Bis-GMA對人類牙髓細胞細胞週期調節、基質金屬蛋白酶表現、及鹼性磷酸酶活性之影響

Abstract

In previous studies, it was found that the highly cytotoxic dental monomer bis-GMA could be released from resin-based dental materials into the oral environment, which could potentiate both systemic and local adverse effects. Dental monomers could induce excessive production of reactive oxygen species (ROS), leading to cell cycle aberrations and cell cycle arrest. Since cell cycles are tightly regulated by various cell cycle regulators, the effect of bis-GMA to the expression of the cell cycle regulators Cdc2, Cdc25C, Myt1, p21, p53, p-Cdc2, p-Cdc25C, and Bad was investigated in order to understand the mechanism of bis-GMA-induced cell cycle aberrations in dental pulp cells. On the other hand, the matrix metalloproteinases (MMPs) are the major proteinases regulating the maintenance of extracellular matrix (ECM) of human dental pulp. The effect of bis-GMA on the expression of MMP-2, MMP-9, and tissue inhibitors of metalloproteinases (TIMP)-2 in human dental pulp cells were investigated. Dental pulp cells were also investigated for their expression of alkaline phosphatase, a differentiative marker for dentinogenesis after treatment of bis-GMA. Human dental pulp cells were treated with increasing concentration of bis-GMA (0 – 0.25mM) for 24 hours, before morphological changes were observed. Total RNA was isolated from these pulp cells for reverse transcription-polymerase chain reaction (RT-PCR). Proteins were also extracted from these pulp cells to run for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Medium from the dental pulp cells were collected for zymography in identification of MMP-2 and MMP-9 released by pulp cells into the conditioned medium. Another group of dental pulp cells were cultured in bis-GMA (0 – 0.075mM) for 5 days for MTT cytotoxicity test and alkaline phosphatase (ALP) staining. Bis-GMA induced morphological changes in human pulp cells, which characteristically exhibited a long spindle shape with extended cellular processes, to a retracted, rounded, and shell-like appearance. Some cells were detached from culture well indicating loss of vitality at higher concentrations. Bis-GMA also inhibited Cdc2 and Cdc25C production, with promotion in protein production of Myt1, p21, and p53. Bis-GMA treatment induced the mRNA expression of p21 with inhibition in Cdc2, cyclin B1, and Cdc25C mRNA expression. These suggested that bis-GMA could induce cell cycle arrest by aberrant regulation of cell cycle-related proteins in dental pulp cells. Bis-GMA was found to induce both the expression of MMP-2 and MMP-9 mRNA and protein level in dental pulp cells, with generalized weaker expression for MMP-9 compared to MMP-2. A stronger gelatinolytic activity of pro-MMP-2 and a weaker activity of pro-MMP-9 were also found in the conditioned medium of dental pulp cells, whereas little active forms of MMP-2 or MMP-9 were detected. Bis-GMA was found to significantly decrease the release of pro-MMP2 in the conditioned media, with no apparent effect on the release of pro-MMP-9. In contrast to MMPs, the inhibitor of MMPs, TIMP-2 protein level was induced by bis-GMA treatment. Therefore, the combined effects of bis-GMA in induction of MMP-2 and MMP-9 mRNA and protein level, the induction in TIMP-2 and reduction in the release of the latent form of MMP-2 could lead to a net excess in MMPs activities promoting accelerated ECM degradation. Our study also found that low concentrations of bis-GMA which showed no significant cytotoxicity on dental pulp cells demonstrated reduced ALP activities. This result suggested that low concentrations of bis-GMA had an inhibitory effect on the differentiative ability of pulp cells for dentinogenesis, which could lead to interferences in dentine formation. At higher concentrations, bis-GMA exhibited a significant cytotoxic effect on dental pulp cells. In conclusion, bis-GMA could lead to aberrations in cellular morphology and cell cycle regulation, imbalanced MMPs activities, and disturbances in differentiative activities of dental pulp cells. In previous studies, dentin bonding agents have been found to cause inflammatory changes, dilation and congestion of blood vessels, production of irregular dentine, and odontoblastic displacement. Composite restorations were also found to be associated with more pulpal breakdown in deep and pulpally exposed cavities in comparison to amalgam restorations. However, further studies in vitro and in vivo were required to investigate effect of bis-GMA to human pulp tissues.