Avaliação dos efeitos osteogênico, genotóxico e antimicrobiano de eludatos do AH Plus reforçados com nanohidroxiapatita isolada e funcionalizada com zinco

Abstract

Nowadays, millions of endodontic treatments of caries and bacterial infections are performed. After pulp infection, all tissues from the root canals are removed, using adequate instrumentation, irrigation and antimicrobial substances. Apexification and obturation are then performed to complete the three-dimensional sealing of the system with endodontic sealers that provide a barrier to periapical tissues, preventing the development of a chronic inflammatory process. Despite their clinical success, commercial sealers do not meet the all the requirements of an ideal endodontic sealer, namely the induction capacity of the periradicular regenerative process that requires the recruitment of mesenchymal stem cells (MSC)/osteoblastic precursor cells and their differentiation into osteoblasts for synthesis of the mineralized matrix. The incorporation of hydroxyapatite (HAp), similar to the mineral phase of bone, into dental materials begins to be a promising approach, to improve the mechanical and biological characteristics of the product. The aim of this study was to verify the osteogenic, genotoxic and antimicrobial effects of eluates of resin-based endodontic cement (AH Plus) reinforced with two concentrations of nanohydroxyapatite (NHAp) or nanohydroxyapatite functionalized with zinc (NHAp-Zn3 and NHAp-Zn5). For this purpose, different cells were used: human mesenchymal stem cells derived from Wharton’s jelly (hMSC-WJ) for osteogenesis assays; CHO-K1 and FMM1 cells for genotoxic and oxidate stress analysis; four bacterial and four fungal strains for antimicrobial studies. MTT results showed that cells in the presence of NHAp, with and without zinc, had reduced viability at 14 days, showing a growth arrest at 7 days. Two dilutions of AH Plus were toxic, however, there was an excellent recovery of cell viability in the presence of NHAp, with and without zinc, with only the most diluted eluate. The mineralization assay, measured by the extracellular matrix staining by Alizarin red and its quantification, showed that NHAp improves osteogenesis, especially in the osteogenic medium, with NHAp-Zn3 in the lowest concentration more effective than NHAp-Zn5. Regarding genotoxicity, the results of the cytokinesis-block micronucleus (CBMN) assay showed no differences between the cell lineages used, with an increase in the frequency of MN only for the most diluted eluate alone and in conjunction with NHAp-Zn3 at the highest concentration. The combination that presented the best osteogenic potential (most diluted eluate of AH Plus with NHAp-Zn3 at the lowest concentration) and the one recommended in the study did not lead to changes in the frequency of micronuclei (MN), nuclear buds (NBud) and nucleoplasmic bridges (NPB). Antimicrobial assays, with Gram positive and negative bacteria and in four different fungi, were performed by the minimum inhibitory concentration (MIC) method, where most combinations were bacteriostatic and fungistatic. All the aforementioned results suggest that NHAp improves osteogenesis in in osteogenic medium without showing genotoxicity indicating that they may be an alternative for bone regeneration therapies in endodontic treatment.