不同孔隙率钽涂层钛合金材料对细胞成骨的影响

Effect of Tantalum-Coated Titanium Alloy with Different Porosities on Cell Osteogenesis

摘要: 目的：研究不同孔隙率钽涂层钛合金材料对SD大鼠原代骨髓间充质干细胞(rBMSC)黏附、增殖及成骨分化的影响并进行对比。方法：3D打印60%、70%和80%三种不同孔隙率的钛合金支架5 × 2 mm，并完成钽涂层的覆盖。在体外提取rBMSC进行原代培养至第三代，各组分别进行细胞黏附、CCK8技术、碱性磷酸酶(ALP)定量检测、茜素红S染色、活死细胞染色及实时定量聚合酶链反应(qRT-PCR)检测成骨基因实验包括Runx-2、Osterix和ALP基因，并以纯钽(孔隙率为0)作为阴性对照，各组间进行对比。结果：细胞黏附实验中60%、70%和80%三种不同孔隙率的钛合金支架黏附性能优于纯钽；CCK8法检测细胞活性在第1、3天各组间无明显差异，第5天80%孔隙率钽涂层钛合金支架的增殖活性最高；第7天检测细胞ALP活性可见80%孔隙率钽涂层钛合金支架的明显高于其他三组；细胞培养第14、21天茜素红S染色80%孔隙率钽涂层钛合金支架均明显高于其他三组；细胞培养第1、3、5天活死细胞染色80%孔隙率钽涂层钛合金支架活细胞数明显增多，死细胞数减少，而其他三组活细胞、死细胞数均增多；qRT-PCR检测Runx-2、Osterix和ALP基因均在80%孔隙率钽涂层钛合金支架组表达最高。结论：相对于纯钽、60%和70%孔隙率钽涂层钛合金支架，80%孔隙率钽涂层钛合金支架能更好地促进rBMSC增殖、黏附和成骨基因诱导。

Abstract: Objective: To study and compare the effects of different porosities tantalum-coated titanium alloy materials on the adhesion, proliferation and osteogenesis differentiation of SD rats’ primary bone marrow mesenchymal stem cells (rBMSCs) in vitro. Methods: 3D printing 5 × 2 mm pure tantalum scaffolds and 60%, 70% and 80% porosities titanium alloy scaffolds coating tantalum coverage by chemical vapor deposition technology (CVD). In vitro, rBMSCs were extracted for primary culture to the third generation, and each group was subjected to cell adhesion, CCK8 technology, alkaline phosphatase (ALP) quantitative detection, alizarin red S staining, live and dead cell staining, and real-time quantitative polymerase chain reaction (qRT-PCR) detection of osteogenetic genes including Runx-2, Osterix and ALP genes. Pure tantalum (the porosity was zero) was regarded as a negative control, and comparisons were made between groups. Results: In cell adhesion experiments, the adhesion performance of tantalum-coated titanium alloy scaffolds with different porosities of 60%, 70% and 80% was better than that of pure tantalum. The CCK8 method detected no significant differences between the groups on days 1 and 3, and the proliferative activity of 80% porosity tantalum-coated titanium alloy scaffolds on day 5 was the highest, and the detection of cell ALP activity on day 7 showed that the 80% porosity tantalum-coated titanium alloy scaffolds were significantly higher than those of the other three groups. On days 14 and 21 of cell culture, alizarin red S stained revealed that 80% porosity tantalum-coated titanium alloy scaffolds were significantly higher than those of the other three groups. On the 1st, 3rd and 5th days of cell culture, the number of live cells stained with 80% porosity tantalum-coated titanium alloy scaffold increased significantly, and the number of dead cells decreased, while the other three groups of living cells and dead cells both increased. qRT-PCR showed that Runx-2, Osterix and ALP gene expression were all highest in 80% porosity tantalum-coated titanium alloy scaffolds. Conclusion: Compared with pure tantalum, 60% and 70% porosities tantalum-coated titanium alloy scaffolds, 80% porosity tantalum-coated titanium alloy scaffolds can better promote rBMSC proliferation, adhesion and osteogenetic gene induction.