Characteristics and biological considerations of newly developed bioactive NanoFA-based dental composite

Abstract

Amalgam use will be phased out in restorative dentistry. The introduction of the Minamata convention offers the perspective of dental composite becoming the main direct restorative material. The currently available tooth-coloured restorative materials are satisfactory with regards to mechanical and optical properties. Moreover, significant innovation has improved the clinical performance of the material, reduced the polymerization shrinkage and enabled conservation of the tooth structure. Most of the available composite materials contain Bis-GMA, which is toxic due to Bisphenol A content. Additionally, they lack bioactive properties. Bioactive composites are materials with bacteriostatic and remineralising potential due to fluoride release. Alternative, Bis-GMA free composite materials that release fluoride are expected to significantly decrease restoration failure caused by secondary caries. Newly developed materials need to be thoroughly tested for cytocompatibility. In addtion, the effect of adding bioactive compounds on the mechanical properties should be investigated. In this project prototypes of novel restorative materials were developed, and their mechanical properties were tested. These were based on UDMA (urethane dimethacrylate) and HEMA (2-hydroxyethyl methacrylate) and silica glass fillers (SiO2). The remineralizing properties were provided by adding nanofluorapatite (NanoFA) nanoparticles as secondary fillers. The degree of polymerization was measured by infra-red spectroscopy (FTIR). The amount of residual monomer after polymerization was quantified by HPLC. The mechanical properties were examined by three-point bending test. Fluoride release was measured using an ion selective electrode. The effect of storage in distilled water, artificial saliva and acidified artificial saliva on composite specimen was monitored and examined under a scanning electron microscope (SEM). The toxicity was assessed by different parameters. The toxicity of individual dental resin monomers to oral gingival fibroblasts (HGF) was assessed by XTT viability assay and RT-qPCR. Finally, the effect of the final material on gingival fibroblasts was characterized by RNA Seq transcriptome analysis. The novel NanoFA filled material achieved a mean degree of conversion of 84.61%. Additionally, the fluoride release of the novel material was significant compared to the NanoFA-free control. The material’s mechanical properties were compromised by storage in iii aqueous environment. Dental resin monomer toxicity was revealed by a significant reduction (P<0.05) in the cell viability at 2 mM of HEMA, while for UDMA a reduction in viability to 60% was found at 1 mM (P<0.05). RT-qPCR expression analysis of DNA repair genes showed a dose dependent expression increase in HGF treated with HEMA. A statistically significant expression change (P<0.001) of four genes (DDX11, IPPK, XRCC2 and RAD50) was noted with 10 mM and 40 mM of HEMA. In contrast, UDMA increased the expression of stress response genes (Cox-2, CES2 and HO-1) at 4 mM (P<0.001). The amount of residual resin monomers quantified by HPLC were below the toxic concentrations. However, RNA Seq analysis revealed signs of toxicity to oral gingival fibroblasts after 24 hours exposure to dental composite materials as manifested by an increased expression of cellular signalling pathways such as oxidoreductase activity, NADPH activity and ferroptosis. To conclude, novel dental composite materials were successfully designed. The new materials have promising performance with regards to high degree of conversion and fluoride release, though mechanical properties need to be improved. The release of residual monomers from the polymerized materials were below the detected toxic concentrations. Nevertheless, further research may improve the material formula with the aim of reducing chemical toxicity to oral gingival fibroblasts.