成骨诱导的犬骨髓间充质干细胞复合Bio-Oss构建组织工程化骨的体外研究

Construction of tissue engineered bone by osteoblasts from canine bone marrow mesenchymal stem cells and Bio-Oss:An in vitro study

目的以骨髓间充质干细胞(MSCs)为种子细胞,以Bio-Oss小牛无机骨颗粒为支架材料,构建MSCs-Bio-Oss组织工程化骨。探讨犬MSCs成骨活性及与Bio-Oss复合构建组织工程化骨的可行性。方法杂种犬MSCs体外分离、培养及成骨诱导分化;取成骨诱导后的MSCs,以106个细胞/ml种植于Bio-Oss无机骨颗粒,轻度负压静置孵育培养。应用倒置相差显微镜、扫描电镜观察其形态学变化,进行细胞表面因子CD44的免疫荧光标记、钙结节染色、ALP定性和定量检测,结果以SPSS12.0统计软件包进行统计学分析。结果MSCs形态较均一,排列紧密,呈纺锤形,CD44表面抗原阳性;诱导分化后,表现出明显的成骨细胞活性,钙结节的茜素红S染色阳性,ALP的Gomori法染色阳性,ALP活性定量检测实验组在诱导分化3、7、14d时,ALP含量分别为(3.307±0.217)U/g、(5.929±0.781)U/g和(9.739±0.547)U/g,与对照组的(0.442±0.087)U/g、(0.581±0.027)U/g和(0.768±0.126)U/g比较有显著差异(P<0.01);MSCs在Bio-Oss表面贴附紧密,生长良好,构建形成组织工程化骨。结论以成骨诱导的犬MSCs作为种子细胞,复合支架材料Bio-Oss构建组织工程化骨是可行的。

PURPOSE： Using the MSCs-Bio-Oss tissue engineered bone which was constructed by MSCs as seed cells and the Bio-Oss calf inorganic bone grains as scaffold materials to determine the canine bone formation activity and the feasibility of Bio-Oss combined MSCs to construct tissue engineered bone. METHODS： Gybrid canine MSCs were dissociated, cultivated, bone formation induced and differentiated into osteoblast in vitro; The bone formation induced MSCs were allowed to grow onto Bio-Oss calf inorganic bone grains at 10^6cell/ml, and then incubated and cultivated, under light pressure without other treatments. Inverted phase contrast microscope and scanning electron microscope were used to observe their morphological changes, immunofluorescent labeling of cell surface factor CD44,calcium nodus staining,qualitative and quantitative detection of ALP were carried out, and SPSS 12.0 software package was used for statistical analysis. RESULTS： The MSCs were uniform with compact alignment and shape of prosenchymatous cells, CD44＇s surface antigen was positive; Osteoblast activity was present after induction and differentiation, alizarin Bordeaux S stain of calcium nodus was positive, ALP＇s Gomori staining was also positive. ALP content in the experimental group were （3.307±0.217）U/g,（5.929±0.781）U/g and （9.739±0.547）U/g respectively at 3^rd day,7^th day, 14^th day after induction and differentiation, which were significantly different （P〈0.01） from the control group： （0.442±0.087）U/g, （0.581±0.027）U/g and （0.768±0.126）U/g; the MSCs stuck compactly on the surface of Bio-Oss, grew well, and formed tissue engineered bone. CONCLUSION： Using canine MSCs which were induced by bone formation as seed cells combined with Bio-Oss as scaffold materials to construct tissue engineered bone is feasible.Supported by Liaoning Provincial Natural Science Foundation（Grant No.20042062）.