Comparison of Biocompatibility of Newly Developed Calcium Silicate-Based Sealers and Epoxy Resin-Based Sealer on Human Periodontal Ligament Stem Cells

Abstract

The aim of this study was to evaluate various biocompatibility of newly introduced calcium silicate-based sealers (CeraSeal and EndoSeal TCS) and epoxy resin-based sealer (AH-Plus) on various aspect on human periodontal ligament stem cells (hPDLSCs). hPDLSCs were acquired from premolars (n = 4) of four subjects, whose ages extended from 16 to 24 years of age. To make media extracted from freshly mixed sealers (fresh media), all unset experimental sealers were mixed with culture medium (DMEM) and incubated for 24 h. To make media extracted from set sealers (setting media), materials were set in disc form for 48 h, and then extracted in DMEM for 24 h. hPDLSCs were cultured in fresh media or setting media according to each experimental condition. The expression of mesenchymal stem cell surface molecules (CD11b, CD19, CD34, CD45, CD73, CD90, CD105, and HLA-DR) was analyzed with flow cytometry (FACS) after culture in setting media. Cell viability was assessed using cell viability assay (Cell Counting Kit-8; CCK-8) after culture in fresh and setting media. To evaluate inflammatory response to the materials, concentrations of IL-6, IL-8, TGF-β in fresh and setting media were analyzed using ELISA kit. The osteogenic potential of hPDLSCs in setting media was quantified with RT-qPCR (Real Time-quantitative Polymerase Chain Reaction) for ALP, OCN, and RUNX2, and visually qualified with ALP (Alkaline Phosphatase) staining and ARS (Alizarin Red S) staining. Cell attachment on material and material surface morphology was evaluated with Scanning Electronic Microscopy. Statistical differences were assessed by analysis of variance followed by the Tukey’s test (p < 0.05). In FACS analysis, mesenchymal stem cell markers showed high level (> 99%) and the hematopoietic markers showed low expression level (<1%) in both Calcium silicate-based sealers and AH-Plus, therefore stemness of hPDLSCs was maintained in all materials. In cell viability test, AH-Plus showed the lowest cell viablity in all experimental periods, and CeraSeal showed significantly higher cell viability than others in fresh media (p < 0.05). In setting media, cell viablity was not significantly different between materials over all time periods. In ELISA test, AH-Plus showed higher expression of pro-inflammatory cytokines (IL-6 and IL-8) than other sealers. In the anti-inflammatory cytokine TGF-β, only CeraSeal maintained a similar level to control in setting media. In RT-qPCR test, AH-Plus showed lower expression level than other material, however EndoSeal TCS showed better expression level than others. As a result of ALP and ARS staining tests, calcium silicate-based sealers were stained similarly to positive control, and AH-Plus was less stained. Finally, scanning electron microscopy studies showed low degree of cell proliferation and differentiation on AH-Plus. However, CeraSeal and EndoSeal TCS showed high degree of cell proliferation and differentiation. Calcium silicate-based sealers are more biocompatible and less cytotoxic than epoxy-resin based sealer. In particular, CeraSeal showed less cytotoxicity than other materials before setting, and EndoSeal TCS showed better osteogenic potential than other materials.