多孔Ti-6Al-4V医用金属植入材料对骨髓间充质干细胞活性和成骨特性的作用  

Effect of the porous Ti-6Al-4V bio-metallic implants on cytoactive and osteogenic properties of bone marrow mesenchymal stem cells

作　　者：杨岚 胡梦蝶 杨成雪 张剑 龙远铸 喻正文 刘建国 Yang Lan;Hu Mengdie;Yang Chengxue;Zhang Jian;Long Yuanzhu;Yu Zhengwen;Liu Jianguo(Key Lab of Oral Disease Research,School of Stomatology,Zunyi Medical University,Zunyi Guizhou 563006,China)

机构地区：[1]遵义医科大学口腔医学院口腔疾病研究重点实验室,贵州遵义563006

出　　处：《遵义医科大学学报》2024年第6期569-576,586,共9页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:22265033);贵州省基础研究计划项目[NO:黔科合基础-ZK(2023)一般497,黔科合基础-ZK(2023)一般536];贵州省卫健委基金项目(NO:gzwkj2024-192)。

摘　　要：目的初步研究多孔(Ti-6Al-4V,TC4)金属支架的成骨分化作用和对SD大鼠骨髓间充质干细胞(rBMSCs)的细胞毒性作用。方法SD大鼠骨髓间充质干细胞(rBMSCs)原代细胞的培育,采用体外全骨髓贴壁的方法进行;rBMSCs表型鉴定采用流式细胞术;实验组TC4支架分别为圆形9、16、64孔,方形25、64孔,对照组为无孔,都采用选择性激光熔化(SLM)制备;在TC4上接种rBMSCs,用扫描电镜(SEM)观察48 h后的细胞形态和黏附状况,利用CCK-8对TC4的细胞毒性作用进行检测;用酶标法定量到第7、14天测碱性磷酸酶(ALP),用实时荧光定量PCR(qRT-PCR)检测骨连接蛋白(on)、Ⅰ型胶原蛋白(col1)这两者成骨基因的表达,于21 d时行茜素红染色。结果扫描电镜显示:小孔径TC4金属支架更利于rBMSCs细胞黏附;CCK-8结果显示:大孔径支架细胞增殖活性更高,圆形9孔为最高(第7天OD平均值0.69);ALP活性测定显示:ALP活性随着孔径减小而提高,圆形和方形64孔组ALP活性最高(第14天圆形64孔、方形64孔组ALP平均值分别为1.75金氏单位/gprot、1.61金氏单位/gprot);qRT-PCR检测显示:成骨基因表达水平随着孔径减小而提高,圆形和方形64孔组on和col1表达水平最高(第14天圆形64孔on和col1平均值分别为2.17、2.29;方形64孔on和col1平均值分别为1.70、1.81);茜素红染色显示:圆形和方形64孔支架组橘红色钙化结节相较于对照组颜色更深,面积更大。结论由SLM制备的多孔TC4支架对rBMSCs无细胞毒性,并且能促进其增殖,较大孔径对rBMSCs增殖有益,较小的孔径更利于rBMSCs黏附及成骨分化。Objective The porous(Ti-6Al-4V,TC4)titanium alloy scaffold was employed to investigate the cell viability,proliferation and osteogenic differentiation behavior of SD rat bone marrow mesenchymal stem cells(rBMSCs).Methods Primary rBMSCs were cultured using the whole bone marrow adherent method.Flow cytometry was employed to identify the phenotype of rBMSCs.TC4 titanium scaffolds were 9,16 and 64 round-like wells and 25 and 64 square-like wells,fabricated by selective laser melting technology(SLM).rBMSCs were seeded into TC4 wells as the experimental groups and TC4 titanium scaffolds without wells was set as the control group.Cell morphology and adherence of rBMSCs cultured for 48 h were observed by scanning electron microscopy(SEM).Cell viability was measured by CCK-8 kit.Alkaline phosphatase(ALP)activity in 7 d and 14 d was measured by enzyme-linked immunosorbent assay.The expression of osteogenic genes including bone connexin(Osteonectin)and collagen type Ⅰ(Col 1 a1)were examined by quantitative real-time fluorescence PCR(qRT-PCR).The rBMSCs osteogenic ability in 21 d was assessed using alizarin red staining.Results rBMSCs in TC4 scaffolds of 64 round-like wells and 64 square-like wells showed the excellent cell adhesion behavior.CCK-8 assay demonstrated that the scaffold with 9 round-like wells promoted rBMSCs proliferation.ALP activity assay showed that all experimental groups exhibited higher ALP activity compared with the control group,scaffolds with both 64 round-like wells and 64 square-like wells showed the highest ALP activity among experimental groups.qRT-PCR revealed that the expression levels of both Osteonectin and Col 1 a1 gradually increased with increasing wells,and 64 round-like and 64 square-like wells showed the highest expression levels of Osteonectin and Col 1 a1.rBMSCs osteogenic differentiation in TC4 scaffolds was significantly higher compared with the control group.Alizarin red staining illustrated that TC4 scaffolds with both 64 round-like wells and 64 square-like wells produced orange

关 键 词：TI-6AL-4V 骨髓间充质干细胞 细胞毒性 成骨诱导 

分 类 号：R318.08[医药卫生—生物医学工程]