Proteomic analysis of cell menbrane of human gingival fibroblasts cultured on different titanium surfaces and evaluation of exogenous antioxidant to control cellular oxidative stress

Abstract

Surface treatments are applied in dental implants in order to increase roughness and optimize osseointegration. Despite the clinical success achieved, its common cervical bone resorption, also called saucerization bone, which exposes the implant cervical region to the soft tissue. This condition can allow movement of toxins and bacteria from the oral cavity into bone. Thus adhesion between the gingival tissue and the implant surface form a barrierthat is extremely important to the successful osseointegration. To date, the influence of the roughness on adhesion of gingival fibroblasts was not investigated by the latest techniques in proteomics. It is also known that nanoparticles of titanium in this region may be released after insertion of the implant, which have cytotoxic potential for triggering of oxidative stress in the cells and thereby modulate the inflammation process. In this context, the administration of natural antioxidants, such as procyanidin, commonly found in many foods, can stimulate cell bioactivity attenuating inflammation when in contact with nanoparticles of titanium. The objective of this study is to evaluate the influence of ttanium surface roughness in the expression of cell membrane proteins of human gingival fibroblasts by proteomic analysis, as well as evaluating the oxidative potential of titanium particles in cells of the gingival tissue. Primary cells of human gingival fibroblasts will be cultured on commercially pure titanium disks grade 4 (12.7 × 2 mm) with minimally surface rough, moderately rough and rough. After 4, 12 and 24 h of culture, cells are collected and cell membrane proteins extracted using a kit cormercial. The proteins are separated by polyacrylamide gel electrophoresis and differential spots related to each surface will be identified by proteomic analysis using Tandem mass spectrometry (LC-MS/MS). In this same period, the cells are stained for visualization by confocal laser scanning microscopy for cell morphology correlate with the expression of membrane proteins. The evaluation of the inflammatory potential of particulates of titanium assay is carried out by reducing the compound diclorofluorescina diacetate (DCFH-DA). To evaluate the effect of procyanidin B2 as treatment the test is used to reduce the tetrazolium salt (MTS), and cell counts with trypan blue. (AU)