兔间充质干细胞在Vanco-AB-FG三维支架中的增殖和分化

Experimental study on proliferation and osteogenic differentiation of rabbit BMSCs in 3D scaffold of vancomycin alginate beads embedded in fibrin gel

目的观察兔间充质干细胞(mesenchymal stem cells derived from bone marrow,BMSCs)在带有万古霉素藻酸盐微球的纤维蛋白凝胶(vancomycin alginate beads embedded in fibrin gel,Vanco-AB-FG)中增殖和成骨分化情况。方法选取健康成年家兔,通过梯度离心结合贴壁培养法分离、纯化BMSCs。实验组:将兔BMSCs溶解于纤维蛋白原溶液中,以注射的方式均匀接种于Vanco-AB-FG内部构建组织工程骨;对照组:将BMSCs直接接种于Vanco-AB-FG复合物表面。测定生长曲线比较两组细胞增殖能力;通过碱性磷酸酶和茜素红染色观察两组BMSCs成骨分化的情况。结果生长曲线结果显示,早期(前7 d)处于三维生长模式的实验组细胞的增殖速度慢于处于二维生长环境的对照组,在5 d和7 d时二者差异有统计学意义(P<0.05);然而,后期(9 d后)二者之间差异无统计学意义(P>0.05)。碱性磷酸酶检测显示实验组细胞的碱性磷酸酶活性在12 d达到峰值,为5.98 pg/(min.mg.cell),明显高于对照组(P<0.05)。茜素红染色显示实验组Vanco-AB-FG复合物的内部和表面均有明显的钙化结节。结论 BMSCs可以在Vanco-AB-FG复合物内增殖和成骨分化。

Objective To observe the proliferation and osteogenic differentiation of mesenchymal stem cells derived from rabbit bone marrow（BMSCs） in the 3D scaffold of vancomycin alginate beads embedded in fibrin gel（Vanco-AB-FG）.Methods BMSCs were derived from healthy adult rabbits through gradient centrifugation and adherent culture.In the experimental group,BMSCs were suspended in a fibrinogen solution,and then injected into Vanco-AB-FG to construct tissue engineered bones.In the control group,BMSCs were seeded on the surface of Vanco-AB-FG.The growth curves of BMSCs in the experimental and control groups were detected in different phases to compare their cell proliferation abilities.Alkaline phosphatase（ALP） activity test and alizarin red staining were utilized to evaluate the osteogenic differentiation of BMSCs in the two groups.Results The growth curves showed that the cell proliferation rate in the experimental group was lower than that in the control group in the earlier phase（1-7 d）.There was a significant difference between them on 5 and 7 d（P0.05）.In the later phase（after 9 d）,there was no significant difference between the two groups.The activity of ALP in the experimental group was significantly higher than that in the control group,especially during 6-15 d（P0.05）.The maximum activity of ALP in the experimental group was 5.98 pg/（min·mg·cell） on 12 d.In the experimental group,calcified nodules stained by alizarin red were observed in the interior and on the surface of the 3D scaffold of Vanco-AB-FG.Conclusion BMSCs can proliferate and undergo osteogenic differentiation in the 3D scaffold of Vanco-AB-FG.