【摘要】 目的观察大鼠骨髓干细胞(BMSCs)诱导分化的软骨细胞,在具有三维孔隙结构的海绵状猪小肠粘膜下层(SIS)表面的生长情况,探讨SIS由薄膜状重塑为海绵状后对软骨样细胞增殖分化的作用,为软骨组织工程提供新型的天然生物衍生材料。方法原代培养SD大鼠BMSCs,流式术检测细胞表面抗原表达;诱导BMSCs分化为软骨细胞,甲苯胺蓝染色鉴定。按Abraham法将猪近段空肠制备成脱细胞的SIS,再将薄膜状的SIS经液氮低温研磨制成微粒,交联后采用冷冻干燥技术重塑形为具有三维孔隙结构的海绵状SIS。将软骨细胞与海绵状SIS共培养:扫描电镜观察细胞生长状态;Western blotting检测共培养组织ColⅡ的表达;DMMB法测量葡萄糖胺聚糖(GAG)表达量。应用材料浸提液培养软骨细胞,相差显微镜观察细胞形态,MTT法检测细胞的增殖。结果原代培养的BMSCs表达干细胞相关抗原,并可分化为软骨细胞,甲苯胺蓝将糖胺多糖染成蓝色。脱细胞的SIS未见有核物质,海绵状的SIS具有大量较均匀的三维孔隙。软骨细胞能在材料孔隙内良好生长,软骨分化指标ColⅡ与GAG表达量明显增加;浸提液培养的软骨细胞增殖能力强。结论重塑形后具有三维孔隙结构的海绵状SIS促进BMSCs来源的软骨细胞增殖和分化,为软骨组织工程提供了新型的天然生物衍生材料。更多还原

【Abstract】 Objective To study the effect of remodeling sponge-like SIS on the proliferation and differentiation of chondrocytes originated from BMSCs. Methods The BMSCs were cultured and identified by flow cytometry, and induced and differentiated into chondrocytes identified by toluidine blue staining. The acellular and monoptychial film-like SIS was prepared with porcine proximal jejunum by Abraham method. Diameter of the homogeneous particles was made with filmlike SIS by milling-in liquid nitrogen cryogenic grinding, and the particles were reshaped to sponge-like SIS with much three-dimensional pores after cross-linked by freeze-drying technology. The chondrocytes were cultured in cavernous SIS, and the cell morphous was observed by scanning electron microscope. The expression of ColⅡwas detected by Western blotting, the content of glycosaminoglycan(GAG) was measured by DMMB method. The proliferation of chondrocytes in material extracts of SIS was evaluated with MTT. Results The primary cultured BMSCs presented proliferation strongly and expressed stem cell-associated antigen positively. The chondrocytes originated from BMSCs were stained by toluidine blue positively, grew well and could secret much more ColⅡ and GAG in cavernous SIS with much regular three-dimensional pores but no cell nucleus. The material extracts of SIS promoted the proliferation of chondrocytes. Conclusion The sponge-like SIS promoted the proliferation and differentiation of chondrocytes originated from BMSCs, might be a novel natural biological material for cartilage tissue engineering.更多还原