膠原蛋白二型支架及硫酸軟骨素對於幹細胞進行軟骨分化之效應

The Chondrogenesis Effects of Type II Collagen Scaffold and Chondroitin Sulfate C by Culture of Mesenchymal Stem Cells

陳威全(Wei-Chuan Chen)

指導教授 ： 朱ㄧ民

國立清華大學/工學院/化學工程學系/博士(2011年)

摘要

本篇論文主要有兩個研究目的，一為探討幹細胞在 type II collagen支架(COL II scaffold)，對軟骨體內修復上的效應; 二是對於多種不同型態之chondroitin sulfate C (CSC)，對幹細胞(MSCs)誘導分化為軟骨細胞(chondrocytes)的效應及CSC-COL II支架對軟骨組織修復的效應。
在第一部分，在三週的誘導之後，先以in vitro狀態下判斷細胞是否具有分化為軟骨細胞的能力，將含有兔子骨髓細胞(rabbit bone marrow stem cells, RBMSCs)之type II collagen支架，再以含有TGF- β3生長因子的培養基進行分化培養。由軟骨分化相關基因的表現、H&E stain 及Alcian Blue stain的結果可以說明，RBMSCs可以被誘導分化為chondrocyte-like細胞。次之探討type II collagen支架軟骨體內修復上的效應。將培養有RBMSCs之type II collagen支架植入此受損軟骨組織中，評估修復的狀況。八週後觀察兔子的關節修復情形可以發現，於外觀上可以看到，受損的部位已經被type II collagen支架填滿。由H&E stain, Alcian Blue stain組織染色及type II collagen, aggrecan免疫組織染色的結果，可以進一步說明修復的組織中，已佈滿具有lacuna結構的軟骨細胞。於二十四週後的結果顯示，外觀已和一般組織相同，由H&E stain可以看出修復的部位，與正常組織很類似。
接著探討CSC對軟骨修復情形的影響。首先比較CSC(free CSC)或oligosaccharide CSC (free oligosaccharide CSC)添加於培養基中培養臍帶血間葉幹細胞(umbilical cord blood-derived MSCs, UMSCs)或將UMSCs培養於經genipin交聯之CSC-COL II支架(crosslinked CSC)，進行軟骨分化的效應。根據組織染色、基因表現及ECM分泌的結果顯示，UMSCs培養於free CSC, free oligosaccharides CSC及crosslinked CSC 支架，都可以被誘導成chondrocytes。以不同CSCs濃度交聯的crosslinked CSC 支架，發現35 ug 的CSC交聯至支架時(T+CCR,H35)，對UMSCs的軟骨分化表現最好。此外低分子量之free oligosaccharide CSC很明顯的相較於高分子量的free CSC更能提升軟骨分化基因的表現，及刺激ECM的分泌累積。進一步將CSC-COL II支架植入紐西蘭白兔體內進行評估。將RBMSC-laden的COL II 支架(T+)或CSC-COL II支架(T+CCR,H35)植入，除利用組織染色評估外，並進一步以Q-PCR約略評估其修復的狀況。研究結果顯示，四週後觀察兔子的關節修復情形可以發現，外觀上T+CCR,H35組與T+組相同，軟骨缺損均被修復。四週的組織染色的結果指出T+CCR,H35組已呈現具有lacuna結構的軟骨細胞，T+組則支架尚未完解分解，說明CSC的確在軟骨修復方面有正面的效應。

關鍵字

軟骨組織 ； type II collagen支架 ； 間葉幹細胞 ； 硫酸軟骨素

並列摘要

Two major research topics were discussed in this thesis. One is evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold (COL II scaffold); the other is to discuss the effects of chondrogenesis by culture of mesenchymal stem cells with various types of chondroitin sulfate C (CSC). Moreover, the defect repair potential of CSC-COL II scaffold was also evaluated this thesis.
In the first section, after 3-week of in vitro induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The rabbits were sacrificed after eight and twenty four weeks. Eight weeks later, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites with¬out apparent inflammation. After twenty four weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo condi¬tions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.
In the second section, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen scaffolds with CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC crosslinked type II scaffolds (crosslinked CSC). According to the analyses of histology stain, gene expression and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC and on the crosslinked CSC scaffolds all would be induced into chondrocytes. We also found crosslinked CSC scaffold had the good response for chondrogenesis when 35 ug of CSC was crosslinked to COL II scaffold (T+CCR,H35). Moreover, free oligosaccharide CSC presented in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Thus, we believed that crosslinked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. The defect repair potential of CSC-COL II scaffold (T+CCR,H35) was also evaluated by rabbit model. Our results showed that T+CCR,H35 scaffold showed better repair ability on cartilage defect than T+ scaffold by histological and immunohistological staining and the recovered cells in the T+CCR,H35 scaffold had lacuna structure. At the meanwhile, T+CCR,H35 scaffold revealed the type II collagen fibers in the defect site. Furthermore, the generated cells in the T+CCR,H35 scaffold on the repair site showed the higher COL II and aggrecan gene expression and lower type I collagen gene expression compared to the cells in the T+ scaffold after twenty-four week transplantation. Thus, we believed that T+CCR,H35 scaffold would be a potential candidate for cartilage defect repair by tissue engineering approach.

並列關鍵字

cartilage defect ； type II collagen scaffold ； mesenchymal stem cells ； chondroitin sulfate C

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