Anti-inflammatory and regeneration properties of iloprost on inflamed human dental pulp cells model: an in vitro study

Abstract

In the clinical situation, pulp exposure can happen during caries removal procedures. Inflammation can occur on the pulp of the exposed area because of infiltration of bacteria from caries or as a result of pulp reaction to caries removal drilling. When the pulp was exposed during the caries removal procedure, traditional methods to preserve the pulp such as pulp capping and pulpotomy were performed. The ideal treatment outcome of the pulp exposure is to regain the primary structure of tubular dentin as well as maintain the vitality and healthiness of the dental pulp Thus, good dental materials or drugs used for vital tooth therapy should have properties of antibacterial, anti-inflammatory, and dentin-pulp tissue regenerative capability. Previous studies suggested that iloprost, the analog of prostacyclin, could induce tertiary dentin, boost angiogenesis, increase blood flow, and promote neovascularization, both on healthy human dental pulp cells, and in rat models. However, the role of iloprost on inflammatory control in dental pulp has not been investigated before.This study aimed to develop an inflamed human dental pulp cell model in vitro and employ the model to investigate the anti-inflammatory and promoting tissue regeneration effect of iloprost. To create the inflammation model in vitro, HDPCs were treated by the cocktail of three cytokines; IL-1β, IFN γ, and TNF α. With three different doses of these cytokines in the ratio of 1:10:100 (IL-1β:1ng/ml, TNF α:10 ng/ml, and IFN γ: 100 ng/ml), the viability of the cells was examined by MTT assay and the level of IL-6 and IL-12 was assessed by RT-qPCR. After 6h and 24h, the expression of IL-6 protein was assessed by ELISA assay. After the mimicked inflamed HDPCs model was obtained, iloprost treatment was performed, using iloprost solution with concentrations of 10-6 mol for 6h and 24h. Then, the viability of the cells was examined by MTT assay and the expression level of IL-6 and IL-12 was measured by RT-qPCR, and IL-6 protein expression was assessed by ELISA assay. The results were compared to the control sample which was treated by cytokines cocktail only. The comparison was analyzed using GraphPad Prism 9, either with one-way ANOVA or Kruskal-Wallis method. The results of this study showed that cytokine cocktail that consist of IL-1β:1ng/ml, TNF α:10 ng/ml, and IFN γ: 100 ng/ml, was not toxic, and could induces the expression of IL-6 robustly and IL-12 on HDPCs. Iloprost treatment was capable to downregulate the expression of IL-6 and IL-12 mRNA but had no significant impact as a cytoprotective agent on HDPCs. In conclusion: This study suggested that cytokine cocktail creates a better acute inflammation model than LPS induced model, and iloprost was capable as an anti-inflammatory agent on inflamed HDPCs. Hence, open the possibility of iloprost to be used as additional vital pulp therapy material.