骨组织工程支架体外血管化初步研究

Angiogenesis of demineralized bone matrix scaffold in vitro

目的观察兔骨髓诱导的内皮细胞用纤维蛋白胶接种到骨组织工程支架上,在体外人工骨血管化效果。方法梯度离心分离兔单个核细胞,直接向内皮细胞方向诱导培养,传代培养至第3代细胞,倒置显微镜观察细胞形态及生长特点,第2代细胞作免疫组化鉴定和内皮细胞功能实验;以107/ml密度稀释于纤维蛋白凝胶中并接种到脱钙骨基质支架上,培养并定期观察细胞在支架上的生长情况,细胞支架复合物切片并作HE染色和电镜观察细胞在支架内的生长。结果诱导的内皮细胞为铺路石样,第2代细胞八因子相关抗原免疫组化为阳性,摄取低密度脂蛋白和结合凝集素实验均阳性;细胞在支架内呈立体生长,细胞在支架内3d后成梭形铺开,6d后自发形成管腔样结构并在支架表面形成内皮样结构。结论骨髓诱导的内皮细胞在脱钙骨支架内可以在体外培养成管腔样内皮样结构。

Objective To observe angiogenesis of induced endothelial cells（ECs） in demineralized bone matrix scaffold seed by fibrin sealant in vitro. Methods The marrow mononuclear cells（MNCs） in rabbit marrow were isolated by the gradient centrifugation and cultivated to isolate and induce ECs. The changes of ECs morphology, growth and proliferation, were observed during the passage cultivation. The immunohistochemical staining of vWF and function of ECs were checked. 10^7cells/ml were seeded into lml demineralized bone matrix scaffold by fibrin sealant, the angiogenesis of induced ECs were observed under microscope, HE and electon microscope. Results Induced ECs were monolayer arrayed to form cobblelike shape. The cells of 2nd passage showed positive staining of vWF, and positive labeling of DiI-LDL and UEA. The cells grew in three-dimensional in demineralized bone matrix scaffold,became fusiform on 3d and emerged tube-form and endothelial-like structure after 6d. Conclusion Induced ECs can emerge tube-form and endothelial-like structure in demineralized bone matrix scaffold in vitro.