电化学脱合金Ti6Al4V基台对人牙龈成纤维细胞的影响

Effect of electrochemically dealloying Ti6Al4V abutments on human gingival fibroblasts

摘要

目的 探讨电化学脱合金Ti6Al4V基台对人牙龈成纤维细胞（human gingival fibroblasts，HGFs）的影响，为种植基台表面改性设计提供实验依据。方法 根据试样不同表面进行分组：NC组（阴性对照，光滑表面无其他处理）、NM-1组（纳米网-1，1 mol/L NaOH电化学脱合金处理1 h，电压2 V）、NM-2组（纳米网-2，5 mol/L NaOH电化学脱合金处理1 h，电压2 V）。扫描电镜（scanning electron microscopy，SEM）观察不同试样表面形貌及HGFs在其表面的黏附状态，接触角测量仪测定试样表面亲水性。CCK-8评估HGFs在不同试样表面的增殖情况;qRT-PCR检测HGFs在不同试样表面Ⅰ型胶原（collagen Ⅰ，COL1A1）、Ⅲ型胶原（collagen Ⅲ，COL3A1）、纤连蛋白1（fibronectin 1，FN1）、黏着斑激酶（focal adhesion kinase，FAK）、黏着斑蛋白（vinculin，VCL）、整合素α2（integrin α2，ITGA2）、整合素β1（integrin β1，ITGB1）等黏附相关基因表达水平;免疫荧光染色，激光共聚焦显微镜（confocal laser scanning microscope，CLSM）观察HGFs在不同试样表面vinculin蛋白表达情况;天狼猩红染色评价HGFs在不同试样表面胶原纤维分泌合成情况。结果 SEM观察到NM-1组和NM-2组表面形成有序均一的三维网状结构，网格直径NM-1组约为30 nm，NM-2组约为150 nm;NM-1组和NM-2组对比NC组，水接触角显著下降（P<0.000 1）;NM-1组细胞增殖能力较NC组显著提高（P<0.01），NM-1组和NM-2组的水接触角及细胞增殖能力差异无统计学意义（P>0.05）。SEM镜下可见HGFs黏附24 h后在各组试样表面均黏附良好，NM-1组和NM-2组细胞与NC组对比伸展面积更大，形态纤长，细胞伪足更发达。qRT-PCR结果显示NM-1组中COL1A1、COL3A1、FN1、FAK、VCL等黏附相关基因表达水平显著高于NC组和NM-2组（P<0.01）。vinculin蛋白免疫荧光结果表明，NM-1组vinculin蛋白表达水平最高，单个细胞黏着斑数量最多（P<0.01）。天狼猩红染色结果显示NM-1组胶原纤维分泌合成量最高（P<0.000 1）。结论 Ti6Al4V经电化学脱合金改性后所构建的三维纳米网状结构有促进HGFs黏附增殖和胶原分泌合成的作用，其中网格直径约为30 nm的NM-1组对HGFs的促进作用明显。

Abstract

Objective To investigate the effects of electrochemically dealloying of Ti6Al4V abutments on human gingival fibroblasts (HGFs) and to provide experimental evidence for surface modification of implant abutments. Methods The samples were divided into an NC group (negative control, no other treatment on a smooth surface), an NM-1 group (nanomesh-1, electrochemical dealloying treatment in 1 mol/L NaOH 1 h on 2 V voltage), and an NM-2 group (nanomesh-2, electrochemical dealloying treatment in 5 mol/L NaOH 1 h on 2 V voltage). The surface morphologies of the samples and the adhesion of HGFs on the sample surfaces were observed with scanning electron microscopy (SEM). The surface hydrophilicities of the samples were measured with a contact angle measuring instrument. The proliferation of HGFs on the different samples were evaluated with CCK-8, and the expression of adhesion-related genes, including collagen Ⅰ (COL1A1), collagen Ⅲ (COL3A1), fibronectin 1 (FN1), focal adhesion kinase (FAK), vinculin (VCL), integrin α2 (ITGA2), and integrin β1 (ITGB1), on the different samples was measured with qRT-PCR. The expression of vinculin on the surfaces of HGFs was observed via confocal laser scanning microscopy (CLSM) after immunofluorescent staining. Collagen fiber secretion and syntheses of HGFs from different samples were evaluated via Sirius red staining. Results SEM revealed the formation of ordered and uniform three-dimensional mesh structures on the surfaces of the NM-1 and NM-2 groups, with grid diameters of approximately 30 nm for the NM-1 group and approximately 150 nm for the NM-2 group. Compared with that of the NC group, the water contact angles of the NM-1 group and NM-2 groups were significantly lower (P<0.000 1). Cell proliferation in the NM-1 group was significantly greater than that in the NC group (P<0.01). Moreover, there was no significant difference in the water contact angles or cell proliferation between the NM-1 group and the NM-2 group. SEM revealed that HGFs were adhered well to the surfaces of all samples, while the HGFs in the NM-1 and NM-2 groups showed more extended areas, longer morphologies, and more developed pseudopodia than did those in the NC group after 24 h. qRT-PCR revealed that the expression levels of the adhesion-related genes COL1A1, COL3A1, FN1, FAK and VCL in the NM-1 group were significantly greater than those in the NC and NM-2 groups (P<0.01). The expression of vinculin protein in the NM-1 group was the highest, and the number of focal adhesions was greatest in the NM-1 group (P<0.01). The results of Sirius red staining showed that the NM-1 group had the highest secretion and syntheses of collagen fibers (P<0.000 1). Conclusion The three-dimensional nanomechanical structure of Ti6Al4V modified by electrochemical dealloying promoted the adhesion, proliferation, collagen fiber secretion and syntheses of HGFs, and electrochemical dealloying of Ti6Al4V with a grid diameter of approximately 30 nm obviously promoted HGF formation.

关键词

电化学脱合金 / Ti6Al4V / 种植体基台 / 表面改性 / 三维网状结构 / 人牙龈成纤维细胞 / 细胞黏附 / 细胞增殖 / 黏着斑

Key words

electrochemical dealloying / Ti6Al4V / implant abutment / surface modification / three-dimensional grid structure / human gingival fibroblasts / cell adhesion / cell proliferation / focal adhesion complex

Author summay

蔡东轩，医师，博士，Email：[e-mail]