BMSCs复合胶原材料三维诱导软骨细胞的初步研究

PRELIMINARY STUDY OF BMSCs SEEDED INTO COLLAGENⅠ-GLYCOSAMINOGLYCAN MATRICES IN-DUCED TOWARD CARTILAGE

目的通过体外培养犬BMSCs,以胶原海绵作为支架材料,体外三维复合培养后,观察向软骨细胞定向诱导的可行性。方法健康家犬10只,体重10～15kg,雌雄不拘。抽取骨髓,体外培养BMSCs,传至第3代,倒置相差显微镜观察细胞形态。实验组将第3代BMSCs以1×106/mL密度接种于胶原海绵支架材料,体外培养48h后,将细胞材料复合物以软骨细胞定向诱导培养基进行诱导,每隔2天换液;对照组将第3代BMSCs以相同密度接种于胶原海绵支架材料,每隔2天以DMEM换液。培养后14d,将细胞材料复合物行HE染色和免疫组织化学染色观察。结果倒置相差显微镜下第3代BMSCs与第2代BMSCs形态基本一致,传代初期细胞伸出伪足,呈梭形或三角形,7d后逐渐以梭形为主,胞体饱满,细胞传代后增长速度明显加快。组织学观察:实验组细胞广泛分布于支架材料孔隙内,多数细胞呈多角形或者不规则形,少数呈短梭形,细胞周围间隙可见基质成分;对照组细胞分布于支架材料孔隙内,呈梭形或圆形,核呈圆形或椭圆形,胞体较大,胞质透亮均匀,细胞核轮廓清楚,呈蓝色。免疫组织化学染色显示,实验组支架材料孔隙内的细胞胞浆可见棕黄色颗粒,细胞周围出现黄染区;对照组无表达。结论BMSCs接种于胶原海绵材料后,经定向软骨诱导后,可向软骨组织方向分化。

Objective To investigate the possibility of BMSCs seeded into collagen Ⅰ -glycosaminoglycan （CG） matrices to form the tissue engineered cartilage through chondrocyte inducing culture. Methods Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment （DHT） cross-linked CG matrices at lx 106 cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen Ⅱ immunohistochemical staining. Results The examinations under the inverted phase contrast microscope indicated the 2nd and 3nd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-like spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen Ⅱ staining appeared around the cells in the experimental group. There was no positive collagen Ⅱ staining appeared in the control group. Conclusion It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.