Effect of electrochemically dealloying Ti6Al4V abutments on human gingival fibroblasts

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.

Keywords

• electrochemical dealloying，
• ti6al4v，
• implant abutment，
• surface modification，
• three-dimensional grid structure，
• human gingival fibroblasts，
• cell adhesion，
• cell proliferation，
• focal adhesion complex，