Avaliação in vitro e in vivo do efeito de nanopartículas de ouro estabilizadas com ácido ascórbico para capeamento pulpar

Abstract

Introduction: Direct pulp capping is a technique that consists of applying a material directly on the exposed pulp tissue and aims to preserve the vitality of the dental element. Gold nanoparticles (AuNPs) can contribute to the treatment of pulp inflammation due to their antioxidant and anti-inflammatory effect. Objective: 1) To characterize ascorbic acid-coated AuNPs and evaluate if they present cytotoxic and anti-inflammatory effects in cell culture; 2) to verify if laser-Doppler flowmetry (LDF) allows the diagnosis of pulp condition in rat molars; and 3) to evaluate if AuNPs interfere with pulp repair in rat molars. Material and methods: Study 1: 3T3 fibroblasts, J774 macrophages and undifferentiated cells of human dental pulp (UCHDP) were treated with AuNPs for 24 and 48 hours, and cell viability was analyzed through the MTT assay for the 3 cell lines and cell morphology analysis for UCHDP. In macrophages, the inflammatory response was induced by LPS and the release of TNF- α was quantified (ELISA) after treatment with AuNPs. Study 2: Using laser-Dopplet flowmetry (LDF) the pulpal blood flow (PBF) of the upper first molars of 10 rats was measured by 2 different operators at 2 times with different techniques (without and with guides), determining the method with better reproducibility through the analysis of the coefficient of variation and the Bland-Altman concordance test together with the t test for one sample. Then, models of pulp necrosis and inflammation in upper first molars of rats were created (n=5), and the PBF values were compared with the histopathological findings in order to obtain the specificity and sensitivity of the method for the diagnosis of inflammation and pulp necrosis through the analysis of ROC curve. Study 3: Pulp exposures were performed in the first molars of rats, which were treated with: sterile cotton (control), calcium hydroxide, and AuNPs for 21 days (n=4 per group). The PBF was measured periodically using LDF and the histopathological analysis considered scores for inflammation severity, pulp tissue integrity and mineralized barrier production. ANOVA with Tukey’s post-test was used to compare parametric numerical data and the Kruskal-Wallis test followed by Dun’s multiple comparisions test for score analysis. Results: AuNPs did not show cytotoxic effects on J774 macrophages, 3T3 fibroblasts and UCHDP. However, after 48h the UCHDP demonstrated morphological changes indicative of apoptosis. Low concentrations of AuNPs negatively regulate the in vitro release of TNF-α in macrophages. The standardization of the LDF method indicated that measurements taken without guides were more reproducible. Teeth with pulp inflammation showed an increase in PBF values of 48% and 52% after 6h and 48h, respectively. In teeth with pulp necrosis the PBF values decreased 44% after 21 days. LDF showed good specificity (90%) and sensitivity (100%) for the diagnosis of pulp necrosis, however, it was inefficient for the diagnosis of pulp inflammation. In the third study, all materials tested for pulp capping decreased PBF immediately after the procedure, with the PBF being restored to normal values during the experimental period, except in the CaOH group, which maintained a drecenth of 21% after 21 days. AuNPs did not promote control of the severity of inflammation and did not induce the formation of a dentin matrix. Conclusions: AuNPs present anti-inflammatory activity in vitro, showing no cytotoxic effect on fibroblasts, macrophages and UCHDP. LDF is able to differentiate vital pulp from non-vital pulp (necrosis) in upper first molars of rats, but the method was neither sensitive nor specific to diagnose pulp inflammation. AuNPs have no additional benefit in the formation of repair dentin and in controlling the inflammation when used alone on exposed pulp tissue as a capping material.