3D打印钛合金多孔材料对体外成骨细胞系MC3T3-E1的生物安全性

Biosafety of 3D printed titanium alloy porous materials to osteoblast cell line MC3T3-E1

目的利用电子束熔融成型技术(EBM)3D打印钛合金多孔支架材料,检测其对成骨细胞(OB)的生物安全性。方法打印3种拓扑单元结构的多孔钛合金材料(2.0、2.25及2.5 mm的Dode-Medium单元结构),制备各组材料在多个时间点的浸提液(浸泡时间设24、48、72、96 h),并将浸提液用于成骨细胞系MC3T3-E1的体外培养。通过MTT实验,计算成骨细胞的相对增殖率(RGR);通过苏木精-伊红(HE)染色,形态学观察成骨细胞的生长状态;通过碱性磷酸酶(ALP)测定,分析成骨细胞的分化活性;通过免疫荧光染色,观察成骨细胞中I型胶原(ColⅠ)的表达水平。结果在各组材料浸提液孵育下,成骨细胞贴壁牢固、增殖活跃,生长状态良好。与DMEM孵育的阴性对照组比较,各材料浸提液组成骨细胞的相对增殖率无显著性差异;与DMSO孵育的阳性对照组比较,各材料浸提液组成骨细胞的相对增殖率显著升高(P<0.01);各组材料浸提液的细胞毒性程度评估均≤1。与DMEM孵育的阴性对照组比较,各材料浸提液组成骨细胞的ALP活性与ColⅠ含量均无显著性差异;与DMSO孵育的阳性对照组比较,各材料浸提液组成骨细胞的ALP活性与ColⅠ含量均显著升高(P<0.01)。结论以EBM技术3D打印钛合金多孔材料,其浸提液不会影响成骨细胞的体外增殖与分化,即具有良好的生物安全性。

Objective To prepare 3D-printed porous materials of titanium alloy by electron beaming melting(EBM)and to confirm its biosafety to osteoblasts(OB).Methods Three topological structures of titanium alloy porous materials(2.0 mm,2.25 mm and 2.5 mm Dode-Medium structure)were prepared by 3D printing technology.The extracts of titanium alloy porous materials were collected at multiple time points(24,48,72,96 h)and used for in vitro culture of osteoblasts cell line MC3T3-E1.The relative growth rate(RGR)of osteoblasts was cal-culated by MTT test.The morphological observation of osteoblasts was performed by HE staining.The activity of ALP was determined for analyzing the differentiate capacity of osteoblasts.The expressional level of ColⅠin osteoblasts was assessed by immunofluorescence staining.Results Following incubation with extracts of titanium alloy porous materials,the osteoblasts were found to adhere and proliferate actively.There was no significant difference in the RGR of osteoblasts between extract group and the negative control group of DMEM.However,the RGR of osteoblasts increased significantly in extract groups compared with that in the positive control group of DMSO(P<0.01).The cytotoxic degree of osteoblasts in extract groups was all less than or equal to 1.There was no significant difference in the level of ALP and ColⅠof osteoblasts between extract groups and the negative control group of DMEM.However,the level of ALP and ColⅠof osteoblasts enhanced significantly in extract groups compared with that in the positive control group of DMSO(P<0.01).Conclusions The extracts of titanium alloy materials do not affect the proliferation and differentiation of osteoblasts in vitro that confirmed its biosafety.