以3种材料为载体体外构建组织工程软骨

In vitro construction of tissue engineered cartilage using three materials

背景:纤维胶原蛋白被公认是非常理想的载体,但其胶性易流动,降解较快,且通透性有待置疑。而海绵状胶原蛋白多微孔,三维立体结构,吸附性好,临时基质与细胞基质相近(Ⅱ型胶原),作者所查文献中作为骨髓间充质干细胞载体用于软骨形成的报道较少。目的:对比观察骨髓间充质干细胞在纤维蛋白海绵、生物蛋白海绵、明胶海绵载体中软骨形成的能力和可行性。设计、时间及地点:体外对比观察实验,于2004-12/2008-08在海南省人民医院中心实验室完成。材料:纤维胶原蛋白海绵由美国Sigma公司提供,生物蛋白海绵(主要成分为Ⅱ型胶原,并吸附定量的碱性成纤维细胞生长因子)由辽宁绿谷制药有限公司提供,明胶海绵由南京金陵制药有限公司提供。方法:抽取猪髂骨骨髓血,用密度梯度离心法分离出有核细胞,在含转化生长因子β1的DMEM培养液中扩增骨髓间充质干细胞,将第3代骨髓间充质干细胞以载体内多点注射和表面点滴法植于Ⅱ型纤维胶原蛋白海绵、生物蛋白海绵及明胶海绵3种载体上,继续以含有转化生长因子β1的DMEM培养液体外培养。主要观察指标:观察细胞的生长情况,于4周取材进行大体组织观察,组织学分析及超微结构观察。结果:骨髓间充质干细胞在体外分离后可在转化生长因子β1培养液下扩增。纤维胶原蛋白海绵组可见少量的软骨基质与细胞;生物蛋白海绵组可见较多的基质与软骨细胞,细胞生长活跃,明胶海绵组可见少量散在的胶原基质,但无细胞生长。生物蛋白海绵组软骨细胞阳性数量多于纤维胶原蛋白海绵组(P<0.01),两组都明显多于明胶海绵组(P<0.01)。结论:骨髓间充质干细胞在生物蛋白海绵载体中软骨形成能力最强,纤维蛋白海绵次之,而明胶海绵与细胞生长不同步,软骨形成能力最差。

BACKGROUND： It is generally accepted that fibrous collagen protein is an ideal vector, limiting by easy fluxion, rapid degradation, and doubtable permeability. However, few reports address sponginess collagen protein, characterizing by multiple micropore, three-dimensional structure, great absorbency, and similar temporary matrix to cell matrix （type Ⅱ collagen）, which is considered as a vector of bone mesenchymal stem cells to act on chondrogenesis. OBJECTIVE： To compare the chondrogenic ability and feasibility of bone mesenchymal stem cells among fibrin sponge, bioprotein sponge, and gelatin sponge vectors. DESIGN, TIME AND SETTING： In vitro contrast study was performed at the Central Laboratory of Hainan People＇s Hospital between December 2004 and August 2008. MATERIALS： Fibrin sponge was provided by Sigma Company, USA; bioprotein sponge, a major component of type Ⅱ collagen, absorbing a certain quantity of basic fibroblast growth factor, was provided by Liaoning Lvgu Pharmaceutical Co., Ltd.; gelatin sponge was provided by Nanjing Jinling Pharmaceutical Co., Ltd. METHODS： Bone marrow blood was drawn from pig ilium to separate karyocytes using density gradient centrifugation. Bone mesenchymal stem cells were amplified in DMEM culture media containing transforming growth factor-β ; thereafter, the third-passage bone mesenchymal stem cells were transplanted on fibrin sponge, bioprotein sponge, and gelatin sponge using multipeint injection and surface drop methods. And then, the samples were in vitro cultured in DMEM culture media containing transforming growth factor- β. MAIN OUTCOME MEASURES： Cell growth, gross histological observation in the fourth week, histological analysis, and ultrastructural observation. RESULTS： After separation in vitro, bone mesenchymal stem cells could be amplified by transforming growth factor- β1. A few of cartilage matrixes and cells were observed in the fibrin sponge vector; a lot of matrixes and chondrocytes were observed in the bioprotein sponge vector, characterizing by active growth; a few of collagen matrixes but no cells were observed in the gelatin sponge vector. Number of positive chondrocytes in the bioprotein sponge vector was significantly greater than fibrin sponge vector （P〈 0.01）, and the numbers in both fibrin sponge and bioprotein sponge vectors were significantly greater than gelatin sponge vector （P 〈 0.01）. CONCLUSION： Chondrogenesis of bone mesenchymal stem cells is the strongest in the bioprotein sponge vector, and then in fibrin sponge vector and gelatin sponge vector.