WE43镁合金的血液相容性和对骨髓间充质干细胞增殖及成骨分化的影响

Hemocompatibility of WE43 magnesium alloy and its effect on cell proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

目的初步探讨WE43镁合金生物材料血液相容性和对SD大鼠骨髓间充质干细胞的细胞增殖及成骨分化的影响。方法溶血率实验、动态凝血时间实验和血小板黏附实验评价WE43镁合金的血液相容性。显微镜观察BMSCs细胞形态,流式细胞仪鉴定细胞。分别采用培养基、WE43镁合金浸提液、TC4钛合金浸提液培养细胞,CCK-8检测细胞增殖情况。采用成骨诱导液、WE43镁合金浸提液+成骨诱导液、TC4钛合金浸提液+成骨诱导液培养细胞,qRT-PCR检测成骨相关基因骨钙素(OCN)、Ⅰ型胶原(COL-Ⅰ)的mRNA表达;茜素红染色检测成骨矿化能力。结果WE43镁合金和TC4钛合金的溶血率均小于5%,符合医用材料溶血率要求,不会产生溶血作用。与TC4钛合金相比,WE43镁合金的溶血率和动态凝血时间并没有明显差异(P>0.05),但WE43镁合金表面黏附的血小板更少且变形程度轻,具有更好的抑制血小板聚集和变形的能力,血液相容性更优。镜下观BMSCs呈纤维状,FCM检测P3-P4代BMSCs膜表面抗原高表达CD29(99.3%)、CD90(99.87%),低表达CD45(1.93%),符合SD大鼠BMSCs的特点。细胞增殖实验中,与空白对照组相比WE43镁合金处理组和TC4钛合金处理组均促进了细胞增殖,且WE43镁合金组的细胞增殖率高于TC4钛合金组(P<0.05)。细胞成骨诱导后,WE43镁合金组OCN、COL-I的mRNA表达高于TC4钛合金(P<0.05),且WE43镁合金组的茜素红染色较TC4钛合金更为明显。结论WE43镁合金有着较好的血液相容性,可以促进BMSCs的细胞增殖和成骨分化。

Objective To investigate hemocompatibility of WE43 magnesium alloy and effect on cell proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods The experiment of hemocompatibility was divided into WE43 magnesium alloy group,TC4 titanium alloy group and blank control group.The hemocompatibility of WE43 was evaluated by hemolysis experiment,measurement of clotting time and observation of platelet adhesion using an electron microscope in vitro.BMSCs were cultured from whole bone marrow adherent method.Morphologies of primary and passageof BMSCs were observed by the optical microscope.Cell surface markers were identified by FCM.BMSCs were cultured by using WE43 and TC4 extracts respectively.CCK-8 assay was employed to detect the BMSCs proliferation.qRT-PCR and Alizarin red staining(ARS)were used to detect the osteogenic differentiation of BMSCs.Results The hemolysis rates of WE43 and TC4 were less than 5%,which satisfied the hemolysis requirements of medical materials.Compared with TC4 group,the rate of hemolysis of WE43 group was 3.7%where there was no significant difference in dynamic coagulation time(P>0.05).Although there was a few platelets adhering the surface of WE43 alloy,platelet aggregation and deformation were inhabited,and the alloy exhibited the excellent hemocompatibility.Fibrous BMSCs were observed by microscopy and FCM results showed that the expression of CD29 and CD90 were 99.3%and 99.87%respectively,while the expression of CD45 was 1.93%.The results were consistent with the characteristics of BMSCs.Compared with the control group,both WE43 and TC4alloys promoted the cell proliferation.The rate of cell proliferation in the WE43 group was higher than that of the TC4 group(P<0.05).OCN and COL-I mRNA expression of WE43 group were higher than that of TC4 group(P<0.05),and ARS result indicated that WE43 group was obvious.Conclusion WE43 magnesium biomaterial shows the excellent hemocompatibility and can promote cell proliferation and osteogenic differentiation of BMSCs.