骨髓间充质干细胞复合骨基质明胶构建组织工程化软骨  

Tissue engineered cartilage constructed by growth factor-induced bone marrow mesenchymal stem cells and allogenic bone matrix gelatin

作　　者：尹战海[1] 韩健 刘淼[1] 曹峻岭[3] 王金堂[1] 王民[1] 韩学哲[1] 

机构地区：[1]西安交通大学第一医院骨科,710061 [2]陕西省泾阳县医院骨科 [3]西安交通大学地方病研究所

出　　处：《中华骨科杂志》2005年第3期170-175,共6页Chinese Journal of Orthopaedics

基　　金：陕西省自然科学基金资助项目(2001SM79)

摘　　要：目的分离并诱导骨髓间充质干细胞(MSCs)成软骨分化,与同种异体骨基质明胶(BMG)结合,在体外构建组织工程软骨。方法分离骨髓MSCs,实验组DMEM培养液中加入rhTGF-β1和rhIGF-Ⅰ,对照组未加入诱导因子。比较实验组和对照组细胞的增殖和成软骨分化情况。制备同种异体BM G,扫描电镜观察,与细胞结合在体外构建组织工程软骨,1、2、3和5周取材作胶原染色、蛋白聚糖染色和扫描电镜观察。结果实验组中M SCs集落形成效率(21/106)明显高于对照组(3/106)(u=3.8783,P<0.01);实验组8例中有7例表达Ⅱ型前胶原m RNA,而对照组仅1例表达(χ2=6.250,P<0.05);实验组细胞培养液中的己糖醛酸逐渐升高,第15d已达到初始浓度的1.38倍,而对照组无明显升高(t=3.933,P<0.01)。制备得到的同种异体BMG为三维立体多孔结构,可塑性好并有适宜的机械强度。胶原、蛋白聚糖染色和扫描电镜观察发现,细胞在BM G表面和孔隙内大量增殖,培养后5周在组织工程软骨块的内部即可见软骨陷窝。结论rhTGF-β1和rhIGF-Ⅰ可促进MSCs增殖和成软骨分化,与同种异体BMG结合后可在体外成功构建组织工程软骨。同种异体BM G符合组织工程软骨基质材料的基本要求,有望成为一种理想的用于关节骨软骨缺损修复的软骨组织工程载体材料。Objective To construct tissue engineered cartilage with bone marrow mesenchymal stem cells (MSCs) and allogeneic bone matrix gelatin (BMG). Methods Bone marrow was aspirated and MSCs were separated by consecutive 0.70(70%)(V/V) Percoll gradient centrifugation and fibronectin adhesion. In experimental group, rhTGF-β1 and rhIGF-Ⅰ were applied into DMEM to induce proliferation and chondrogenic transformation, while in control group only DMEM was used. Colony forming efficiency (CFE) of MSCs was calculated, procollagen α1(Ⅱ)mRNA expression in cells was detected by RT-PCR, hexuronic acid in culture medium was measured by carbazole-sulfuric acid method. Allogeneic bone matrix gelatin (BMG) was prepared by successive defatting and decalcification, observed by scanning electronic microscope. Chondrocyte progenitor cells induced from MSCs were seeded into BMG to construct tissue engineered cartilage. Culture masses were sectioned and stained by Masson's trichrome. Results CFE was 21/106 in experimental group, significantly higher than that of control group, which was 3/106 (u=3.878, P<0.01). Expression of articular cartilage specific procollagen α1(Ⅱ)mRNA was 7/8 in experimental group, while 1/8 in control group(χ2=6.250, P<0.05). Hexuronic acid in culture medium of experimental group rised gradually, reaching 1.38 time of initial concentraton at the 15th day, while there was no obvious rise in control group. Allogeneic BMG prepared was a three-dimensional highly-porous structure with good plasticity and suitable mechanical hardness. Tissue engineered cartilage with fine cartilage lacunae and comparable hardness could be observed 5 weeks after tissue engineering. Conclusion Proliferation and expression of chondrocytic phenotype by MSCs can be induced by the synergistic action of rhTGF-β1 and rhIGF-I. Allogeneic BMG may satisfy the demands of scaffold for tissue-engineered cartilage.

关 键 词：组织工程软骨 对照组 同种异体 成软骨分化 骨基质明胶 体外 骨髓间充质干细胞 扫描电镜观察 DMEM 骨块 

分 类 号：R687[医药卫生—骨科学]