氧化锆多孔表面显微形貌对成骨细胞增殖及分化的影响

Effect of porous zirconia ceramics on proliferation and differentiation of osteoblasts

目的:探索两种氧化锆多孔表面显微形貌对小鼠前成骨细胞增殖、分化的影响。方法:根据加工及造孔方式不同,将氧化锆试件分为4组,分别为切削终烧结组(milled sintering group,M-Ctrl)、切削多孔组(milled porous group,M-Porous)、3D打印终烧结组(3D printed sintering group,3D-Ctrl)和3D打印多孔组(3D printed porous group,3D-Porous)。通过扫描电镜(scanning electron microscope,SEM)、激光显微形貌测量显微镜进行表面显微形貌分析,用接触角测量仪测量静态接触角,通过能量色散X射线仪进行表面元素分析。将小鼠胚胎成骨细胞前体细胞MC3T3-E1接种于试件表面,SEM观察细胞培养第1、7天的黏附形态;使用细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)测量细胞培养第1、3、5天的增殖情况;采用实时荧光定量聚合酶链式反应检测分化诱导第14天时碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(typeⅠcollagen,Colla1)、Runt相关转录因子2(Runt-related transcription factor-2,Runx2)及骨钙素(osteocalcin,OCN)mRNA的相对表达量。结果:3D-Porous组孔径[(419.72±6.99)μm]及孔深度[(560.38±8.55)μm]均显著大于M-Porous组的孔径[(300.55±155.65)μm]及孔深度[(69.97±31.38)μm,(P<0.05)],且3D-Porous组圆孔形状更规则,分布更均匀。各组静态接触角均小于90°,其中3D-Ctrl组的静态接触角(73.83°±5.34°)与M-Porous组(72.7°±2.72°)最大,两组间差异无统计学意义(P>0.05)。M-Porous及3D-Porous组表面均可见细胞在孔内黏附,且培养第3、5天时两组试件表面细胞增殖活性均显著高于M-Ctrl组及3D-Ctrl组(P<0.05)。细胞分化诱导第14天时3D-Porous组ALP、Colla1、Runx2、OCN mRNA的相对表达量均显著低于M-Ctrl组及3D-Ctrl组(P<0.05)。M-Porous组Colla1、Runx2、OCN mRNA的相对表达量均显著高于3D-Porous组(P<0.05)。结论:氧化锆表面多孔形貌能促进小鼠前成骨细胞MC3T3-E1的增殖及黏附,但对其成骨向分化有一定抑制作用。

Objective:To investigate the effect of porous surface morphology of zirconia on the proliferation and differentiation of osteoblasts.Methods:According to different manufacturing and pore-forming methods,the zirconia specimens were divided into 4 groups,including milled sintering group(M-Ctrl),milled porous group(M-Porous),3D printed sintering group(3D-Ctrl)and 3D printed porous group(3D-Porous).The surface micromorphology,surface roughness,contact angle and surface elements of specimens in each group were detected by scanning electron microscope(SEM),3D laser microscope,contact angle measuring device and energy-dispersion X-ray analysis,respectively.MC3T3-E1 cells were cultured on 4 groups of zirconia discs.The cell morphology of MC3T3-E1 cells on zirconia discs was eva-luated on 1 and 7 days by SEM.The cell proliferation was detected on 1,3 and 5 days by cell counting kit-8(CCK-8).After osteogenic induction for 14 days,the relative mRNA expression of alkaline phosphatase(ALP),typeⅠcollagen(Colla1),Runt-related transcription factor-2(Runx2)and osteocalcin(OCN)in MC3T3-E1 cells were detected by real-time quantitative polymerase chain reaction.Results:The pore size[(419.72±6.99)μm]and pore depth[(560.38±8.55)μm]of 3D-Porous group were significantly larger than the pore size[(300.55±155.65)μm]and pore depth[(69.97±31.38)μm]of M-Porous group(P<0.05).The surface of 3D-Porous group appeared with more regular round pores than that of M-Porous group.The contact angles of all the groups were less than 90°.The contact angles of 3D-Ctrl(73.83°±5.34°)and M-Porous group(72.7°±2.72°)were the largest,with no significant difference between them(P>0.05).Cells adhered inside the pores in M-Porous and 3D-Porous groups,and the proliferation activities of them were significantly higher than those of M-Ctrl and 3D-Ctrl groups after 3 and 5 days’culture(P<0.05).After 14 days’incubation,ALP,Colla1,Runx2 and OCN mRNA expression in 3D-Porous groups were significantly lower than those of M-Ctrl and 3D-Ctrl groups(P<0.05).Colla1,Runx2 and OCN mRNA expressions in M-Porous group were higher than those of 3D-Porous group(P<0.05).Conclusion:The porous surface morphology of zirconia can promote the proliferation and adhesion but inhibit the differentiation of MC3T3-E1 cells.