鈣磷化物混幾丁聚醣/明膠支架之生物活性和骨母細胞相容性

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研究生：	曾建豪 Chien-hao Tseng
論文名稱：	鈣磷化物混幾丁聚醣/明膠支架之生物活性和骨母細胞相容性 Bioactivity and osteoblast cytocompatibility of Ca/P-doped chitosan/gelatin macroporous scaffolds
指導教授：	楊銘乾 Ming-chien Yang
口試委員:	李振綱 Cheng-kang Lee 楊禎明 Jen-ming Yang
學位類別：	碩士 Master
系所名稱：	工程學院 - 材料科學與工程系 Department of Materials Science and Engineering
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	77
中文關鍵詞：	骨母、生物相容性、支架、共成法、鈣磷化物、明膠、幾丁聚醣
外文關鍵詞：	osteoblast, scaffold, cytocompatibility, in-suit preparation, Ca/P compound, Gelatin, Chitosan
相關次數：	點閱：241 下載：0
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本研究利用共成(in-situ)的方法以氯化鈣(CaCl2)和磷酸銨((NH4)2HPO4)在明膠(gelatin)溶液中製作出鈣磷化物，再將此明膠溶液和幾丁聚醣(chitosan)溶液混和，以冷凍乾燥法製成多孔性支架。支架性質測定包括其孔隙度、壓縮模數、膨潤度，並用熱重分析儀(TGA)和電子顯微鏡(SEM)分別觀察其熱性質及微結構的變化。生物活性(bioactivity)則係將支架浸泡於模擬體液(SBF)後的變化來分析。生物相容性則以骨母細胞(osteoblast)進行體外培養，利用MTS方法觀察細胞增生狀況，並以鹼性磷酸活性(ALP)和蛋白質生成的變化來觀察細胞活性。本研究發現利用此法製作的支架具有70~250μm的孔徑且互通的孔洞，孔隙度則在83~93%之間，且支架的機械性質隨鈣磷化物添加量增加而增加，孔隙度和膨潤度則相反；而增加明膠所產生的變化則和磷酸鈣相反。添加磷酸鈣後的支架生物活性明顯優於未添加的支架。細胞增生測試結果則顯示有添加鈣磷化物的支架較適合骨母細胞成長，但卻會抑制骨母細胞活性的表現。這結果顯示增強骨母細胞增生並不需伴隨著較佳的細胞活性和鹼性磷酸生成。

Ca/P compound-doped chitosan (CHS)/gelatin (GEL) composite scaffolds were prepared in-situ by mixing calcium chloride (CaCl2), ammonium phosphate ((NH4)2HPO4) and GEL with CHS to form homogeneous solution. Afterward, the solution was molded, lyophilized, and freeze-dried. Thereby, the three- dimensional and macroporous scaffolds were fabricated. These scaffolds were characterized with porosity, compressing factor, and swelling ratio, as well as thermogravimetric analysis (TGA) and scanning electronic microscopy (SEM). Furthermore, the bioactivity was evaluated by the variation of calcium and phosphorous elements in the simulated body fluid (SBF) and Ca/P crystals formation in scaffold. The cytocompatibility was also investigated in-vitro with osteoblast proliferation, MTS colorimetricy assay, alkaline phosphatase (ALP), and total protein generation. The results showed that the scaffolds exhibited porous structure of about 70~250μm in-size and 83~93% porosity. The mechanical strength increased with the increase of content of Ca/P compound, however, the swelling ratio was correspondingly decreased. The content of gelatin in the scaffold showed effect opposite to that of Ca/P compound. Furthermore, the Ca/P-doped scaffolds resulted in enhancing bioactivity and provided environments for the cell proliferation, while curtailing the cell viability and ALP secretion. The un-expected results implied that the improving osteoblast proliferation could not necessarily lead to better cell viability and ALP performance.

