| HEMA is an important component in dental composite resins and dentin adhesive materials used in dental restorations, and the smallest molecular weight hydrophilic monomer, which penetrates easily the hybrid layer through dentin molecular sieve characteristics that mediated dentin collagen and hydrophobic resin material combination. Total etching adhesive system containing HEMA can penetrate into the acid etched dentin in order to prevent collagen fiber collapse. Objectively, it maintains the stability of the adhesive interface and has advantage in the bio-mechanical. The pulpo-dentinal complex has the special structure, that is the dentinal tubule near pulp exist nerve endings, which is susceptible to stimulation of adhesive monomer. Dentin adhesive contacts with dentin long-term and close by hybrid layer, which is impossible to achieve complete polymerization. The eluted monomers can directly contact the exposed collagen fibre and even enter the pulp chamber through the dentinal tubule to make contact with the odontoblasts on the surface and the pulp cells in the deep layer. Residual monomers decrease not only the mechanical properties of repair materials, but also have the adverse effects for the biocompatibility. At present, the effects of dentin adhesive monomers on MMPs in human pulpo-dentinal complex have been scarcely examined. Therefore, in this study, the matrix monomer HEMA, which is common in dentin adhesive systems, was employed to treat human pulp cells and odontoblast-like cells. The effects of HEMA on MMPs in human pulp cells and odontoblast-like cells were assessed, providing experimental evidence for the relationship between the resin materials and the activity of MMPs in pulpo-dentinal complex.Objective:This study is designed to explore the effects of HEMA, a dentin adhesive monomer, on MMPs in human dental pulp and odontoblast-like cells.Methods:Human dental pulp cells were obtained using tissue explant technique in vitro, and then the third to fifth generation pulp cells were cultured in media containing different concentrations of HEMA for 24,48,72 h, respectively. Cell proliferation was measured by MTT assay. After the pulp cells were treated with HEMA for 48 h, the morphological changes of cells were observed by the inverted fluorescence microscope. After the pulp cells were treated with DMEM medium containing 100 or 400 μg/ml of HEMA for 48 h, the quantity of migrating cells was tested, and the expression of MMP-2 and MMP-9 was analysed by Western Blot. The pulp cells were induced to differentiate about 3-4 weeks, RT-PCR tested the gene expression of DSPP, BSP, ALP. After the pulp cells were successfully induced to differentiate into odontoblast-like cells, the activity of HEMA on MMP-2,-9 was tested by gelatin zymography.Results:The proliferation ability of Human dental pulp cells decreased when exposed to HEMA in both time-and concentration-dependent manner. Cell activity value at 24 h was statistically higher than those at 48 h and 72 h. Different time points of the cell viability values were significantly lower than that of their respective blank control group (P<0.05). After the pulp cells were treated with HEMA for 48 h, the cells in the control group grew well. With the increase of HEMA concentration, the density of cells was significantly lower, and the growth of the cells was inhibited. When the HEMA concentration was increased to 800 μg/ml, the cells started to become round, showing signs of apoptosis and cell debris. In the 1500 μg/ml group, a large number of death cells were observed. And the number of migrating Human dental pulp cells was significantly reduced in HEMA groups at different concentrations, compared with that of blank control group (P<0.05). Simultaneously, with the increase of HEMA concentration, compared with the blank control group, the expression of MMP-2、-9 decreased significantly. The pulp cells were successfully induced to differentiate into odontoblast-like cells, after a 24-hour incubation by HEMA, a dose-dependent inhibition was observed, and HEMA showed a strong inhibitory effect at the concentration of 10%, whereas the activity of MMP-2 proenzyme was not completely inhibited. The inhibitory effects of HEMA on the precursor and activated form of MMP-2 as well as the precursor of MMP-9 were analysed with linear regression, with the coefficient determined as r2=0.921 (P<0.05); the difference was statistically significant. MMP-2 and MMP-9 showed a good linear fit with HEMA.Conclusion:In this study, the clinical significance of the inhibitory effects of HEMA on MMPs in pulpo-dentinal complex were presented in two aspects:1) MMPs in pulp cells, odontoblasts and dentin are homologous. The inhibition of the activity of MMPs by HEMA may effectively protect the degradation of type I collagen during dentin adhesive or bonding operations, which can objectively maintain the stability of the resin-dentin bonding interface and improve the dentin adhesive durability.2) HEMA can not only inhibit the activity of MMPs in pulp cells but also limit the migration of dental pulp cells toward odontoblasts, thereby affecting the formation of reparative dentin. Therefore, HEMA affects the formation of reparative dentin while maintaining the stability of the bonding interface. |
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