三维血管化成骨微组织的构建及其促进牵张成骨的研究

Distraction osteogenesis has become an effective technique in treating craniofacial defects or growth deficiencies. Its limitations involves an extended treatment time, which increases risks of complications or failure. Recent studies showed that cell-based therapies might be promising in resolving this difficult problem. Our previous study has showed that the osteogenic cell sheet fragements could improve cell retention and thus facilitate bone regeneration in vivo. we also found injection of cell sheet fragements has superior bone regeneration capacity than injection of the dissociated cells. However, the commonly used 2-dimensional culture system usually resulted in poor cellular phenotypes and could not mimic the physiological function of cells in the body. Based on these facts, we hypothesize that the use of microtissue generated in a 3-dimensional culture system will bypass the problems associated with 2-dimensional culture and exogenous scaffold materials, and then improve bone formation and shorten treatment period of distraction osteogenesis. . To test the hypothesis, we will first fabricate microtissue from adult stem cells using 3-dimensional culture system. Its diameter, histological morphology, cell viability, cell migration, fusion, osteogenic capacity, and endothelial potential will be investigated. Furthermore, we will try to generate scaffold-free, vascularized, and osteogenic microtissue using cell co-culture method. And then we will transplant the complex microtissue into the distraction site in animal model to investigate if distraction osteogenesis can be enhanced. Finally, the molecular mechanisms of microtissue in enhancing bone formation will be addressed in our work. The strategy may be promising in shortening treatment time of distraction osteogenesis if the hypothesis is proved to be practical.

牵张成骨是治疗颅颌骨缺损和畸形的有效技术，但治疗周期长，限制了其推广应用。细胞治疗策略有望解决这一难题。我们前期发现：二维培养获得的间充质干细胞聚集体可提高细胞移植后的滞留率，较单细胞的成骨潜能更高。最近研究表明三维细胞聚集体类似组织芽，能模拟细胞的体内生长环境，更利于维持细胞表型、活性和功能。基于此我们设想：通过三维培养将成骨相关细胞构建成无支架的微组织，可以克服二维培养和外源性载体所伴随的问题。如移植到牵张区，将显著增加成骨效率并缩短治疗周期。本项目拟将成骨分化、血管内皮分化、及未分化的间充质干细胞三维共培养，构建血管化成骨微组织，体外研究其可注射性和生物学特征；然后将微组织注射于兔和羊的下颌骨牵张区，从组织形态学、细胞学和分子生物学水平，论证其促进成骨的可行性和有效性，明确最佳应用方式，揭示其加快新骨形成的作用机理，进而为临床上解决牵张成骨治疗周期过长的问题提供新策略和理论依据。

牵张成骨是治疗颅颌骨缺损和畸形的有效技术，但治疗周期长，限制了其推广应用。细胞治疗策略有望解决这一难题，最近研究表明三维细胞聚集体类似组织芽，能模拟细胞的体内生长环境，更利于维持细胞表型、活性和功能。基于此将一定数量的骨髓间充质干细胞细胞接种于超低粘附96孔板内，三维培养构建成无支架的微组织。与传统的二维单层培养相比, 三维培养细胞结构保留了细胞自分泌的细胞外基质，增强了细胞-基质及细胞间相互作用，血管生成生长因子和细胞因子的表达显著增加，进而增强促血管生成特性，增加移植细胞的滞留率和生存率，进而将成骨分化干细胞聚集体经皮下注射到裸鼠中以后, 观察到了显著更大和更致密的异位骨形成。本项目进一步将成骨分化、血管内皮分化、及未分化的间充质干细胞三维共培养，构建血管化成骨微组织，体外研究其可注射性和生物学特征；然后将微组织注射于兔下颌骨牵张区，从组织形态学、细胞学和分子生物学水平，论证其促进成骨的可行性和有效性，揭示其加快新骨形成的作用机理，进而为临床上解决牵张成骨治疗周期过长的问题提供新策略和理论依据。