thesis. Surprisingly, unlike long-bone defect model, the alveolar bone was preserved and regenerated when the extraction socket was implanted with pure chitosan sponge. It could be concluded that osteoconductivity of chitosan has much to do with the implanted site. Pure chitosan sponge has great potential to be used as a socket filler to prevent adsorption of alveolar bone after tooth extraction. In spite of the promising performance of the chitosan scaffolds, they could not be applied in the load-bearing bone defects because polymer themselves are mechanically too weak. Consequently, a final aim was to prepare chitosan/hydroxyapatite composites designed to mimic the properties of bone, which itself is a composite. The mechanical properties were significantly improved as hydroxyapatite was incorporated. In vivo animal studies in male New Zealand white rabbits showed that composite scaffolds provided a suitable structure for new cellular infiltration throughout the scaffold pore structure. Composite scaffolds also supported the vascularization of new tissue within the defect site, as well as newly mineralized bone tissue at the margins of the defect. In summary, this dissertation demonstrates the successful generation of a biomimetic scaffold capable of localizing growth factor delivery which indicates significant potentials in tissue engineering and regenerative medicine.ABSTRACT (IN CHINESE) II ABSTRACT (IN ENGLISH) IV CONTENTS VII LIST OF FIGURES XIII LIST OF TABLES XX LIST OF TABLES XX CHAPTER 1 1 INTRODUCTION, SUMMARY AND AIM OF THE THESIS 1 1.1. INTRODUCTION 1 1.2. SUMMARY OF THE THESIS 2 1.3 AIM OF THE THESIS 3 CHAPTER 2 5 STRUCTURE, FUNCTION AND PHYSIOLOGY OF BONE 5 2.1 BONE 5 2.1.1 Characteristics of bone 6 2.1.2 Seven functions of bones 8 2.1.3 Five types of bones 9 2.1.4 Bone cells 11 2.1.5 Matrix 12 2.1.5. Formation 13 2.2 OSSEOUS TISSUE 15 2.2.1 Formation 15 2.2.2 Types 15 2.2.3 Functions of osseous tissue 16 2.2.4 Osseous tissue versus bones 16 2.3 BONE HEALING 18 2.3.1 Physiology and process of healing 18 2.3.2 Phases of fracture healing 19 2.3.3 Inadequate healing or formation 21 2.3.4 Medical Treatments 22 2.3.5 Osseointegration 22 CHAPTER 3 23 BONE TISSUE ENGINEERING 23 3.1. INTRODUCTION 23 3.2. CLINICAL NEEDS IN THE BONE REPLACEMENT AND REGENERATION FIELD 24 3.2.1 Methods to Augment Deficient Bone 24 3.2.2 .Autograft 25 3.2.3. Allograft 25 3.2.4. Metals and ceramics 26 3.3. TISSUE ENGINEERING 26 3.3.1. Definition 26 3.3.2. Scaffolds – Temporary Matrices for Bone Growth 27 3.3.3 Scaffolds Essential Properties 28 3.3.4 Biomaterials used as Bone TE Scaffolds 30 3.4. SCAFFOLD DESIGN AND FABRICATION 38 3.4.1 Solvent-Casting and Particulate Leaching Technique 39 3.4.2 Gas-Foaming Process 40 3.4.3 Emulsion Freeze Drying 41 3.4.4 Electrospinning Technique 42 3.4.5 Rapid-Prototyping Techniques 42 3.4.6 Thermally Induced Phase Separation (TIPS) 43 3.4.7 Surface Modification 44 3.5 OSTEOACTIVE AGENTS 45 3.5.1 Bone morphogenetic protein 45 3.5.2 BMP signal transduction 47 3.5.3 Recombinant BMPs 48 3.5.4 Functions of BMPs 49 3.5.5 BMP carriers 50 3.6 ANIMAL MODELS 51 CHAPTER 4 55 CHITOSAN MEMBRANE WITH CHEMICALLY-IMMOBILIZED RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2 FOR ENHANCED GUIDED BONE REGENERATION 55 4.1 INTRODUCTION 56 4.2 MATERIALS AND METHODS 60 4.2.1 Materials 60 4.2.2 Preparation of the chitosan membrane 60 4.2.3 Chemical immobilization of rhBMP-2 61 4.2.4 Quantitative determination of immobilized rhBMP-2 61 4.2.5 Biologiacl activity of immobilized rhBMP-2; cell attachment and proliferation on membranes containing immobilized rhBMP-2 63 4.2.6 Rabbit critical-sized defect model 64 4.2.7 Statistical analysis 66 4.3 RESULTS 67 4.3.1 Membrane morphology 67 4.3.2. Chemical immobilization of rhBMP-2 68 4.3.3 In vitro growth factor release behavior; chemical immobilization versus physical adsorption 69 4.3.4 In vitro cell culture 72 4.3.5.. Cell morphology 72 4.3.6. In vivo bone regeneration 79 4.4 DISCUSSION 83 4.4.1 Definition of GTR 83 4.4.2 Membrane for GTR 84 4.4.3. Advantage of chemical immobilization 84 4.4.4. In vitro MG-63 cell interaction 85 4.4.5. In vivo animal model 86 4.5 CONCLUSION 87 CHAPTER 5 89 PREPARATION OF MACROPOROUS CHITOSAN