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研究生: 柯智升
Ko, Chih-Sheng
論文名稱: 利用多孔性Collagen II/chondroitin sulfate/hyaluronic acid載體誘導間葉幹細胞應用於組織工程軟骨之研究
Chondrogenic differentiation of human umbilical cord blood mesenchymal stem cells in cross-linking collagen II / schondroitin sulfate / hyaluronic acid sponge
指導教授: 朱一民
Chu, I-Ming
口試委員:
學位類別: 博士
Doctor
系所名稱: 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年: 2008
畢業學年度: 97
語文別: 中文
論文頁數: 133
中文關鍵詞: 組織工程軟骨膠原蛋白第二型醣胺素硫酸化軟骨素透明質酸人類臍帶血間葉幹細胞
外文關鍵詞: umbilical cord blood mesenchymal stem cells, cross-linked collagen type II sponges, chondrogensis, genipin, chondroitin sulfate, hyaluronan
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  • 由於關節軟骨組織有限的修復能力,對於罹患關節疾病的病人來言,發展組織工程軟骨是必須的。組織工程的軟骨是利用生物相容性和生物可分解性的載體培養軟骨細胞,製備近似可供植入的軟骨組織。天然關節軟骨主要由膠原蛋白第二型和較大的蛋白醣聚合體(proteoglycans,簡稱PGs)與透明質酸(hyaluronan 簡稱HA)組成,而蛋白醣單體最主要的醣胺素(glycosaminoglycans,簡稱GAGs)是硫酸化軟骨素(chondroitin sulfate,簡稱CS)。本研究已由牛氣管純化出膠原蛋白第二型,並利用不同冷凍溫度(-20℃、-80℃與-196℃)與冷凍乾燥法製備具多孔性的支架,孔洞大小分佈範圍為20–260μm,孔隙度為95%±1。再利用交聯劑genipin (GP)將CS和膠原蛋白第二型纖維及HA交聯,以製備三種支架COL II、COL II/CS (CCS)與COL II/CS/HA (CCH),交聯後雖然有塌陷的現象,但仍具有孔洞結構,且具高含水率(132□145%)及高孔隙度(95□92%)特性,孔洞大小範圍為106–173□m。此外支架經過交聯後,增加了機械強度與變性溫度、提升抵抗酵素消化的能力與抗原性降低等。至於支架中CS與HA含量,CCS中CS含量為17.8±1.2 (μg/matrix),而CCH中CS與HA的含量分別為15.6±2.3 (μg/ matrix)與22.55±1.8 (μg/ matrix)。本研究中我們評估COL II、COL II/CS與COL II/CS/HA支架對人類軟骨細胞的增生與分化影響。由組織切片可觀察到,軟骨細胞均勻遍佈在所有支架之中,並顯示圓形的型態。根據DNA與GAGs含量,CCH支架分別是21.3 □g/ matrix與150.15□g/ matrix皆高於COL II支架與CCS支架。此外,於統計學上CCH中軟骨細胞的基因表現,如aggrecan、 type II collagen、COMP 與 type X collagen gene等,相較於COL II支架,具有顯著的差異。再者,根據alcian blue染色與免疫組織染色的結果,CCH支架上有較多PGs、collagen type II與aggrecan的沈積。實驗結果顯示,CCH支架具有產生組織工程軟骨的潛能。
    為了解決工程軟骨上細胞來源的限制,本研究評估人類臍帶血間葉幹細胞(human umbilical cord blood mesenchymal stem cells (UMSCs)),於COL II支架上分化成為軟骨細胞的潛能。並評估植入的幹細胞數目與COL II支架,對於UMSCs增生與分化的影響。幹細胞數目對分化之影響,由實驗結果顯示,相較於其他兩組細胞數目(6.05 ×104 cells/ml與6.05 ×105 cells/ml),高細胞數目組(1.21 ×106 cells/ml) DNA與GAGs之含量及軟骨細胞特定基因的表現,隨誘導分化時間明顯達到最高值。表示幹細胞數目影響間葉幹細胞分化能力。
    至於COL II支架對UMSCs分化之影響,根據實驗結果顯示,支架內細胞產生PGs和軟骨細胞特定基因之表現與軟骨細胞標誌ECM沈積。其表示交聯後的COL II支架,仍具有促進UMSCs分化成軟骨細胞的功能。然而,UMSCs於IM (induction medium)系統中培養, 其PGs之生化合成量、軟骨細胞特定基因之表現與標誌ECM的累積,皆高於BM系統。這結果可能意味著COL II支架與生長因子之間,存在syngerism的作用。