誌謝	I
摘要	III
ABSTRACT	IV
目錄	V
圖索引	IX
表索引	XI
第1章	緒論	1
1.1	前言	1
1.2	實驗目的	2
第2章	文獻回顧	3
2.1	骨科材料的簡介	3
2.1.1	天然骨科材料	3
2.1.2	人造骨科材料	4
2.2	組織工程	6
2.3	生物支架簡介	6
2.4	幾丁聚醣簡介	9
2.5	幾丁質、幾丁聚醣在生醫材料的應用	11
2.6	明膠簡介	11
2.7	骨骼的組成	13
2.7.1	骨重塑與骨母細胞	13
第3章	材料和實驗方法	17
3.1	藥品	17
3.2	儀器	19
3.3	實驗流程	20
3.4	多孔性支架之製備	21
3.4.1	chitosan溶液之配製	21
3.4.2	明膠/鈣磷化物共成溶液的配製	21
3.4.3	支架的製備	21
3.5	膨潤度測試	22
3.6	孔隙度(porosity)	22
3.7	壓縮模數	23
3.8	支架熱性質分析	23
3.9	支架結構分析	24
3.10	支架表面元素分析	24
3.11	生物活性試驗	24
3.11.1	模擬體液的浸泡試驗	24
3.12	骨母細胞培養測試	25
3.12.1	細胞培養	25
3.12.2	將細胞植入支架	25
3.12.3	細胞微型態的觀察	26
3.12.4	細胞增生能力測定(MTS assay)	26
3.12.5	細胞活性測試 (鹼性磷酸活性和蛋白質生成)	26
第4章	結果與討論	28
4.1	支架的基本性質	28
4.1.1	膨潤度測試	28
4.1.2	孔隙度測試	29
4.1.3	壓縮模數	30
4.1.4	支架熱性質	31
4.1.5	電子顯微鏡	32
4.1.6	ESCA元素分析	36
4.1.7	討論	37
4.2	支架生物活性的測試	38
4.3	支架生物相容性的測試	44
4.3.1	骨母細胞貼附支架後的微型態觀察	44
4.3.2	骨母細胞增生測試	46
4.3.3	細胞蛋白質生成測試	48
4.3.4	鹼性磷酸測試	50
4.3.5	細胞培養的討論	52
第5章	結論	54
第6章	文獻	55
附錄	60


圖索引
Figure 2 1 （a）幾丁聚醣，為β-1，4-D-葡萄糖胺（β-1，4-D-glucosamine）聚合物；（b）幾丁質，為β-1，4-D-N乙醯葡萄糖胺（β-1，4-D-acetyl glucosamine）聚合物。	10
Figure 2-2  Gelatin的基本結構式	12
Figure 2-3 骨母細胞的發展(in vitro development of the osteoblast phenotype) [18]	15
Figure 2-4 骨母細胞分化的各個過程和其蛋白質表現(mRNA expression in osteoblasts differentiation)[19]	15
Figure 4-1 各支架浸於去離子水中一天的膨潤度 (n=6)	28
Figure 4-2 各支架的壓縮模數 (n＝6)	30
Figure 4-3 支架的熱重量分析圖	31
Figure 4-4 支架孔洞的SEM圖	33
Figure 4-5 各支架表面微結構變化圖	35
Figure 4-6 支架浸泡SBF後的變化	40
Figure 4-7 支架浸泡後的SEM圖	41
Figure 4-8支架浸泡在SBF後，SBF中含鈣的變化	42
Figure 4-9 支架浸泡在SBF後，SBF中含磷的變化	42
Figure 4-10  314支架浸泡3天SBF後的EDS元素分析	43
Figure 4-11支架經過3天細胞培養後細胞貼附的情形	45
Figure 4-12  MTS測試反應式	46
Figure 4-12 各支架的細胞增生速率(n=3)	47
Figure 4-13 相同體積下各支架所產生蛋白質量(n=3)	49
Figure 4-14 等量的細胞在各支架上所產生的蛋白質量的比較(n=3)	49
Figure 4-16  鹼性磷酸脢測試反應式	50
Figure 4-15 相同體積下各支架所產生ALP活性(n=3)	51
Figure 4-16 等量的細胞在各支架上所產生的ALP活性的比較(n=3)	51


表索引
Table 3-1 各支架的成分比例	22
Table 4-1 各支架的孔隙度(n=6)	29
Table 4-2 各支架的孔洞大小(n＝20)	34
Table 4-3 支架的表面元素分析	36
Figure 4-4 各20℃下各支架溶液的黏度(n=5)	37
                                

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