同种异体脂肪干细胞复合脱钙骨支架材料修复兔胫骨缺损的实验研究

目的探讨同种异体脂肪干细胞复合脱钙骨支架材料修复兔胫骨缺损的实验研究。方法购自北京生物有限公司(动物实验中心)新西兰大白兔8对,其中2对白兔进行脂肪干细胞分离培养,并采用反复滴加方法将细胞均匀分布于支架材料上,获取脂肪干细胞复合脱钙骨材料。其中6对构建兔胫骨骨缺损模型,造模后即刻将单纯脱钙骨材料植入右侧缺损区设为对照组,将诱导后的兔脂肪干细胞-脱钙骨支架复合物植入左侧缺损区设为实验组。于术后12周采用空气栓塞处死大白兔,获取大白兔双侧后肢胫骨修复骨及其周围软组织,行Micro-CT和组织学检测,了解实验组和对照组白兔骨组织生长情况,兔脂肪干细胞-脱钙骨材料成骨表型测定体内降解情况和组织学检测结果。结果大体观察显示实验组白兔后肢骨缺损区可见骨样组织修复,对照组白兔为纤维样组织充填未见骨样组织覆盖;三维CT影像学显示实验组白兔骨缺损断端和材料接合部已充分融合且骨轮廓、光滑的骨塑形清晰可见,对照组白兔骨缺损处多为纤维性连接;HE染色下可见实验组白兔有典型再生骨组织、大量骨陷窝、骨细胞及部分骨小梁结构,对照组白兔为胶原纤维样组织和残留脱钙骨基质、无大片再生骨样组织。两组白兔Ⅱ型胶原免疫组织化学染色均呈阳性,细胞胞浆及胞外基质呈棕黄色,胞核呈蓝色,证实修复细胞表达Ⅱ型胶原,为软骨细胞。实验组和对照组白兔修复组织评分分别为(2.41±0.40)、(2.70±0.45)分,实验组白兔组织修复得分高于对照组白兔,差异有显著性(P<0.05)。结论兔脂肪干细胞接种于脱钙骨材料,经成骨诱导后具有较强成骨分化能力,能较好地修复兔胫骨骨缺损,具有较高临床疗效。

Experimental study of rabbit tibia defect with allogeneic adipose stem cell composite decalcified bone scaffold

Objective To investigate the experimental study of rabbit tibia defects with allogeneic adipose stem cells composite decalcified bone scaffold.Methods Eight pairs of New Zealand white rabbits were purchased from Beijing Biotechnology Co.,Ltd.(animal experiment center),where isolation and culture of adipose stem cells were performed in two pairs of white rabbits,and the cells were evenly distributed on the scaffold by repeated dropping method to obtain the composite decalcified bone materials of adipose stem cells;the remaining 6 pairs were used to establish rabbit tibia bone defect models.After that,simple decalcified bone material was immediately implanted into the right defect area as the control group,and the induced rabbit adipose stem cell-decalcified bone scaffold complex was implanted into the left defect area as the experimental group.At 12 weeks after operation,the rabbits were sacrificed by air embolization,and the bilateral posterior tibia bones of the white rabbits were used to repair the bone and surrounding soft tissues.Micro-CT and histological examination were performed to understand the bone growth of rabbits in the experimental group and the control group.Rabbit fat Stem cell-decalcified bone material osteogenic phenotype was used to determine in vivo degradation and histological test results.Results Gross observation showed that the bone-like tissue repair in the posterior bone defect area of the rabbits was observed in the experimental group,and the rabbits in the control group were the fibrous tissue filling with no bone-like tissue coverage.Three-dimensional CT imaging showed that the bone defect broken end and material joints of white rabbits in the experimental group were fully fused with clearly visible bone contour and smooth bone shape,while the bone defect of the white rabbit in the control group was mostly fibrous.Under HE staining,the rabbits in the experimental group had typical regenerated bone tissue,a large number of bone lacuna,bone cells,and some trabecular bone structure,whereas the rabbits in the control group had collagen fibrous tissue and residual decalcified bone matrix,as well as no large regenerated bone-like tissue.Immunohistochemical staining of type II collagen in both groups of rabbits was positive.The cytoplasm and extracellular matrix of the two groups were brownish yellow,and the nucleus was blue,which confirmed that the repair cells expressed type II collagen and were chondrocytes.The repaired tissue scores of the rabbits in the experimental group and the control group were(2.41±0.40)and(2.70±0.45),respectively;the tissue repair scores in the experimental group were higher than those in the control group(P<0.05).Conclusion Rabbit adipose stem cells are inoculated from decalcified bone material and have strong osteogenic differentiation ability after osteogenic induction.The repair of rabbit tibia bone defects has high clinical efficacy.