兔胫骨骨膜细胞膜片的体外构建

Construction of Rabbit Tibial Periosteal Cell Sheets in Vitro

目的体外采用维生素C构建兔胫骨骨膜细胞膜片并检测其基本生物学特性。方法培养兔胫骨骨膜细胞,采用维生素C诱导构建兔胫骨骨膜细胞膜片。采用显微镜对膜片进行观察,苏木精-伊红(H-E)及Masson染色检测膜片的结构及胞外基质;细胞活死荧光染色观察膜片的活力。结果体外成功分离培养兔胫骨骨膜细胞,CCK-8检测结果显示骨膜细胞增殖曲线呈S型增长,结晶紫染色显示其具有克隆形成能力,并具有多向分化能力。采用维生素C连续诱导14 d,可获得半透明乳白色膜样结构。H-E及Masson染色显示膜片由多层细胞及胞外基质构成,细胞活死荧光染色显示膜片由大量绿染的活细胞构成。结论维生素C可成功诱导兔胫骨骨膜细胞膜片,有望应用于骨缺损再生修复。

Objective To explore the effects of Vitamin C treatment of constructing rabbit tibial periosteal cell sheets in vitro and characterize their basic biological characteristics. Methods The rabbit tibial periosteal cells were isolated and cultured in DMEM supplemented with 10% fetal bovine serum. The rabbit tibial periosteal cell sheet formation was observed under an inverted microscope. In order to assess the morphology of cells sheets and extracellular matrix, hematoxylin and eosin(H-E) and Masson staining were employed. The viability of the cell sheet was evaluated by the live and dead cell assay. Results Rabbit tibial periosteal cells could be successfully isolated and cultured in vitro. The result of CCK8 showed that the growth curve of cells was S-type. Crystal violet staining indicated that periosteal cells culturd in vitro had clony formation ability. And it possessed multifunctional differentiation potential. Furthermore, isolated cells could construct the rabbit tibia PCs sheet when incorporated with Vitamin C for 14 days. The constructed rabbit tibial periosteal cell sheets were translucent and composed of film-like structure. H-E and Masson staining showed that the cell sheets were predominant of rabbit tibial periosteal cells and abundant with ECM. When observed with live and dead fluorescence staining, cell sheets were presented with a higher percentage of live cells. Conclusion The rabbit tibia periosteal cell sheet can be successfully constructed by utilizing Vitamin C, which could be a novel path for bone defect repair.