    由不同支架影響UMSCs分化的結果顯示,CCH支架促進幹細胞的分化,collagen type II與aggrecan mRNA的表現量,相較於COL I與chitosan支架,具有顯著的差異。至於ECM生成量也相對較高。於In vivo實驗中,由組織切片的結果顯示,CCH支架生物相容性非常的好。根據細胞基因表現與軟骨標誌ECM之螢光染色的結果,我們可以推論CCH支架,對於以幹細胞修復關節軟骨組織的治療,是個非常良好的載體。


    摘要 I 目錄……………………………………………………………………..VI 圖目錄……………………………………………………..................... IX 表目錄………………………………………………………………....XV 第一章研究動機與架構 1 第二章 文獻回顧 3 2.1組織工程淵源 3 2.2組織工程骨架所需具備的特性 3 2.3組織工程骨架之製備方式 4 2.4軟骨(cartilage)的種類 6 2.5退化性關節炎(osteoarthritis, OA) 8 2.6組織工程之關節軟骨 11 2.7幹細胞之簡介與軟骨組織工程上之運用 12 2.8幹細胞來源………………………………………………………...15 2.8.1胚胎幹細胞應用於軟骨組織工程………………………………..16 2.8.2臍帶血間葉幹細胞運用於軟骨組織工程………………………..16 2.8.3骨髓間葉幹細胞運用於軟骨組織工程…………………………..17 2.9幹細胞未來發展…………………………………………………..17 第三章 研究方法與材料 27 3.1 純化膠原蛋白第二型 27 3.2 多孔性膠原蛋白載體的製備 27 3.3 交聯膠原蛋白載體 28 3.4 SEM測試…………………………………………………………28 3.5 膨潤測試 29 3.6 孔隙度測試 29 3.7機械性質評估 29 3.8變性溫度(Denaturation temperature, Td)分析 30 3.9膠原蛋白支架的降解測試………………………………………….30 3.10交聯指數(fixation index)…………………………………………..30 3.11支架中CS與HA含量的測定…………………………………….31 3.12 DNA與glycosaminoglycans (GAGs)含量分析............................31 3.13組織與免疫組織化學染色………………………………………...32 3.14蘇木紫與伊紅染色(H&E stain)…………………………………..33 3.15普魯士藍染色(Alcian blue)……………………………………….33 3.16 RNA抽取與cDNA合成………………………………………….34 3.17及時定量PCR (Quantifcation Real-Time PCR)…………………..35 3.18軟骨細胞的培養…………………………………………………...36 3.19臍帶血間葉幹細胞培養…………………………………………...37 3.20 UMSCs植入支架中之細胞密度對誘導成軟骨細胞之影響…….39 3.21不同支架對UMSCs誘導分化之影響…………………………….39 3.22統計學的分析…………………………...........................................39 第四章結果與討論-軟骨組織工程…………………………………….42 4.1純化膠原蛋白第二型和載體製備 42 4.2 3-D複合支架之製備 42 4.3交聯後的第二型膠原蛋白支架之特性 44 4.4膠原蛋白第一型與第二型支架對軟骨細胞之影響 45 4.5不同支架對人類軟骨細胞之影響………………………………….46 4.5.1 DNA與GAGs含量測定…………………………………………46 4.5.2軟骨細胞的基因表現……………………………………………..46 4.5.3分析支架中新生的ECM與細胞型態……………………………48 第五章結果與討論-幹細胞誘導與分化…………………………….....76 5-1於pellet系統人類臍帶血間葉幹細胞分化成軟骨細胞…………..76 5-2植入支架中的UMSCs細胞密度對軟骨分化形成之影響………..77 5-3機械性質的測試對於植入不同細胞密度的支架…………………78 5-4交聯的膠原蛋白第二型支架對UMSCs分化之影響……………..79 5-5支架之切片染色與collagen type II、aggrecan免疫染色…………..82 5-6不同支架對UMSCs誘導分化之影響……………………………..83 5-6.1 DNA與GAGs含量………………………………………………83 5-6.2 基因表現分析……………………………………………………84 5-6.3支架之切片染色與collagen type II、aggrecan免疫染色………...85 5-7 In vivo之評估………………………………………………………85 第六章 結論與未來展望 122 第七章 參考文獻 126 圖目錄 圖2-1 Proteoglycan aggregate示意圖 22 圖2-2 關節軟骨細胞分佈圖 23 圖2-3 膝蓋關節結構圖 24 圖2-4 軟骨硫素(chondroitin sulphate)的三種異構物 25 圖2-5 