臺灣博碩士論文加值系統

本研究主要分為兩個方向，首先改善膠原蛋白微粒之製程以製備低利徑之微粒，接著進入動物實驗測試膠原蛋白微粒於硬骨修補的潛力，並應用於骨質植體做為填充材料。另一個方向是探討奈米尺寸之膠原蛋白基材對類硬骨細胞移動行為的影響，未來期能藉由調控硬骨植入材料之表面特性來改變材料周圍硬骨組織內細胞的行為表現。
於第一部份實驗中，利用均質機之攪拌乳化法可成功製備出數十微米之膠原蛋白微粒。在改變了數種參數後發現，提高均質機轉速、降低水油相比、提高界面活性劑濃度、降低膠原蛋白溶液之濃度，皆可降低膠原蛋白微粒之粒徑，我門亦可藉由調控上述參數來製作特殊粒徑之微粒。而利用奈米等級之氫氧基磷灰石結晶顆粒，可依需求製作出不同蛋白質/陶瓷比例之膠原蛋白微粒，且陶瓷呈現均勻分佈。
第二部份，在將膠原蛋白微粒應用於骨填補材料的實驗，結果顯示，無論在微粒表面或是包覆於微粒內部，骨髓幹細胞都能貼附生長並增生，並保有分化能力。此外，若以褐藻酸包覆系統製備膠原蛋白微粒，起始的細胞濃度以106 cell/ml會有較好的細胞活性與增生幅度。動物實驗部份，以骨髓幹細胞結合膠原蛋白/氫氧基磷灰石微粒之硬骨修復程度較佳，螢光標定分析也顯示幹細胞有參與硬骨組織的修補。總結來說，運用含骨髓幹細胞之膠原蛋白微粒作為硬骨組織填補材料，是種可行的策略，值得做更進一步的探討。
第三部份，膠原蛋白微粒應用於製作骨質植體的動物實驗亦在本研究中有所探討。實驗設計以定期注射PRP進入植體作為變因。根據放射線學分析結果，實驗組與對照組皆有鈣化組織生成，且隨著植入時間增加而提高鈣化程度。其中，實驗組的鈣化速度較對照組為高，但到二十四週時差異會被拉近。而注射PRP之實驗組植體在早期ALP活性明顯較對照組高，推測PRP所含生長因子讓骨質植體有較高的新骨生成作用發生。根據第二十四週實驗組組織切片H&E染色可以觀察到膠原蛋白/氫氧基磷灰石微粒會逐漸被分解而被新生組織所取代，細部也可觀察到新生血管的產生及成骨細胞的作用，甚至有少量的類骨髓組織生成。此部分動物實驗結果證實，在白兔模型上製作預先成形之骨質植體是可行的。
最後，人類類成骨細胞MG63在不同尺寸(直徑為50-200 nm, 200-500 nm, and 500-1000 nm)膠原蛋白電紡纖維基材上的行為亦得到探討。在膠原蛋白電紡纖維上，MG63細胞的增生較在TCPS及膠原蛋白單體塗佈之玻璃表面上高出約70%。另一方面，細胞之移動性會隨著纖維尺寸的增加而降低。在膠原蛋白基材上，MG63細胞也會有較明顯的張力纖維形成。此外，MG63細胞在不同纖維上的貼附及延展型態亦有所不同。而纖維尺寸的改變對細胞移動性的影響大於對增生的影響。實驗顯示，提供適合的基材表面粗操度可以促進MG63細胞與基材的互動。此結果提供有利資訊於改進以膠原蛋白為基材之生醫材料。

In this study, we have prepared sub-micron collagen microspheres by employing homogenized emulsion process. By controling the homogenizer speed, water to oil ratio, surfactant and collagen concentrations, microspheres of specific size can be obtained. With nanoscale hydroxyapatite crystals, microspheres of various collagen/HAp weight ratios were fabricated with ceramics particles well dispersed in the collagen matrix.
For testing the potential application of collagen microspheres in bone repair, MSCs（mesenchymal stem cells） were cultured on the surface or inside the microspheres. The MSCs cultured with these microspheres maintained their differentiation ability into bone-like tissue. Results of in vivo study showed that MSCs and microspheric composite participated in bone repair and promote healing rate. We conclude that MSCs/microsphere composite has potential in clinical application.
Prefabricated bone segment was examined next. By incorporating vessel bundle into PTFE chamber pre-loaded with collagen microspheres, prefabricated bone graft was made and implanted into rabbits for in vivo study. Newly formed calcified tissue in the bone graft was histologically confirmed. The calcification degree in bone segments of the experiment group was higher at 4 weeks which was probably promoted by PRP injection, but showed no difference as compared with the control group after 24 weeks. Neovascularization and bone remodeling were observed, and the bone marrow like tissue was developed in the bone graft at 24 weeks after implantation. We conclude that the combination of collagen/HAp microspheres, autogenous PRP and a vessel bundle within a artificial vessel is a promising approach for the prefabrication of vascularized bone.
Finally, the behaviors of human osteoblast-like MG63 cells cultured on electrospun collagen fibers of three different sizes (50-200 nm, 200-500 nm, and 500-1000 nm in diameter) were investigated. The growth of MG63 cells on all three electrospun collagen fibers are about 70% higher than those cultured on monomeric collagen and TCPS. The migration speed of MG63 cells, on the other hand, decreased as the diameter of nanofibers increased. There were more distinct actin stress fibers formed in MG63 cells when the cells cultured on collagen substrates as compared with TCPS. In addition, MG63 cells displayed different adhesion and spreading patterns on different sizes of collagen fibers. Size variation of collagen nanofibers apparently has more impact on cell migration distance and cell morphology as compared with cell growth. It was demonstrated that collagen nanofibers promoted MG63 cell interaction with matrices by providing a suitably rough nanometer surface. The results of this study present important information for the development of collagen based biomaterials.

