不同比例软骨细胞与间充质干细胞对构建组织工程软骨的影响

目的以不同比例将大鼠软骨细胞及骨髓间充质干细胞（bonemarrowstromalceu，BMSCs）依次体外及体内混合培养，探讨二者构建组织工程软骨的最佳比例。方法分别取1个月及1d龄SD大鼠的BMSCs及关节软骨，原代软骨细胞与第2代BMSCs混合比例为全软骨细胞组、3：1、1：1、1：3、全BMSCs组，共五组。以终浓度4×10^7／mI．种于聚乳酸一聚羟基乙酸支架上，体外培养4周后，植入裸鼠皮下继续培养，8周后对标本进行大体观察、组织学切片、Ⅱ型胶原免疫荧光染色及检测氨基糖胺多糖的含量。结果软骨细胞与BMSCs混合比例3：1组较其他各组所构建出的组织工程软骨体积大，厚度厚，有光泽；组织染色见软骨细胞增殖较旺盛，被大量细胞外基质包围，分布均匀，并可见软骨陷窝；Ⅱ型胶原免疫荧光染色见细胞内和细胞外基质发出的红色荧光较明亮；3：1组的组织块氨基糖胺多糖含量（470．38±29．78）μg高于其他各组（P〈O．05）。结论软骨细胞与BMSCs混合培养有助于节省软骨细胞，二者混合浓度比例以3：1最好。

Experimental Study on the Cocuiture of Bone Marrow Stromal Cells with Chondrocytes at Different Ratios to Construct Engineered Cartilage

Objective To find out the best ratio of Bone marrow stromal cells （BMSCs）mixed with chondrocytes in constructing engineered cartilage in vitro and in vivo. Methods To culture chondrocytes and BMSCs from SD rat of newborn and 1 month respectively. Primary chondrocytes and BMSCs of the second generation were mixed at dif- ferent ratios （chondrocytes/BMSCs .- 3/1,1/1,1/3,pure chondrocytes and pure BMSCs）and then seeded onto PLGA scaffoldat the final concentration of 4 X 107/mL. After 4 weeks in vitro,the specimens were implanted into nude rats subcutaneous for 8 weeks. Gross observation, histological check, immunofluorescence test and glycosaminoglycan （GAG） quantification were applied to evaluate the results. Results Compared with other groups ,the engineered car- tilages of 3/1 group had the following characteristics ：greater volume ,thicker thickness ,smoother surface. Histological check shows better cell proliferation in the 3/1 group,in which chondrocytes are surrounded by mass matrix. Histolo- gy and immunofluorescenee demonstrated that the 3/1 group specimens grew much better than the others, which showed a compact tissue structure and the engineered cartilage matrix staining were stronger than the others. The GAG content in 3/1 group specimens were higher than the other specimens with significant difference（P〈0. 05）. Conclusion Coculture of chondrocytes mixed with BMSCs at the ratio of 3/1 to construct engineered cartilage can save a large number of chondrocytes.