為mosaicplasty手術,將非受力部位的osteochondral graft移植到受力缺損處之示意圖 26 圖2-6 為間葉幹細胞分化成各種細胞組織之示意圖 26 圖4-1 (a)交聯前膠原蛋白骨架,(b)經genipin交聯後膠原蛋白骨架 .56 圖4-2 膠原蛋白第二型SDS-PAGE電泳 57 圖4-3 (a)膠原蛋白二型之alcian blue染色,(b)經GP交聯後COL II/CS/HA骨架之alcian blue染色 58 圖4-4 組織免疫染色(a) collagen type II,(b) collagen type I (negative control) 59 圖4-5 (a)第一型與(b)第二型膠原蛋白之SEM圖 60 圖4-6 冰凍溫度(a)-20℃、(b)-80℃、(c)-196℃之SEM 61 圖4-7膠原蛋白第一型(COL I)與第二型(COL II)支架對人類軟骨細胞之影響, (a)支架內的細胞數與(b)支架內的GAGs含量 62 圖4-8於膠原蛋白第一型(COL I)與第二型(COL II)支架上培養四星期後,人類軟骨細胞之基因表現……………………………………….. ………………………...63 圖4-9各種膠原蛋白第二型支架之SEM截面圖,(a)未交聯膠原蛋白第二型支架,(b)交聯的膠原蛋白第二型支架,(c)交聯的膠原蛋白第二型支架含有CS,(d) 交聯的膠原蛋白第二型支架含有CS與HA. ….64 圖4-10 (a)人類軟骨細胞經過十四天培養,各種膠原蛋白第二型支架中DNA之含量(COL II: COL II 支架; CCS: COL II/CS支架; CCH: COL II/CS/HA支架) (n=3).............................................................................................................................65 圖4-10 (b) 人類軟骨細胞經過十四天培養,各種膠原蛋白第二型支架中GAGs之含量(COL II: COL II 支架; CCS: COL II/CS支架; CCH: COL II/CS/HA支架) (n=3)………………………………………………………………………………….65 圖4-11 (a)人類軟骨細胞於支架上培養十四天後其aggrecan之基因表現 66 圖4-11 (b)人類軟骨細胞於支架上培養十四天後其collagen type II之基因表現..66 圖4-11 (c)人類軟骨細胞於支架上培養十四天後其Sox9之基因表現……………67 圖4-11 (d)人類軟骨細胞於支架上培養十四天後其collagen type I之基因表現…67 圖4-11 (e)人類軟骨細胞於支架上培養十四天後其collagen type X之基因表現..68 圖4-11 (f)人類軟骨細胞於支架上培養十四天後其COMP之基因表現…………68 圖4-11 (g)人類軟骨細胞於支架上培養十四天後其collagen type IX之基因表現.69 圖4-11 (h)人類軟骨細胞於支架上培養十四天後其HCLP之基因表現………….69 圖4-12 (a)經過十四天培養COL II支架切片之H&E染色………………………..70 圖4-12 (b)經過十四天培養COL II支架切片之alcian blue染色………………….70 圖4-12 (c)經過十四天培養COL II/CS支架切片之H&E染色……………………71 圖4-12 (d)經過十四天培養COL II/CS支架切片之alcian blue染色……………...71 圖4-12 (e)經過十四天培養COL II/CS/HA支架切之H&E染色………………….72 圖4-12 (f)經過十四天培養COL II/CS/HA支架切片之alcian blue染色………….72 圖4-13 (a)經過十四天培養COL II支架切片之collagen type II免疫染色……….73 圖4-13 (b)經過十四天培養COL II/CS支架切片之collagen type II免疫染色……73 圖4-13 (c)經過十四天培養COL II/CS/HA支架切片之collagen type II免疫染色.74 圖4-14 (a)經過十四天培養COL II支架切片之aggrecan免疫螢光染色………….74 圖4-14 (b)經過十四天培養COL II/CS支架切片之aggrecan免疫螢光染色……..75 圖4-14 (c)經過十四天培養COL II/CS/HA支架切片之aggrecan免疫螢光染色…75 圖5-1 (A) UMSCs經過誘導分化後分析其軟骨細胞之特定性基因表現,分別比較平面(mono)與3-D pellet培養之差異……………………………………………….91 圖5-1 (B) UMSCs pellet 於IM培養基中,經過3星期誘導分化之組織切片 (I) Hematoxylin and eosin (H&E) 染色,(II) Alcian blue 染色,(III) 免疫組織染色collagen type II……………………………………………………………………….92 圖5-2 (a)植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其隨時間變化之DNA含量(n=3)………………………………………………………………...93 圖5-2 (b)植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其隨時間變化之GAGs含量(n=3)………………………………………………………………..93 圖5-2 (c)植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其隨時間變化之生化合成率(GAGs含量/DNA含量) (n=3)……………………………………94 圖5-3 (a)植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其軟骨細胞之特定基因表現量(n=3)…………………………………………………………….94 圖5-3 (b)植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其軟骨細胞之特定基因表(n=3)………………………………………………………………….95 圖5-3 (c)植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其硬骨細胞之特定基因表現量(n=3)…………………………………………………………….95 圖5-4植入不同細胞密度之UMSCs於膠原蛋白載體培養21天,其機械性質Young’s modulus之測定(n=5)……………………………………………………….96 圖5-5 (a) UMSCs與人類軟骨細胞(N-HACs)培養於膠原蛋白支架中,其隨時間變化之DNA含量(n=3)………………………………………………………………...97 圖5-5 (b) UMSCs與人類軟骨細胞(N-HACs)培養於膠原蛋白支架中,其隨時間變化之GAGs含量(n=3)………………………………………………………………..97 圖5-5 (c) UMSCs與人類軟骨細胞(N-HACs)培養於膠原蛋白支架中其隨時間變化之生化合成率(GAGs含量/DNA含量) (n=3)……………………………………98 圖5-6 (a) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因collagen type II (COL II)之表現量…………………………………………….…98 圖5-6 (b) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因aggrecan (agg)之表現量………………………………………………………..…99 圖5-6 (c) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因COMP之表現量…………………………………………………………………..99 圖5-6 (d) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因Sox 9之表現量…………………………………………………………………100 圖5-6 (e) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞去分化特定基因collagen type I (COL I)之表現量……………………………………..…100 圖5-6 (f) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因collagen type X (COL X)之表現量……………………………………………...101 圖5-6 (g) UMSCs與N-HACs於膠原蛋白載體中培養21天,其硬骨細胞特定基因osteocalcin (OS)之表現量……………………………………………………….101 圖5-6 (h) UMSCs與N-HACs於膠原蛋白載體中培養21天,其脂肪細胞特定基因adipocyte-specific fatty acid binding protein (aP2)之表現量…………………...102 圖5-6 (i) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因collagen type IX (COL IX)之表現量……………………………………………….102 圖5-6 (j) UMSCs與N-HACs於膠原蛋白載體中培養21天,其軟骨細胞特定基因human cartilage link protein (HCLP)之表現量…………………………………….103 圖5-7 (a)於BM與IM系統中經過誘導分化的UMSCs,其膠原蛋白支架之H&E染色…………………………………………………………………………………104 圖5-7 (b)於BM與IM系統中經過誘導分化的UMSCs,其膠原蛋白支架之alcian blue染色…………………………………………………………………………….105 圖5-7 (c)於BM與IM系統中經過誘導分化的UMSCs,其collagen type