组织工程骨表面微观形貌和生物矿化的实验研究

目的：用绿色荧光蛋白( GFP)标记结合扫描电镜和X射线能谱分析( SEM/EDS)技术对组织工程骨的表面微观形貌和生物矿化进行观测,以评价脱钙骨( DBM )支架体外构建组织工程骨的生物性能。方法用重组腺病毒介导GFP基因转染兔骨髓间充质干细胞( BMSCs)进行示踪标记,细胞与DBM复合经成骨诱导后,通过倒置荧光显微镜对细胞生长情况进行即时观察,结合SEM/EDS技术,观测组织工程骨的表面微观形貌和生物矿化。结果在倒置荧光显微镜下见细胞在DBM支架上能较好的黏附、重叠生长和增殖,体外培养至14 d 时,细胞内 GFP 有较高水平瞬时表达。SEM见DBM呈疏松多孔结构,孔隙直径为300~600μm,孔隙率达90%。 SEM下观察组织工程骨,见细胞在DBM网孔内表面贴壁生长,分泌基质旺盛,并可见粗糙的生物矿化物覆盖支架。 EDS显示其表面为钙、磷沉积物,其钙磷比( Ca/P)为1．46。结论 DBM体外构建组织工程骨有非常好的生物性能,GFP标记结合SEM/EDS技术可以作为DBM体外构建组织工程骨较好的评价手段。

Experimental study on surface microscopic morphology and biological mineralization of tissue engineering bone

Objective Green fluorescent protein( GFP) labeling, scanning electron microscope and energy disper-sive spectrometer ( SEM/EDS) were applied to observe surface microstructure and biological mineralization of tissue engineering bone for the purpose of evaluating the decalcified bone matrix ( DBM ) scaffold materials of biological properties in tissue engineering bone. Methods The rabbit bone-marrow mesenchymal stem cells ( BMSCs) were marked by Ad-GFP. Real-time growth of the cells was observed by an inverted fluorescence microscope after the os-teoinductive culture on to DBM, and surface microstructure and biological mineralization of tissue engineering bone were observed by SEM/EDS. Results The cells on surface of DBM had a good adhesion, overlap growth, as ob-served by an inverted fluorescence microscope, and a higher level of transient expression of GFP was confirmed af-ter 14 days in vitro culture. SEM image showed that DBM had a porous structure, with pore diameter ranging from 300 to 600μm, and a porosity rate was around 90%. The tissue engineering bone showed that cells grew adherently on the surface of DBM, matrix secretion was strong, and the DBM was covered by rough biological mineralization. X-ray photoelectron spectroscopy showed that the surface of rough biological mineralization consisted of Calcium, Phosphorus sediment,and its Calcium and Phosphorus ratio ( Ca/P) was 1. 46. Conclusion Tissue engineering bone constructed by DBM scaffold materials in vitro has excellent biological properties, and combined application of GFP labeling, SEM and X-ray photoelectron spectroscopy is a feasible method for evaluating DBM scaffold material in tissue engineering bone.