真皮BMPR-IB+细胞在骨修复重建中的作用

Lack of stable sources of the seed cells (osteoblast) is the obstacle preventing wide clinic application of the Tissue-Engineered bone, and the key resolution is to build up a standardized seed cells bank of bone. The dermal cells in the prepuce harvested by circumcision are the potential and ideal source of the osteoblast cells bank, because these cells are close to be homogeneous and contain a certain percentage of the osteoblast. However, there are many none-osteoblast cells existing in the abandoned prepuce, which inhibit the bone formation. Developing a strategy to enrich of the osteoblast from the dermal cells in the prepuce could turn waste into treasure..In the previous studies, the proposer found that the dermal cells with the positive expression of Bone Morphogenetic Protein Receptor (BMPR-IB+) have the abilities of stable expression, high proliferative potential and high osteogenic differentiation potential in vitro. Therefore, BMPR-IB can be used as a cellular membrane marker to enrich the dermal osteoblast in the prepuce..Based on the previous studies, the proposer will focus on the osteogentic ability of the BMPR by multiple ways, such as RNA interference, cell-tracking, allogenic skin-tissue microsurgical transfer and reconstruction of bone defect. Our research will answer the following questions:(1) Why the BMPR-IB positive dermal cells have high osteogenic differentiation potential in vitro, and what is the mechanism? (2) Whether the BMPR-IB positive dermal cells can cure autogenic and allogenic bone defect in vivo? (3) How about the long following-up of the bone reconstruction by the BMPR-IB positive dermal cells and where the BMPR-IB positive dermal cells transfer?.All of the answers may form the technical foundation and basic principle for building up the standard allogenic bone seed bank.

种子细胞来源问题一直是限制组织工程骨规模化临床应用的瓶颈，解决的关键是建立标准化的骨种子细胞库。幼儿包皮环切所得皮肤来源稳定、个体差异小且含有一定比例的成骨潜能细胞，有望变废为宝成为理想的骨再生细胞来源，但其所含的大量非成骨潜能细胞干扰了骨再生效果，如何富集其成骨潜能细胞是关键。申请人前期研究发现：包皮来源细胞中骨形成蛋白受体阳性（ BMPR-IB+）细胞表达率稳定、增殖力强且体外成骨能力强。骨形成蛋白受体作为细胞表面标志，可用来富集包皮来源的成骨潜能细胞。本课题拟在此基础上，通过RNA干扰、细胞标记、皮瓣移植和骨缺损修复等手段，阐明以下几个关键问题：（1）真皮BMPR-IB+细胞为什么体外成骨能力强？机理何在？（2）真皮BMPR-IB+细胞能否成功修复自体和同种异体骨缺损？（3）骨修复长期疗效如何？细胞最终转归？.这些问题的阐明将为建立标准化同种异体骨种子细胞库提供技术基础和理论依据。

目的 以细胞标记技术为手段，研究真皮BMPR-IB+GFP+细胞在体内的分布和迁移转归的特点；建立起比较成熟的真皮BMPR-IB+细胞分选方案，体外鉴定其成骨分化能力；研究BMPR-IB+细胞在异位（皮下）骨形成能力、骨缺损修复能力和最终转归。.方法 采用免疫磁珠分选针对细胞表面骨形成蛋白I型受体B亚型（BMPR-IB）进行分选，分选后的细胞培养后鉴定其成骨分化能力；使用珊瑚制成组织工程支架，检验珊瑚支架的基础性能包括孔隙率和孔径；复合细胞后通过扫描电镜观察细胞材料间的生物相容性；构建裸鼠颅骨缺损模型，用复合细胞的珊瑚支架予以修复。.结果 用免疫磁珠方法可以成功分选得到BMPR-IB+细胞，体外诱导成骨后，定性的碱性磷酸酶、钙结节染色和定量检测都证实其具有极强的成骨能力；珊瑚材料与细胞具有良好的生物相容性，细胞能在其上生长增殖；构建的组织工程支架能修复裸鼠颅骨缺损。.结论 BMPR-IB+细胞可通过磁珠分选获得，在体外验证其有极强成骨能力，与珊瑚材料构建的组织工程支架能够成功修复裸鼠颅骨缺损。