II之組織免疫染色………………………………………………………………………....106 圖5-8 (a)於BM系統中經過誘導分化的UMSCs,其aggrecan之免疫螢光染色染色……………………………………………………………………………………107 圖5-8 (b)於IM系統中經過誘導分化的UMSCs,其aggrecan之免疫螢光染色染色…………………………………………………………………………………....107 圖5-9 (a) UMSCs於不同種類的支架上,經過四星期誘導分化後其隨時間變化 DNA之含量(n=3)…………………………………………………………………..108 圖5-9 (b) UMSCs於不同種類的支架上,經過四星期誘導分化後其隨時間變化 GAGs之含量(n=3)………………………………………………………………....108 圖5-9 (c) UMSCs於不同種類的支架上,經過四星期誘導分化後其隨時間變化之生化合成率(GAGs含量/DNA含量) (n=3)………………………………………..109 圖5-10 (a) UMSCs於不同種類的支架中誘導分化28天,其軟骨細胞特定基因aggrecan之表現量(n=3)……………………………………………………………109 圖5-10 (b) UMSCs於不同種類的支架中誘導分化28天,其軟骨細胞特定基因collagen type II之表現量(n=3)…………………………………………………….110 圖5-10 (c) UMSCs於不同種類的支架中誘導分化28天,其軟骨細胞去分化之特定基因collagen type I之表現量(n=3)……………………………………………..110 圖5-10 (d) UMSCs於不同種類的支架中誘導分化28天,其軟骨細胞特定基因collagen type X之表現量(n=3)…………………………………………………….111 圖5-10 (e) UMSCs於不同種類的支架中誘導分化28天,其硬骨細胞特定基因ALP之表現量(n=3)……………………………………………………………..….111 圖5-10 (f) UMSCs於不同種類的支架中誘導分化28天,其硬骨細胞特定基因OS之表現量(n=3)……………………………………………………………………...112 圖5-11 UMSCs於CCH支架中培養28天之組織切片染色(a) H&E染色; (b)alcian blue染色…………………………………………………………………………….113 圖5-12 UMSCs於COL I支架中培養28天之組織切片染色(a) H&E染色; (b)alcian blue染色…………………………………………………………………………….114 圖5-13 UMSCs於chitosan支架中培養28天之組織切片染色(a) H&E染色;(b) alcian blue染色……………………………………………………………………..115 圖5-14 (a) UMSCs於COL II/CS/HA支架中誘導分化後28天之collagen type II (COLII)免疫螢光染色……………………………………………………………...116 圖5-14 (b) UMSCs於COL I支架中誘導分化後28天之COL II免疫螢光染色…116 圖5-14 (c) UMSCs於chitosan支架中誘導分化後28天之COL II免疫螢光染色.117 圖5-15 (a) UMSCs於COL II/CS/HA支架中誘導分化後28天之aggrecan免疫螢色染色………………………………………………………………………………117 圖5-15 (b) UMSCs於COL I支架中誘導分化後28天之aggrecan免疫螢光染色.118 圖5-15 (c) UMSCs於chitosan支架中誘導分化後28天之aggrecan免疫螢光染色……………………………………………………………………………………118 圖5-16 (a)植入裸鼠背四星期後,CCH支架上細胞基因之表現…………………119 圖5-16 (b)植入裸鼠背四星期後,CCH支架上細胞基因之表現………………....119 圖5-17 (a)植入裸鼠背四星期後,CCH支架之組織切片染色H&E……………120 圖5-17 (b)植入裸鼠背四星期後,CCH支架之組織切片alcian blue染色……….120 圖5-18 (a)植入裸鼠背四星期後,CCH支架之collagen type II 免疫螢光染色…121 圖5-18 (b)植入裸鼠背四星期後,CCH支架之Aggrecan免疫螢光染色………...121 表目錄 表2-1 細胞激素或生長因子對軟骨細胞增生與分化之影響 19 表2-2 各種軟骨細胞外間質之組成 19 表2-3 一般組織內的膠原蛋白種類 20 表2-4 Proteoglycans的種類 21 表3-1各種培養基與成分……………………………………………………………40 表4-1冰凍溫度對於膠原蛋白孔洞大小之影響………………………………….. 53 表4-2 交聯後各種膠原蛋白第二型支架之物化性質……………………………..54 表4-3交聯後各種膠原蛋白第二型支架之物化性質 55

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