The Functional Analysis of RVX-208 in Human Dental Pulp Cells: An Initiative for the Application of a Bioactive Compound, RV-208, on Direct Pulp Capping

<p> Purpose: Calcium hydroxide and mineral trioxide aggregate (MTA) are the most widely used for direct pulp capping. However, these agents induce the large pulp necrosis beneath the materials. Therefore, there is a need to develop non-toxic agents for pulp capping. RVX-208, a bioactive compound, plays important roles in inflammation and bone metabolism by epigenetic regulation of the expression of various genes. However, the functions of RVX-208 in dental pulp cells are still unknown. Thus, in this study, we investigated the toxicity and functions of RVX-208 on dental pulp cells to evaluate whether it offers the necessary features as a good next generation candidate for direct pulp capping materials.</p><p> Methods: The first premolars were extracted from patients undergoing orthodontic treatment at the Osaka University Dental Hospital. Then, dental pulp was extracted from the premolars and human dental pulp cells (hDPC) were isolated by primary outgrowth cultures. The cytotoxicity of RVX-208 on hDPC was examined by probidium iodide (PI) staining. The effect of RVX-208 on proliferation of hDPC was analyzed using Bromodeoxyuridine (BrdU) assay. In addition, the effect of RVX-208 on calcification nodules produced by cytodifferentiation of hDPC was evaluated by alizarin staining. Furthermore, the effect of RVX-208 on mRNA expression of odontoblast-related genes during cytodifferentiation of hDPC was examined by real-time PCR.</p><p> Results: The frequency of occurrence of PI⁺ cells was not significantly different between hDPC in the presence of 1, 10, 100 and 0 nmol/l of RVX-208. BrdU assay revealed that RVX-208 (0-100 nmol/l) did not affect the proliferation of hDPC. In contrast, on day 18 of cytodifferentiation of hDPC, alizarin staining showed that RVX-208 increased the calcification nodules of hDPC in a concentration-dependent manner. In addition, real-time PCR analysis revealed that DSPP and DMP1 mRNA expression was up-regulated in hDPC treated with RVX-208 during cytodifferentiation, whereas ALPL and COL1A1 mRNA expression was down-regulated in these cells. On day 6 of cytodifferentiation of hDPC, there was no significant difference in RUNX2 mRNA expression between hDPC treated with or without RVX-208. In contrast, on day 18, RUNX2 mRNA expression was significantly down-regulated in hDPC treated with 100 nmol/l of RVX-208, compared to that treated without RVX-208.</p><p> Conclusion: A bioactive compound, RVX-208, enhanced cytodifferentiation and calcification of dental pulp cells. RVX-208 might be a good candidate for a safe and effective biological direct pulp capping agent.</p>