誌謝 I
中文摘要 II
英文摘要 IV
目錄 VI
圖目錄 IX
表目錄 XII
第一章 以膠原蛋白微粒為基材做為硬骨組織修補材料之介紹 1
1.1 前言 1
1.2膠原蛋白微粒做為硬骨組織修補材料的介紹 2
1.2.1 骨組織的結構與修復 2
1.2.2 骨填補材料 3
1.2.3 第一型膠原蛋白 5
1.2.4氫氧基磷灰石的簡介 9
1.2.5 製備膠原蛋白微粒之方式 10
1.3細胞包覆系統（CELL ENCAPSULATION） 11
1.4 骨髓幹細胞（MSCS）的介紹 14
1.5 預先製成之骨植體 16
1.5.1 預先製成之骨植體的介紹 16
1.5.2 富含血小板血漿的介紹 18
1.5.3 硬骨生成指標 19
1.6 研究動機 20
第二章 膠原蛋白基材表面結構之尺寸對類骨母細胞（MG63）遷移行為的影響 22
2.1 細胞貼附及遷移的機制 22
2.2細胞與基材的交互作用 24
2.3 不同尺寸之基材表面對貼附型細胞遷移行為的影響 25
2.4 靜電紡織 26
2.4.1 靜電紡織原理 26
2.4.2 靜電紡織的生醫應用 27
2.4.3 膠原蛋白靜電紡織 28
2.5 人類類成骨細胞（MG63）的介紹 28
2.6 研究動機 29
第三章 材料與研究方法 30
3.1 膠原蛋白/氫氧基磷灰石微粒製備之實驗方法 30
3.1.1 第一型膠原蛋白之製備 30
3.1.2 第二型膠原蛋白之製備 30
3.1.3 乳化法製備膠原蛋白/氫氧基磷灰石微粒 31
3.1.4 低粒徑膠原蛋白/氫氧基磷灰石微粒之製備 31
3.1.5 微粒之粒徑分析 32
3.1.6 掃瞄式電子顯微鏡（SEM）樣本之製備 32
3.1.7 膠原蛋白/氫氧基磷灰石微粒之無菌處理 32
3.1.8 骨髓幹細胞之分離與培養 32
3.1.9 骨髓幹細胞於微粒表面之培養 34
3.1.10 細胞計數（cell count）與細胞活性（MTT）測試 34
3.1.11 活細胞之螢光染色 34
3.2 膠原蛋白微粒應用於細胞載體之實驗方法 35
3.2.1 膠原蛋白無菌處理 35
3.2.2 包覆細胞（MSCs）於膠原蛋白微粒之製備 35
3.2.3 膠原蛋白微粒之細胞包覆率測定 36
3.2.4雷射共軛焦顯微鏡（Confocal microscope）樣品之製備 36
3.2.5 冷凍切片樣品製備 37
3.2.6掃瞄式電子顯微鏡（SEM）樣本之製備 37
3.2.7 以螢光染劑（PKH26）標定活細胞之實驗方法 37
3.2.8 MSCs細胞之誘導 38
3.2.9 EDS/X-ray樣品之製備 38
3.2.10 臨界尺寸骨缺陷修補之動物實驗 38
3.2.11 鹼性磷酸酶酵素（ALP）活性 40
3.2.12 組織切片製備步驟 40
3.3 膠原蛋白微粒應用於預先成型骨植體之實驗方法 40
3.3.1 預先成型骨植體的製備 40
3.3.2 預先成型骨植體的動物實驗方法 40
3.3.3 富含血小板血漿的製備 41
3.3.4 放射學分析及新骨生成分析方法 42
3.3.5 鈣化程度分析方法 42
3.3.6 鹼性磷酸酵素（ALP）活性 43
3.3.7 骨鈣素（osteocalcin）含量分析 43
3.4 人類類成骨細胞（MG63）貼附於以靜電紡織製備之膠原蛋白基材的實驗方法 43
3.4.1 以電氣紡織製備膠原蛋白奈米纖維 43
3.4.2 以Rhodamin對膠原蛋白電紡纖維做螢光染色 44
3.4.3 以增強型綠螢光蛋白（eGFP）質粒轉染MG63細胞 44
3.4.4 MG63細胞於膠原蛋白電紡纖維上之培養 44
3.4.5 活細胞動態觀察 44
3.4.6 細胞移動距離的計算 45
3.4.7 細胞之螢光染色 45
3.4.8 細胞肌動蛋白表現的半定量方法 46
3.4.9 掃瞄式電子顯微鏡（SEM）樣本之製備 46
3.5 實驗藥品及設備 46
第四章 小粒徑膠原蛋白/氫氧基磷灰石微粒製備之結果與討論 48
4.1 實驗結果 48
4.2 討論 60
第五章 膠原蛋白微粒應用於細胞載體作為骨缺陷修補材料之結果與討論 62
5.1 實驗結果 62
5.2 討論 75
第六章 填充膠原蛋白微粒之預先成型骨植體配合富含血小板血漿（PRP）注射的動物實驗之結果與討論 76
6.1 實驗結果 76
6.2 討論 84
第七章 人類類骨母細胞(MG63)於膠原蛋白電紡基材上的貼附及遷移之結果與討論 86
7.1 實驗結果 86
7.2 討論 93
第八章 結論及未來方向 95
第九章 參考文獻 97

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