臺灣博碩士論文加值系統

關節軟骨由於本身軟骨細胞的再生能力低且缺乏血流供應等因素導致自我修復能力不佳。現有的治療方式仍因軟骨數量有限、新生軟骨無法與原生軟骨密合以致無法長期荷重使得關節軟骨再生仍是目前臨床上相當具挑戰性的課題。雖然許多策略以組織工程的方式修復關節軟骨缺陷，但結果不盡理想。本研究發展膠原蛋白/三鈣磷酸鹽微粒合成移植物並以此微粒粉末交聯明膠製備支架。觀察微粒發現三鈣磷酸鹽顆粒均勻分佈於膠原蛋白纖維基質中。透過穿透式電子顯微鏡可觀察到膠原蛋白纖維的特徵明暗帶，顯示三鈣磷酸鹽顆粒不會妨礙膠原蛋白的重組。膠原蛋白/三鈣磷酸鹽微粒粉末交聯明膠製成的支架為一高孔洞結構，孔洞大小分佈於 50~150μm 之間。本研究同時以骨軟骨缺陷動物實驗模式探討利用 HA-CMC 阻隔膜防止新生骨組織生長到軟骨部的潛力。手術後一個月，新生軟骨與周邊原生軟骨及新生軟骨下骨部有良好的整合性，新生組織近似透明軟骨，但軟骨細胞的排列方式不同於原生軟骨呈柱狀排列。手術後三個月，新生軟骨與周邊原生軟骨及新生軟骨下骨部亦有良好的整合性。此外，軟骨下骨板非常完整，新生軟骨形態近似透明軟骨且軟骨細胞呈柱狀排列。在對照組方面，(1) 無填入材料及 (2) 填入支架並覆蓋骨膜得到較差的組織修復，缺陷填滿纖維軟骨及纖維組織。總結來說，利用 HA-CMC 阻隔膜於骨軟骨缺陷修復可防止新生骨組織生長到軟骨部以得到較佳的修復成效。

Poor spontaneous healing of articular cartilage is mainly due to the low proliferation rate of chondrocyte and insufficient blood supply. The pitfalls of treatment nowadays such as autologous chondrocyte transplantation include limited cartilage donor site available, poor integration between native and regenerative cartilage tissues, and inability to withstand a long term physiological loading. Although strategies of repairing articular cartilage defects with tissue engineering approach have been documented, none is satisfactory. We here developed a synthetic graft consisted of collagen/β-TCP microspheres, collagen/β-TCP microsphere powder and glutaraldehyde-crosslinked gelatin scaffold. The microspheres we fabricated contain β-TCP particles distributed homogenously in the collagen fiber matrix. And the characteristic banding pattern of collagen fibers as shown by transmission electron microscope suggests that the reconstitution of collagen was not hindered by the β-TCP particulates. The scaffold comprised of collagen/β-TCP microsphere powder and glutaraldehyde-crosslinked gelatin exhibits a highly porous structure with pore sizes ranging from 50 to 150 μm. By conducting animal experiment of osteochondral defect model, we explore the potential of using HA-CMC film to prevent regenerated bone tissue from growing into the cartilaginous compartment. At one month post-operation, the regenerated cartilage layer exhibited good structural integration to the adjacent native cartilage and regenerated subchondral bone. This regenerated tissue layer is similar to hyaline cartilage, but its chondrocytes were not arranged in a columnar fashion as observed in the native cartilage. By three months, most of the regenerated cartilage layer showed good integration into regenerated subchondral bone and adjacent native cartilage. In addition, the subchondral plates were intact, morphologically similar to the hyaline cartilage and its chondrocytes showed columnar organization. Poor tissue repair was found in the control groups of either (1) nontreated, or (2) filled with scaffold then convered with periosteum. In these two cases, defect sites were filled with fibrocartilage and fibrous tissue. In conclusion, HA-CMC film can be used to prevent new bone tissue regenerated into the cartilaginous compartment for a better osteochondral defect repairing.

