摘要
背景:成骨细胞缺乏是骨组织工程面临的关键问题,而间充质干细胞联合成骨细胞移植能够获得理想效果。目的:观察脂肪间充质干细胞与成骨细胞复合羟基磷灰石/壳聚糖/聚乳酸支架材料在体内的异位成骨效应。方法:酶消化法和贴壁法分离原代脂肪间充质干细胞、成骨细胞,并进行鉴定。取成骨细胞、脂肪间充质干细胞、成骨细胞+脂肪间充质干细胞混合细胞(比例为1︰1),分别复合羟基磷灰石/壳聚糖/聚乳酸骨修复材料,体外复合培养48 h后,植入SD大鼠背部皮下,作为实验组,以单纯材料植入作为对照组。术后8周取出标本,进行大体观察、组织学观察并计算新骨生成率。结果与结论:(1)脂肪间充质干细胞经成脂、成软骨、成骨诱导后,油红O染色、甲苯胺蓝染色和茜素红染色均为阳性;流式细胞仪检测脂肪间充质干细胞中CD147、CD90、CD105、CD44呈阳性表达(>80%),而CD117、CD34、CD31、CD45则呈阴性表达(<5%);(2)第3代成骨细胞茜素红染色、碱性磷酸酶染色均为阳性;(3)植入8周后大体观察可见材料中有软组织长入,难以分离;(4)植入8周后组织学观察可见各组均有新骨形成,与其他材料组比较,脂肪间充质干细胞+成骨细胞+羟基磷灰石/壳聚糖/聚乳酸组成骨分数最高,差异有显著性意义(P<0.05);(5)结果表明,脂肪间充质干细胞可促进成骨细胞复合羟基磷灰石/壳聚糖/聚乳酸支架材料的异位成骨。
BACKGROUND: Osteoblast deficiency is a key problem in bone tissue engineering, and transplantation of mesenchymal stem cells combined with osteoblast can achieve ideal results. OBJECTIVE: To investigate the in vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells(ADSCs) and osteoblasts(OB) combined with hydroxyapatite(HA)/chitosan(CS)/poly(L-latic acid)(PLLA). METHODS: ADSCs and OB were obtained by adherent method and enzymatic digestion method. ADSCs, OB and the mixture of ADSCs and OB(at a mixture ratio of 1:1) were cultured with HA/CS/PLLA, respectively. After 48 hours of in vitro culture, cell-scaffold complexes were subcutaneously implanted into the back of Sprague-Dawley rats in corresponding groups, and HA/CS/PLLA without cells was implanted as control group. The rats in each group were killed at 8 weeks postoperatively. The macroscopic and histopathological observations were performed to assess the ectopic osteogenesis potential. RESULTS AND CONCLUSION:(1) After adipogenic, chondrogenic and osteogenic induction, ADSCs were positive for oil red O, toluidine blue and alizarin red staining. Results from flow cytometry showed that ADSCs were positive for CD147, CD90, CD105 and CD44 with the rate of positivity > 80%, but negative for CD117, CD34, CD131, CD45 with the rate of positivity < 5%.(2) Passage 3 OB were positive for both alizarin red staining and alkaline phosphatase staining.(3) At 8 weeks after implantation, soft tissues grew into the complexes under gross observation.(4) At 8 weeks after implantation, ectopic bone formation was visible in each group. The bone formation was more visible in the ADSCs-OB/HA/CS/PLLA group than the other groups with significant differences(P < 0.05). To conclude, ADSCs can promote the ectopic bone formation of OB in vivo in combination with HA/CS/PLLA scaffold.
引文
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