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系統識別號 U0026-1102201514575800
論文名稱(中文) 製備幾丁聚醣/鍶鈣磷三維支架應用於骨組織工程
論文名稱(英文) Three Dimensional Nanofibrous Scaffold of Chitosan/Strontium-Substituted Calcium Phosphate for Bone Tissue Engineering
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 103
學期 1
出版年 104
研究生(中文) 蔡尚旂
研究生(英文) Shang-Chi Tsai
學號 p86024043
學位類別 碩士
語文別 英文
論文頁數 96頁
口試委員 指導教授-李澤民
指導教授-張志涵
口試委員-林睿哲
口試委員-陳炳宏
中文關鍵字 電紡絲  幾丁聚醣  鍶置換氫氧基磷灰石  化學沉澱法  水熱法 
英文關鍵字 Electrospinning  Chitosan  Sr-substituted hydroxyapatite  Chemical precipitation method  Hydrothermal method 
學科別分類
中文摘要 幾丁聚醣(Chitosan)是一種具有以下許多優點的高分子,例如:良好的生物相容性、生物可降解性及容易操作等,因此非常適合應用於組織工程的材料。在組織工程的應用,幾丁聚醣已被用來作為支架培養骨細胞、軟骨細胞、皮膚組織、神經細胞和肝細胞等,其中又以培養骨細胞和軟骨細胞的應用最多,這是因為幾丁聚醣具有較好的機械性質、促進傷口癒合及抗菌的特性,因此被視為良好的培養基材。
氫氧基磷灰石(hydroxyapatite, HA)是人體骨骼的主要成分之一,並具有良好的生物相容性、骨引導性、無毒及刺激性,為優良的生醫植入材。鍶是一種人體必需的微量元素,98%存在於骨頭中,可以促進造骨細胞分化以及抑制破骨細胞吸收。鍶置換氫氧基磷灰石(Sr-substituted hydroxyapatite, SrHA),在體內研究指出,鍶可以刺激骨細胞生長使骨頭快速癒合。本研究使用了化學沉澱法與水熱法來合成HA及SrHA,利用穿透式電子顯微鏡觀察鍶鈣磷化合物的型態,並透過能量散射光譜儀、傅立葉轉換紅外光譜儀及X光繞射儀可知合成的鍶鈣磷化合物主要由氫氧基磷灰石組成,且鍶離子有參雜入其結構。
生醫陶瓷或是高分子各具有優缺點,此研究運用幾丁聚醣(chitosan)和HA及SrHA來作為骨組織修復的支架材料,採用目前熱門且新穎的電紡絲技術,製備出含鍶鈣磷化合物的幾丁聚醣奈米纖維支架模擬3D細胞外基質(Extracellular matrix)的結構。利用電子顯微鏡及穿透式電子顯微鏡觀察顯示鍶鈣磷化合物均勻分散於電紡絲的內部與外部,透過能量散射光譜、X光繞射分析及傅立葉轉換紅外光譜證明支架中鍶鈣磷化合物的存在。將試片浸泡於模擬體液中14天之後,利用電子顯微鏡觀察試片表面顯示類骨磷灰石的沉積,並藉由感應耦合電漿質譜分析儀量測鍶離子於模擬體液中的釋放。於體外細胞培養(MC3T3-E1),經由細胞增生與分化實驗得知,含SrHA的電紡絲支架最能促進細胞的增生與分化,適合應用於骨組織的修復。
英文摘要 Because of the biocompatibility, biodegradability, and better mechanical properties, chitosan is a good choice for tissue engineering materials. In the applications of tissue engineering, chitosan has been used for cell cultures. For example, bone, cartilage, skin, nerve and so on. Among the above, bone and cartilage cells are the most used in the cell culture. Chitosan has many attractive properties to be good culture template owing to the promoting wound healing and antibiotic.