目錄
中文摘要 I
Abstract II
目錄 IV
圖目錄 VIII
表目錄 XIII
第一章 緒論 1
1-1 前言 1
1-2 關節軟骨 2
1-3 軟骨細胞外基質 4
1-4 關節軟骨缺陷疾病與分類 6
1-5 軟骨缺損治療方法 7
1-5-1 非侵入性治療 7
1-5-2 顯微灌洗術及關節清術 7
1-5-3 骨髓刺激修復 8
1-5-4 自體骨軟骨移植 8
1-5-5 自體軟骨細胞移植 9
1-6 軟骨組織工程 10
1-7 利用複合載體修補骨軟骨缺損 12
1-7-1 複合載體組合策略 12
1-7-2 細胞植入載體策略 13
1-7-3 設計複合載體修補骨軟骨缺損 14
1-8 應用膠原蛋白微粒於軟骨下骨部缺陷修復 15
1-9 膠原蛋白 16
1-10 β-三鈣磷酸鹽 19
1-11 透明質酸 19
1-12 應用骨膜於軟骨新生 22
1-13 研究動機 24
第二章 實驗藥品與儀器 25
2-1 實驗藥品 25
2-2 實驗儀器 27
第三章 實驗方法 28
3-1 乳化法製備膠原蛋白/三鈣磷酸鹽微粒 28
3-1-1 掃描式電子顯微鏡樣本製備 28
3-1-2 穿透式電子顯微鏡樣本製備 29
3-1-3 膠原蛋白/三鈣磷酸鹽微粒無菌處理 29
3-2 膠原蛋白/三鈣磷酸鹽微粒粉末交聯明膠製備支架 30
3-2-1 膠原蛋白/三鈣磷酸鹽微粒交聯明膠支架剖面結構觀察 30
3-3 HA-CMC 阻隔膜製備 30
3-4 HA膠體製備 31
3-5 骨軟骨缺陷修復動物實驗 31
3-6 組織切片及化學染色分析 33
3-6-1 組織切片製作 33
3-6-2 蘇木紫-伊紅染色 34
3-6-3 Alcian blue 染色 35
3-7 動物實驗評估與統計方式 36
第四章 結果與討論 38
4-1 膠原蛋白/三鈣磷酸鹽微粒掃瞄式電子顯微鏡分析 38
4-2 膠原蛋白/三鈣磷酸鹽微粒穿透式電子顯微鏡分析 39
4-3 膠原蛋白/三鈣磷酸鹽微粒粉末交聯明膠製備支架 40
4-4 膠原蛋白/三鈣磷酸鹽微粒粉末交聯明膠支架剖面結構觀察 41
4-5 骨軟骨缺陷動物實驗組織化學分析 43
4-5-1 骨軟骨缺陷修復於手術後一個月之組織化學分析 43
4-5-1-1 以支架、HA-CMC 阻隔膜、HA 膠體和骨膜修復 43
4-5-1-2 以支架和骨膜修復 46
4-5-1-3 無填入任何材料 49
4-5-1-4 Pineda score 統計分析 52
4-5-2 骨軟骨缺陷修復於手術後二個月之組織化學分析 53
4-5-2-1 以支架、HA-CMC 阻隔膜、HA 膠體和骨膜修復 53
4-5-2-2 以支架和骨膜修復 56
4-5-2-3 無填入任何材料 59
4-5-2-4 Pineda score 統計分析 62
4-5-3 骨軟骨缺陷修復於手術後三個月之組織化學分析 63
4-5-3-1 以支架、HA-CMC 阻隔膜、HA 膠體和骨膜修復 63
4-5-3-2 以支架和骨膜修復 66
4-5-3-3 無填入任何材料 69
4-5-3-4 Pineda score 統計分析 72
4-5-4 不同時間點 Pineda score 統計比較 73
第五章 結論 74
第六章 參考文獻 75

