可注射壳聚糖水凝胶复合犬骨髓基质细胞在裸鼠体内的组织学观察

The hostological observation of injectable chitosan hydrogel combined with dog bone marrow stromal cells in nude mice

目的:研究壳聚糖水凝胶(Chitosan,CS)-犬骨髓基质细胞(bone marrow stromal cells,BMSCs)复合物体内异位成骨能力,探讨其作为可注射组织工程骨的可能性。方法:利用密度梯度离心法获得犬BMSCs,含10%胎牛血清的L-DMEM培养液进行原代培养并进行成骨诱导培养。合成壳聚糖温敏水凝胶,以5×106/mL的终细胞密度悬浮BMSCs,将注射于裸鼠背部皮下,8周后取材,行大体观察、组织学检查、免疫组化染色观测骨组织的形成情况。结果:BMSCs/CS复合物于裸鼠皮下形成质地较硬的结节,组织学及免疫组化结果证实有骨样结构形成,单纯CS凝胶无骨样结构形成。结论:BMSCs与CS水凝胶可以分别作为种子细胞和支架载体构建可注射的骨组织。

Objective：To study the the ectopic osteogenesis ability of injectable chitosan（CS）hydrogel combined with bone marrow stromal cells（BMSCs）in vivo and the probability that it may be used as tissue engineered bone.Method：Dog bone marrow stromal cells（BMSCs） were obtained by density gradient centrifugation method,cultured in DMEM medium with 10 % fetal bovine serum（FBS）.And then the BMSCs were introduced by the osteogenetic medium.Chitosan hydrogel was prepared and the BMSCs/CS composite was injected into the dorsal subcutaneous tissue of nude mice with final cellular density of 5×106/mL.The specimens were harvested at 8 weeks,and the ability of osteogenesis in vivo was investigated by the gross observation,HE,immunohistochemistry.Result：Eight weeks after injection hard nodulus formed subcutaneously.Histologica1 examination revealed that there was bone-like tissue in the experimental group.And osteopontin（OPN）and Type I Collagen（Col I）expressed positively.But there was no bone-like tissue formed in the control group.Conclusion：This study demonstrates that the chitosan and the bone marrow stromal cells can be successfully used in a constructing technique for the injectable tissue engineered bone.