Bio—gide胶原膜体外复合培养犬骨髓间充质干细胞的软骨分化

背景：应用组织工程方法修复股骨头坏死后的软骨损伤应解决的2个关键因素是种子细胞和支架材料。目的：探讨Bio-gide胶原膜体外与犬骨髓间充质干细胞复合成软骨的可行性。方法：采用全骨髓血离心法结合贴壁筛选法体外分离培养比格犬骨髓间充质干细胞，观察细胞形态学变化，以细胞表面抗原进行鉴定。采用成软骨诱导培养基诱导第3代骨髓间充质干细胞向软骨细胞分化作为实验组，设置普通培养基培养对照组，MTT比色法测试软骨细胞生长曲线，并行Ⅱ型胶原免疫组织化学和甲苯胺蓝染色等生物学鉴定。将第3代骨髓间充质干细胞与Bio-gide胶原膜体外复合培养行倒置相差显微镜、扫描电镜观察。结果与结论：采用全骨髓血离心法结合贴壁筛选法获得了高纯度高活性的骨髓间充质干细胞，生长状态良好，扩增能力强，并成功诱导分化为软骨细胞。大量骨髓间充质干细胞在Bio-gide胶原膜上贴附增殖并复层生长，细胞与Bio-gide胶原膜逐渐融为一体；在胶原膜的断面可见胞体伸出突起相互连接包绕胶原膜，其间有明显细胞基质分泌。表明犬骨髓间充质干细胞可在Bio-gide胶原膜上生长并向软骨细胞分化。

Chondrocyte differentiation of dog bone marrow mesenchymal stem cells co-cultured with Bio-gide collagen membrane in vitro

BACKGROUND：Seed cells and scaffold are two key factors for cartilage defects after osteonecrosis of femoral head using tissue-engineered method. OBJECTIVE：To explore the feasibility of Bio-gide col agen membrane combined with dog bone marrow mesenchymal stem cells into chondrocytes. METHODS：Bone marrow mesenchymal stem cells were isolated from beagle dogs by whole bone marrow blood centrifugation method and adherence screening method in vitro and cultured. Morphological changes in cells were observed, and identification was done using cellsurface antigens. Bone marrow mesenchymal stem cells of passage 3 were induced by chondrocyte induction medium to differentiate into chondrocytes （experimental group）. cells cultured in normal medium were considered as control group. 3-（4, 5-Dimethylthiazol-2-yl） 2, 5-diphenyl tetrazolium bromide assay was used to measure growth curve of chondrocytes. cells underwent typeⅡcol agen immunohistochemistry and toluidine blue staining. The coculture of bone marrow mesenchymal stem cells at passage 3 and Bio-gide col agen membrane were observed under an inverted phase contrast microscope and scanning electron microscope. RESULTS AND CONCLUSION： Bone marrow mesenchymal stem cells with high purity and high viability were obtained by whole bone marrow blood centrifugation method and adherence screening method. cells grew wel and had strong amplified ability, and successful y differentiated into chondrocytes. Numerous bone marrow mesenchymal stem cells adhered on the Bio-gide col agen membrane, showing a tendency of multi-layer growth. cells and Bio-gide col agen membrane seem to blend into an integrant part. Cel processes appeared and connected each other and gradual y wrapped the Bio-gide col agen membrane, with the presence of obvious cel matrix secretion. These results suggested that bone marrow mesenchymal stem cells can grow and differentiate into chondrocytes on the Bio-gide col agen membrane.