体内成骨过程中人脂肪基质细胞对新骨形成的促进作用

Effect of human adipose-derived stromal cells on osteogenesis in vivo

目的:探索以人脂肪基质细胞(human adipose-derived stromal cells,hASCs)为基础的组织工程骨在体内成骨过程中hASCs发挥的作用,为深入研究其体内成骨机制奠定基础。方法:酶消化法分离原代hASCs,进行传代培养。体内实验使用16只裸鼠,于其背部正中做皮肤切口,向两侧分离出4个皮下植入腔,分别植入:(1)空白;(2)单纯β-磷酸三钙(β-tricalcium phosphate,β-TCP)支架(支架对照组);(3)β-TCP支架+人成纤维细胞(阴性细胞对照组);(4)β-TCP支架+hASCs(实验组)。植入后1周、2周、4周、6周进行取材,每时间点取4只裸鼠,其中2只裸鼠的标本经处理后进行扫描电镜(scanning electron microscope,SEM)观察,另外2只裸鼠的标本制成组织学切片,进行HE染色分析。结果:SEM观察可见实验组植入2周后,hASCs周围即出现大量细胞外基质,至4周时细胞外基质开始出现明显的矿化,6周时可见矿化基质进一步增多。HE染色分析可见实验组植入2周后hASCs即开始分泌嗜酸性较强的均质细胞外基质,至4周时细胞外基质中出现了强嗜酸性的类骨组织,6周时新生类骨组织面积明显增大,结构更加典型。在其他组均未见矿化基质和类骨组织生成。结论:人脂肪基质细胞在体内成骨过程中发挥了关键的作用,包括分泌大量细胞外基质,促进细胞外基质矿化和引导新生类骨组织形成。

Objective：To explore the effect of human adipose-derived stromal cells（hASCs） on the osteogenesis during the process of bone formation in vivo,and to lay the foundation of further investigations on the mechanism of in vivo osteogenesis of hASCs.Methods：hASCs were isolated from adipose tissue by the method of collagenase digestion,and were routinely proliferated and passaged.In the in vivo study 16 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice：（1） blank;（2） β-tricalcium phosphate（β-TCP） scaffold only（scaffold control group）;（3） β-TCP scaffold with human fibroblasts（negative cell control group）;（4） β-TCP scaffold with hASCs（test group）.After 1 week,2 weeks,4 weeks and 6 weeks of implantation,samples from the 4 nude mice were collected at each time point.Scanning electron microscope（SEM） observation and histological staining were performed to evaluate the in vivo osteogenesis of hASCs.Results：SEM images showed that large amount of extracellular matrix（ECM） could be observed around hASCs in test group after 2 weeks of implantation.At the time point of 4 weeks,mineral deposit was found in ECM.At the time point of 6 weeks,the mineral deposit was observed to increase significantly.HE staining showed that the ECM with eosinophilic staining could be observed around hASCs after 2 weeks of implantation.At the time point of 4 weeks,newly-formed bone-like tissue could be found in ECM around the scaffold materials.At the time point of 6 weeks,more bone-like tissues were observed in ECM with typical structure of bone tissue.In comparison,no obvious mineralization and bone-like tissue were found in other groups.Conclusion：hASCs play important roles in the process of osteogenesis in vivo,including secretion of large amount of ECM,acceleration of the mineralization of ECM and guidance for the formation of bone-like tissues.