钛及氧化锆表面微沟槽结构对人牙龈成纤维细胞生物学行为的影响

Effects of different implant materials and surface microgrooves on the biological behavior of gingival fibroblasts

目的探讨钛及氧化锆表面微沟槽结构对人牙龈成纤维细胞(human gingival fibroblast,HGF)生物学行为的影响,以期为种植体穿龈部分寻找合适的材料及表面处理方法。方法利用精细加工切片系统在钛和氧化锆试件表面制备微沟槽,沟槽顶宽60μm,沟槽宽60μm,沟槽顶到沟槽底深度10μm。实验分为光滑钛组、光滑氧化锆组、微沟槽钛组和微沟槽氧化锆组(每组5个试件),用扫描电镜、原子力显微镜、接触角测量仪、能量色散X射线分析观察各组试件表面形貌(沟槽宽度及深度)并测量表面粗糙度、静态接触角以及进行元素分析。通过免疫荧光染色和激光扫描共聚焦显微镜观察HGF的黏附形态,利用细胞活性试剂盒检测培养6 h,1、3、5、7 d的细胞增殖情况,通过实时荧光定量PCR检测细胞黏附早期(6 h)和晚期(3 d)整合素α5、β1和Ⅰ型胶原mRNA表达水平。结果光滑钛组和光滑氧化锆组表面粗糙度分别为(63.23±2.55)和(26.78±3.11)nm。微沟槽氧化锆组的静态接触角最小,为51.2°±2.0°。两个沟槽组表面HGF均顺着沟槽排列,培养6 h至7 d的各时间点微沟槽氧化锆组HGF增殖均显著多于其他组(P<0.05)。黏附早期微沟槽氧化锆组整合素α5 mRNA表达量最大(P<0.05),各组整合素β1及Ⅰ型胶原mRNA表达量差异均无统计学意义(P>0.05);黏附晚期两个沟槽组整合素α5、β1和Ⅰ型胶原mRNA表达量均显著大于两个光滑组(P<0.05)。结论氧化锆表面粗糙度较小,其中微沟槽氧化锆亲水性较好,可引导HGF顺沟槽排列,有利于HGF黏附、增殖及结构蛋白和功能蛋白的表达。

Objective To study the effect of microgroove surface modification of titanium and zirconia on the biological behavior of gingival fibroblasts in order to find suitable surface materials for the transmucosal part of the dental implant.Methods Twenty specimens were divided into four groups:smooth titanium(Ti-S),smooth zirconia(ZC-S),microgroove titanium(Ti-MG)and microgroove zirconia(ZC-MG)(five specimens in each group).Microgroove modification of titanium and zirconia surfaces was carried out by using fine machining chip system in the last two groups.The width of groove ridge was 60μm,the width of groove was 60μm,the depth of groove was 10μm.The surface morphologies(the groove width and depth)were observed by scanning electron microscope(SEM),the surface roughness,static contact angle and elemental of specimens in each group were detected by SEM,atomic force microscope(AFM),optical contact angle measuring device and energy-dispersion X-ray analysis(EDX).Morphology of human gingival fibroblast(HGF)that arranged along the groove was analyzed using laser scanning confocal microscope by immunofluorescence staining.Differences in cell proliferation were analyzed and compared using cell counting kit.Expression level of intergrinα5,β1 and collagenⅠmRNA were compared among different groups by quantitative real-time PCR for 6 h and 3 d.Results The surface roughness of smooth titanium group and smooth zirconia group was(63.23±2.55)and(26.78±3.11)nm,respectively.Microgroove zirconia group showed the best hydrophilicity:the static contact angle was 51.2°±2.0°.HGF was arranged along the groove surface,and cell proliferation results showed that proliferation on microgroove zirconia was more significant than that on other groups from 6 h to 7 d(P<0.05).Intergrinα5 mRNA has the highest expression in microgroove zirconia(P<0.05)in the early adhesion(6 h),and there was no significant difference in the surface expression of intergrinβ1 and collagenⅠmRNA in the early adhesion(6 h)of each group.However,in the late adhesion(3 d),intergrinα5,β1 and collagenⅠmRNA expression in microgroove surface groups were higher than those of the smooth groups(P<0.05).Conclusions Microgroove zirconia surface has small roughness and good hydrophilicity,which can guide HGF to line up in the groove,and this is beneficial to the HGF proliferation and the expression of structural proteins and functional proteins。