人脐血间充质干细胞修复颅骨缺损的实验研究

Repair of calvarial defects with human umbilical cord blood derived mesenchymal stem cells and demineralized bone matrix in athymic rats

目的应用人脐血间充质干细胞（umbilical cord blood derived mesenchymal stem cells，UCB—MSCs）复合脱钙骨材料构建组织工程化骨，修复裸大鼠颅骨标准缺损。方法体外扩增培养、成骨诱导人UCB—MSCs，采用AlizarinRed染色和钙离子半定量的方法测定细胞成骨分化能力。将第2代细胞接种在脱钙骨支架材料上继续诱导培养，扫描电镜检测细胞在材料上的生长状况。制备裸大鼠双侧颅骨全层标准缺损（直径5mm），一侧以细胞材料复合物修复作为实验侧（n=8）；另一侧以单纯脱钙骨材料修复作为对照侧（n=8）。术后6、12周取材，分别通过大体形态观察、显微CT（Micro—CT）、组织学方法检测颅骨缺损的修复效果。结果UCB—MSCs体外能够诱导分化为成骨细胞，且在脱钙骨支架材料上生长良好。Micro—CT检测显示术后6周实验侧有部分新生骨组织形成，12周时骨缺损修复率达（78．19±6．45）％，脱钙骨材料降解完全；对照侧6周时无明显新骨生成，12周时材料完全降解，骨缺损未修复。组织学检测显示12周时实验侧有较多成熟骨生成，为骨性愈合；对照侧骨缺损为纤维愈合。结论成骨诱导的人UCB—MSCs复合脱钙骨材料构建的组织工程化骨可修复裸大鼠颅骨全层标准缺损，有望成为新的组织工程骨种子细胞来源。

Objective To investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells （UCB-MSCs） and demineralized bone matrix （DBM） scaffolds to repair criticalsized calvarial defects in athymic rats. Methods Human UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects （5 mm in diameter） of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs （experimental group） or with DBM scaffolds alone （control group）. Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis. Results Micru-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of （78. 19 ± 6.45 ）% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group. Conclusions Tissue-engineered bone comoosed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.