Hydroxyapatite (HA) is one of the major compositions in human bone. Due to its good biocompatibility, osteoconductivity, non-toxic, and non-irritating, hydroxyapatite is a good material of implants. Strontium (Sr) is a trace element in human body and 98% of the total content exists in the skeleton. It can enhance the number and activity of osteoblasts as well as reduce the number and activity of osteoclasts. Therefore, Sr-substituted hydroxyapatite (SrHA) also can improve bone formation and inhibit bone resorption. In this study, HA and SrHA are synthesized under chemical precipitation method and hydrothermal method. Microstructure was observed with transmission electron microscopy (TEM). Through the analyses of energy dispersive x-ray spectroscopy (EDS), X-ray diffraction (XRD), and Fourier transform infrared spectrometer (FTIR), the calcium phosphate with syntheses was mainly hydroxyapatite and combined with strontium.
Bioceramics and polymers have some defects. Consequently, chitosan, HA and SrHA are used to be bone repair materials in this study. Besides, the popular and novel technique "Electrospinning" was used to fabricate chitosan nanofibrous scaffolds containing calcium phosphate. Electrospining provided porous structures which are quite similar to the extracellular matrix (ECM). With field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM), the images showed the calcium phosphate particles were well-dispersed in the nanofibers. With the spectrums of energy dispersive x-ray (EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR), the existence of calcium phosphate was proved. After 14-day immersion in SBF, SEM images showed the deposition of apatite on the surface and the release of strontium ion in SBF was observed through inductively coupled plasma-mass (ICP) analysis. In vitro test, by Alamar blue and ALP assay, cells (MC3T3-E1) proliferate and differentiate the most at the scaffolds containing SrHA and the scaffolds are suitable for bone tissue repair.
論文目次 Chapter 1 Introduction 1
1.1 Background 1
1.2 Bone tissue engineering 2
1.2.1 Biomaterials 2
1.2.1.1 Nature bone graft 3
1.2.1.2 Artificial bone graft 5
1.2.2 Scaffolds in bone tissue engineering 6
1.2.2.1 Essential properties 7
1.2.2.2 Categories 7
1.2.2.3 Preparation techniques 8
1.3 Electrospinning 9
1.4 Chitosan 10
1.5 Hydroxyapatite 11
1.5.1 Ion substitution 11
1.5.2 Strontium substitution 12
1.5.3 Synthesis 13
1.5.3.1 Chemical precipitation method 14
1.5.3.2 Hydrothermal method 15
1.6 Research motivation 15
Chapter 2 Materials and Methods 17
2.1 Chemicals 17
2.2 Experimental instruments 18
2.3 Preparation of specimens 19
2.3.1 Experimental procedure 19
2.3.2 Synthesis of hydroxyapatite 20
2.3.2.1 Chemical precipitation method 20
2.3.2.2 Hydrothermal method 20
2.3.3 Electrospinning 21
2.4 Specimens characteristic analysis 22
2.4.1 Characteristic of HA and SrHA powder 22
2.4.1.1 Microstructure 22
2.4.1.2 Chemical composition 22
2.4.1.3 Phase composition 22
2.4.1.4 Functional groups 23
2.4.2 Characteristic of scaffolds 23
2.4.2.1 Surface morphology 23
2.4.2.2 Chemical composition 23
2.4.2.3 Microstructure 23
2.4.2.4 Phase composition 24
2.4.2.5 Functional groups 24
2.4.2.6 Bioactivity and ion release 24
2.5 In vitro tests 25
2.5.1 Samples sterilization 25
2.5.2 Cell culture 25
2.5.3 Cell morphology 25
2.5.4 Cell proliferation 27
2.5.5 Cell differentiation 28
2.6 Statistical analysis 29
Chapter 3 Results 31
3.1 Hydroxyapatite characteristic analysis 31
3.1.1 Microstructure 31
3.1.2 Chemical composition 31
3.1.3 Phase composition 32
3.1.4 Functional groups 32
3.2 Nanofibrous scaffolds characteristic analysis 33
3.2.1 Surface morphology 33
3.2.2 Chemical composition 33
3.2.3 Microstructure 34
3.2.4 Phase composition 34
3.2.5 Functional groups 34
3.2.6 Bioactivity and ion release 35
3.3 In vitro tests 36
3.3.1 Cell morphology 36
3.3.2 Cell proliferation 36
3.3.3 Cell differentiation 37
Chapter 4 Discussion 38
Chapter 5 Conclusion 43
Reference 46
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