SCAFFOLDS FOR BONE TISSUE ENGINEERING 89 5.1 INTRODUCTION 90 5.2 EXPERIMENTS 93 5.2.1 Preparation of CS scaffolds 93 5.2.2 Porosity 94 5.2.3 Pore size distribution 94 5.2.4 In vitro degradation test 95 5.2.5 Morphology Observation 96 5.2.6 Mechanical Testing 96 5.2.7 Rabbit critical-sized defect model 96 5.2.8 Statistical analysis 97 5.3 RESULTS 100 5.3.1 Freeze-drying method vs. phase separation/solvent extraction method 100 5.3.2 Effect of quenching temperature on the microstructure and pore size 103 5.3.3 Effect of polymer concentration on microstructure 105 5.3.5. Porosity 109 5.3.7 In vitro degradation 113 5.3.8 Rabbit critical-sized radius defect model 114 5.4 DISCUSSION 120 5.4.1 Thermally induced phase separation method 120 5.4.2 Anisotropic structure 122 5.4.3 Different pore microstructures for different uses 123 5.4.4 Effect of quenching temperature on the microstructure and porosity 124 5.4.5 Effect of phase separation on mechanical property 125 5.4.6. Effect of porosity on the scaffold mechanical property 125 5.4.7 . Effect of porosity and pore size in vitro 126 5.4.8. Effect of porosity in vivo 128 5.4.9. Effect of pore sizes in vivo 129 5.4.10 In vitro degradation 131 5.5 CONCLUSION 133 CHAPTER 6 135 CHITOSAN SPONGE FOR HEALING OF ALVEOLAR BONE AFTER TOOTH EXTRACTION 135 6.1 INTRODUCTION 136 6.2 EXPERIMENTS 139 6.2.1 Preparation of CS sponges 139 6.2.2 Animal model and surgery 139 6.2.3 Euthanasia and tissue harvest 139 6.2.4 Tissue fixation, decalcification, embedding, and sectioning 140 6.3 RESULTS 143 6.3.1 Characteristics of chitosan sponge 143 6.3.2 Radiographic examination 143 6.3.3 Macroscopic appearance of the harvested specimen 144 6.3.4 Histological analysis 144 6.4 DISCUSSION 152 6.4.1 Degradation of CS sponge in the rat alveolar bone defect 152 6.4.2 Dental implant in clinical 153 6.4.3 Importance of alveolar bone after tooth extraction for dental implant 154 6.4.4 Reduction of alveolar bone resorption and regeneration of alveolar bone 154 6.4.5 Successful healing of a extraction socket by chitosan sponge without applying the growth factor 156 6.5 SUMMARY 157 CHAPTER 7 158 CHEMICAL IMMOBILIZATION OF RHBMP-2 ON MACROPOROUS CHITOSAN SPONGES FOR HEALING OF CRITICAL-SIZED LONG BONE DEFECTS 158 7.1 INTRODUCTION 159 7.2 EXPERIMENTS 162 7.2.1 Fabrication of macroporous chitosan sponge 162 7.2.2 Chemical immobilization of rhBMP-2 162 7.2.3 In vivo animal model 162 7.3 RESULTS 162 7.3.1 Bone healing efficiency of defects with various treatments 163 7.3.2 Macro-appearance of the harvested bone specimen of rhBMP-2 grafted CS sponge implanted defect. 163 7.3.3 Radiographic examination 166 7.3.4 Histological examination 172 7.4 DISCUSSION 173 7.4.1 Macroporous chitosan sponge for rhBMP-2 immobilization 173 7.4.2 Possible mechanism of rhBMP-2 grafted scaffolds for inducing bone regeneration 173 7.4.3 Carriers for growth factor 174 7.5 CONCLUSION 175 CHAPTER 8 177 MACROPOROUS HYDROXYAPATITE-ENHANCED-CHITOSAN SCAFFOLDS AS BONE CONDUCTIVE MATERIALS 177 8.1 INTRODUCTION 178 8.2 EXPERIMENTS 180 8.2.1 Preparation of CS and CS/HAP composite scaffolds 180 8.2.2 Porosity 180 8.2.3 Pore size distribution 180 8.2.4 In vitro degradation test 181 8.2.5 Morphology Observation 181 8.2.6 Mechanical Testing 181 8.2.7 Rabbit critical-sized defect model 181 8.3 RESULTS 181 8.3.1Morphology of CS/HAP composite scaffold 181 8.3.2 Pore size distribution 185 8.3.3 Porosity 186 8.3.4 Mechanical property 188 8.3.5 In vitro degradation 192 8.3.6 In vivo rabbit radius critical-sized defect model: Radiographic examination 193 8.3.7 Histological investigation 199 8.4 DISCUSSION 204 8.4.1 Why chitosan/hydroxyapatite composite? 204 8.4.2 Fabrication method 205 8.4.3 Effect of HAP on pore structure and porosity 205 8.4.4 Healing process of fractured bone 206 8.4.5 Effect of HAP content on the bone regeneration 207 8.5 CONCLUSION 208 CHAPTER 9 209 CONCLUSION AND FUTURE CONSIDERATIONS 209 9.1 CONCLUSION 209 9.2 FUTURE WORK 212 REFERENCE 217 PUBLICATIONS 24911110597 bytesapplication/pdfen-US幾丁聚醣骨生長因子化學固定氫氧基磷灰石骨組織工程chitosanbone growth factorchemical immobilizationhydroxyapatitetissue engineering[SDGs]SDG3[SDGs]SDG11thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52298/1/ntu-96-F88524037-1.pdf