磷酸钙骨水泥／骨形态蛋白人工骨复合骨髓基质细胞构建组织工程骨

Constructing tissue-engineered bone with calcium phosphate cement/bone morphogenetic protein artificial bone and bone marrow mesenchymal stem cells

背景:体外实验证明磷酸钙骨水泥/骨形态蛋白人工骨具有较强的骨诱导作用,但是磷酸钙骨水泥/骨形态蛋白人工骨能否作为骨髓基质细胞的生长支架报道较少。目的:验证磷酸钙骨水泥/骨形态蛋白人工骨做为骨组织工程支架材料的可行性。设计、时间及地点:观察实验,于2008-03/2009-03在上海市第九人民医院口腔组织工程实验室完成。材料:磷酸钙骨水泥/骨形态蛋白人工骨产品由瑞邦公司提供,大孔径200～500μm,小孔径1～5μm,孔隙率为70%。两岁的健康毕格犬1只。方法:抽取成年毕格犬的骨髓,贴壁法获得骨髓基质细胞,经成骨诱导培养液体外培养、扩增、诱导后观察细胞增殖情况。将培养的第2代细胞接种于多孔型活性磷酸钙骨水泥,进行超微结构观察,并将多孔型活性磷酸钙骨水泥/骨髓基质细胞复合物植入毕格犬背部皮下,2,4周后进行组织学检测。主要观察指标:倒置相差纤维镜下观察细胞的生长、增殖情况;碱性磷酸酶染色、Von Kossa染色、骨钙素免疫细胞化学染色鉴定骨细胞的形成标志;扫描电镜观察细胞材料复合情况;苏木精-伊红染色观察体内异位成骨情况。结果:碱性磷酸酶染色呈阳性;Von Kossa染色可见钙结节形成;骨钙素免疫细胞化学染色呈阳性;超微结构观察可见细胞生长附着于材料网孔内表面;组织学检测提示4周时复合物内有新骨形成。结论:多孔型活性磷酸钙骨水泥/骨髓基质细胞复合物显示良好的成骨活性,磷酸钙骨水泥/骨形态蛋白人工骨可以用于骨组织工程支架材料。

BACKGROUND： In vitro studies have shown that calcium phosphate cement/bone morphogenetic protein artificial bone has strong bone induction effects, but whether calcium phosphate cement/bone morphogenetic protein artificial bone can be used as growth stent of bone marrow stromal cells （BMSCs） is still unknown. OBJECTIVE： To study the probability of calcium phosphate cement/bone morphogenetic protein artificial bone that be used as a scaffold for bone tissue engineering. DESIGN, TIME AND SETTING： This observational study was performed at the Lab of Stomatology and Tissue Engineering, Shanghai Ninth People＇s Hospital from March, 2008 to March, 2009. MATERIALS： Calcium phosphate cement/bone morphogenetic protein artificial bone was supplied by Ruibang Company, with big aperture of 200-500 μm, small aperture of 1～5μ m, and porosity rate of 70%. A healthy beagle dog of two years old was used in this study. METHODS： After drawing out the bone marrow from the Beagle Dog, the BMSCs were separated by adherent methods, and induced with the conditional medium, in vitro cultured, amplified. Cell proliferation was observed following induction. The induced passage 2 cells were mixed to active calcium phosphate cement form a complex. Ultrastructure was observed. The complex was implanted into the subcutaneous parts of the beagle dog. Histological observation were taken after 2 and 4 weeks. MAIN OUTCOME MEASURES： The growth and proliferation of BMSCs were observed with inverted contrast phase microscope. The alkaline phosphatase staining, Von Kossa staining, Osteocalsin immunocytochemical staining were applied to identify the bone cells. The adherence of cells and active calcium phosphate cement was observed with scanning electron microscope. Hematoxylimeosin staining was applied to observe the bone. RESULTS： Alkaline phosphatase staining was positive. Von Kossa staining proofed the formation of mineralization nodules. Immunocytochemical staining showed the expression of osteocalcin. Scanning electron micrograph showed the cells adhered to the inner surface of the active calcium phosphate cement. Hematoxylin-eosin staining showed the new bone formation. CONCLUSION： Complex （active calcium phosphate cement/BMSCs incubated in vitro） showed new bone formation. The calcium phosphate cement/bone morphogenetic protein artificial bone can be used as a scaffold for bone tissue engineering.