圖目錄
圖 1-1 關節軟骨分層結構 3
圖 1-2 關節軟骨細胞外基質組成成份 4
圖 1-3 Aggrecan 結構圖 5
圖 1-4 關節軟骨中 proteoglycan 結構及膨潤性質示意圖 6
圖 1-5 軟骨缺損分類 7
圖 1-6 自體骨軟骨移植 8
圖 1-7 自體軟骨細胞移植 9
圖 1-8 膠原蛋白分子及組成纖維示意圖 18
圖 1-9 膠原蛋白重組現象 18
圖 1-10 透明質酸化學結構式 20
圖 1-11 骨膜之組織結構 23
圖 3-1 乳化法製備膠原蛋白/三鈣磷酸鹽微粒 28
圖 3-2 骨軟骨缺陷修復動物實驗步驟 32
圖 4-1 以 SEM 觀察膠原蛋白/三鈣磷酸鹽微粒表面結構 39
圖 4-2 以 TEM 觀察膠原蛋白/三鈣磷酸鹽微粒內部重組結構 40
圖 4-3 膠原蛋白/三鈣磷酸鹽微粒粉末交聯明膠支架外觀結構 41
圖 4-4 膠原蛋白/三鈣磷酸鹽微粒粉末交聯明膠支架孔洞分佈 42
圖 4-5 膠原蛋白/三鈣磷酸鹽微粒粉末於支架中分佈情形 42
圖 4-6 巨觀觀察缺陷修復情形：實驗組，手術後一個月，植入支架、HA-CMC
阻隔膜、HA 膠體和骨膜 44
圖 4-7 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後一個月組織切片以 HE 染色得到的結果 (x40) 44
圖 4-8 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後一個月組織切片以 HE 染色得到的結果 (x200) 45
圖 4-9 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後一個月組織切片以 Alcian blue 染色得到的結果 (x40) 45
圖 4-10 巨觀觀察缺陷修復情形：對照組，手術後一個月，植入支架和骨膜 46
圖 4-11 植入支架和骨膜於兔子膝關節，手術後一個月組織切片以 HE 染色得到的結果 (x40) 47
圖 4-12 植入支架和骨膜於兔子膝關節，手術後一個月組織切片以 HE 染色得到的結果 (x200) 47
圖 4-13 植入支架和骨膜於兔子膝關節，手術後一個月組織切片以 Alcian blue 染色得到的結果 (x40) 47
圖 4-14 巨觀觀察缺陷修復情形：對照組，手術後一個月，無填入任何材料 49
圖 4-15 無填入任何材料於兔子膝關節，手術後一個月組織切片以 HE 染色得到的結果 (x40) 50
圖 4-16 無填入任何材料於兔子膝關節，手術後一個月組織切片以 HE 染色得到的結果 (x200) 50
圖 4-17 無填入任何材料於兔子膝關節，手術後一個月組織切片以 Alcian blue 染色得到的結果 (x40) 51
圖 4-18 手術後一個月以 Pineda score 分析統計軟骨修復成效 52
圖 4-19 巨觀觀察缺陷修復情形：實驗組，手術後二個月，植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜 53
圖 4-20 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後二個月組織切片以 HE 染色得到的結果 (x40) 54
圖 4-21 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後二個月組織切片以 HE 染色得到的結果 (x200) 54
圖 4-22 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後二個月組織切片以 Alcian blue 染色得到的結果 (x40) 55
圖 4-23 巨觀觀察缺陷修復情形：對照組，手術後二個月，植入支架和骨膜 56
圖 4-24 植入支架和骨膜於兔子膝關節，手術後二個月組織切片以 HE 染色得到的結果 (x40) 57
圖 4-25 植入支架和骨膜於兔子膝關節，手術後二個月組織切片以 HE 染色得到的結果(x200) 57
圖 4-26 植入支架和骨膜於兔子膝關節，手術後二個月組織切片以 Alcian blue 染色得到的結果 (x40) 58
圖 4-27 巨觀觀察缺陷修復情形：對照組，手術後二個月，無填入任何材料 59
圖 4-28 無填入任何材料於兔子膝關節，手術後二個月組織切片以 HE 染色得到的結果 (x40) 60
圖 4-29 無填入任何材料於兔子膝關節，手術後二個月組織切片以 HE 染色得到的結果 (x100) 60
圖 4-30 無填入任何材料於兔子膝關節，手術後二個月組織切片以 Alcian blue 染色得到的結果 (x40) 61
圖 4-31 手術後二個月以 Pineda score 分析統計軟骨修復成效 62
圖 4-32 巨觀觀察缺陷修復情形：實驗組，手術後三個月，植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜 63
圖 4-33 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後三個月組織切片以 HE 染色得到的結果 (x40) 64
圖 4-34 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後三個月組織切片以 HE 染色得到的結果 (x200) 64
圖 4-35 植入支架、HA-CMC 阻隔膜、HA 膠體和骨膜於兔子膝關節，手術後三個月組織切片以 Alcian blue 染色得到的結果 (x40) 65
圖 4-36 巨觀觀察缺陷修復情形：對照組，手術後三個月，植入支架和骨膜 66
圖 4-37 植入支架和骨膜於兔子膝關節，手術後三個月組織切片以 HE 染色得到的結果 (x40) 67
圖 4-38 植入支架和骨膜於兔子膝關節，手術後三個月組織切片以 HE 染色得到的結果 (x200) 67
圖 4-39 植入支架和骨膜於兔子膝關節，手術後三個月組織切片以 Alcian blue 染色得到的結果 (x40) 68
圖 4-40 巨觀觀察缺陷修復情形：對照組，手術後三個月，無填入任何材料 69
圖 4-41 無填入任何材料於兔子膝關節，手術後三個月組織切片以 HE 染色得到的結果 (x40) 70
圖 4-42 無填入任何材料於兔子膝關節，手術後三個月組織切片以 HE 染色得到的結果 (x100) 70
圖 4-43 無填入任何材料於兔子膝關節，手術後三個月組織切片以 Alcian blue 染色得到的結果 (x40) 71
圖 4-44 手術後三個月以 Pineda score 分析統計軟骨修復成效 72
圖 4-45 以 Pineda score 分析比較不同時間點軟骨修復成效 73

表目錄
表 1-1 GAG 種類 5
表 3-1 修正後的 Pineda score 37

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