不同比例EPCs和BMSCs共培养体系对组织工程骨血管化和成骨的量效关系及机制研究

The present of Tissue engineered bone(TEB) provides a novel treatment method for bone defect repair, it has tremendous potential medical applications. Vascularization is crucial for successfully constructing tissue engineered bone. The co-culture strategy of endothelial progenitor cells combined with bone marrow mesenchymal stem cells is one of the hotspot in the field of constructing vascularized tissue engineered bone. But influences of the seeding ratio of EPCs and BMSCs co-culture system on the effect of vascularization and bone formation in vivo have not been studied. And little is known about the dose-effect relationship of vascularization and bone formation. According to our previous research we speculate that bone formation is not linear with the vascularizaion, there is a dose-effect relationship between vascularization and bone formation of TEB. To verify this hypothesis and expose the dose-effect relationship between vascularization and bone formation in vivo, we will establish animal models with different degree vascularized TEBs using different ratios of EPCs and BMSCs co-culture system. The effect of vascularization will be measured and calculated at different time point by Micro CT according to the previous research. And the effect of bone formation will be detected by CT scan, histology and Real-Time PCR. Our research will provide experimental and theoretical basis for constructing vascularized TEB by co-culture strategy in the future. This project has direct application value.

组织工程骨（TEB）的出现为修复骨缺损提供了新的治疗方法，具有巨大应用前景。血管化是成功构建大尺寸TEB的关键因素。EPCs与BMSCs共培养策略是当前血管化TEB研究的热点。然而，EPCs与BMSCs共培养体系接种比例的不同对TEB体内血管化和成骨的影响及血管化和成骨之间量效关系的研究未见报道。根据申请者前期研究推测，TEB的血管化效果和成骨效果之间并非呈线性促进的关系，很可能存在一定的量效关系。为验证这个假说，本项目在前期研究的基础上，通过不同比例的EPCs和BMSCs共培养体系构建不同血管化程度的TEB动物模型；分别在不同时间点，通过前期研究所掌握的Micro-CT技术测算各实验组TEB的血管化效果；采用活体CT、组织学、RT-PCR检测成骨效果；揭示TEB体内血管化和成骨之间的量效关系并初步探讨VEGF在其中的作用机制，为今后采用共培养策略构建血管化的TEB提供实验基础和理论依据。

组织工程骨（TEB）的出现为修复骨缺损提供了新的治疗方法，具有巨大应用前景。血管化是成功构建大尺寸TEB的关键因素。EPCs与BMSCs共培养策略是当前血管化TEB研究的热点。本项目通过将不同比例内皮祖细胞（EPCs）与骨髓间充质干细胞（BMSCs）进行共培养，分析BMSCs的生长活性，成骨分化与成骨效果等，以得到最优的共培养比例；构建BMSCs的VEGF的过表达及沉默模型，以最优比例共培养EPCs与BMSCs，加入干扰后，测定BMSCs的成骨相关基因表达，成骨分化与成骨效果等指标，以明确EPCs对BMSCs成骨促进作用的相关机制；进行动物实验验证以最优比例接种的支架中可获得较好的血管化。从以上研究中，我们可得到EPCs与BMSCs的比例为0.5:1时，可获得较好的成骨效果，其机制与VEGF分子机制相关，且动物实验中，此最优比例接种的支架也得到了较好的血管化。本研究丰富了共培养策略构建血管化组织工程骨的理论，筛选出了EPCs与BMSCs体内构建血管化组织工程骨的最优比，为今后采共培养构建组织工程骨提供实验与理论依据，具有直接的应用价值。