Biocompatibility, bioactivity and immunomodulatory properties of three calcium silicate-based sealers: an in vitro study on hPDLSCs

Abstract

Objectives: To assess the biocompatibility, bioactivity, and immunomodulatory properties of three new calcium silicate cement-based sealers: Ceraseal (CS), Totalfill BC Sealer (TFbc) and WellRoot ST (WR-ST) on human periodontal ligament stem cells (hPDLSCs).

Materials and methods: HPDLSCs were isolated from extracted third molars from healthy patients. Eluates (1:1, 1:2, and 1:4 ratio) and sample discs of CS, TFbc and WR-ST after setting were prepared. A series of assays were performed: cell characterization, cell metabolic activity (MTT assay) cell attachment and morphology (SEM assay), cell migration (wound-healing assay), cytoskeleton organization (phaloidin-based assay); IL-6 and IL-8 release (ELISA); differentiation marker expression (RT-qPCR assay), and cell mineralization (Alizarin Red S staining). HPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media were used as a comparison. Statistical significance was established at p < 0.05.

Results: All the tested sealers exhibited similar results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group. CS and TFbc exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. CS and TFbc also showed a significantly higher calcified nodule formation than the negative and positive control groups. Both the marker expression and calcified nodule formation were significantly higher in CS-treated cells than TFbc treated cells. WR-ST exhibited similar results to the control group. CS and TFbc-treated cells exhibited a significant downregulation of IL-6 after 72 h of culture compared to the negative control group (p < 0.05).

Conclusion: All the tested sealers exhibited an adequate cytocompatibility. CS significantly enhances cell differentiation by upregulating the expression of key genes associated with bone and cementum formation. Additionally, CS was observed to facilitate the mineralization of the extracellular matrix effectively. In contrast, the effects of TFbc and WR-ST on these processes were less pronounced compared to CS. Furthermore, both CS and TFbc exhibited an anti-inflammatory potential, contributing to their potential therapeutic benefits in regenerative endodontics.

Clinical relevance: This is the first study to compare the biological properties and immunomodulatory potential of Ceraseal, Totalfill BC Sealer, and WellRoot ST. The results act as supporting evidence for their use in root canal treatment.

Keywords: Anti-inflammatory; Bioacompatibility; Bioactivity; Ceraseal; Totalfill BC sealer; WellRoot ST.

Fig. 1

(A) Results from hPDLSC characterization. Marker expression is presented as a percentage (%). (B) Results from the cell metabollic assay (MTT) for the 1:1, 1:2 and 1:4 eluates of the tested sealers (TFbc, WR-ST, and CS) after 24, 48, and 72 h of culture with hPDLSCs. Data are presented absorbance values (570 nm) compared to the negative control group. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

(A) Results from the cell migration assay (wound healing) for the 1:1, 1:2 and 1:4 eluates of the tested sealers (TFbc, WR-ST, and CS) after 24, 48, and 72 h of culture with hPDLSCs. Graphical results are presented as percentages of open wound areas compared to the negative control group. ***p < 0.001. (B) Results from the hPDLSC cytoskeleton staining (phaloidin-based assay) after 72 h of culture with 1:1 testes sealers (TFbc, WR-ST, and CS). Scale bar: 100 μm

Fig. 3

(A) SEM images (cell adhesion and morphology assay) after 72 h of culture of hPDLSCs seeded onto the surface of the tested sealer discs (TFbc, WR-ST, and CS). Magnifications: 100X, 300X, and 1500X. Scale bars: 400 μm, 100 μm, and 20 μm. (B) Results from the ELISA to assess hPDLSC expression of IL-6 and IL-8 after 72 h of culture with the testes sealers (TFbc, WR-ST, and CS) compared to the negative control. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

(A) RT-qPCR results for hPDLSCs marker expression after 3, 7, 14 and 21 days of culture with the tested sealers (TFbc, WR-ST, and CS), relative to the negative and positive control groups. *p < 0.05; **p < 0.01; ***p < 0.001. (B) Results from the Alizarin Red S staining assay of hPDLSCs after 21 days of culture with the tested sealers(TFbc, WR-ST, and CS), relative to the negative and positive control groups. *p < 0.05; **p < 0.01; ***p < 0.001. In both panels, asterisks above the bars indicate a significant difference with the negative control group; asterisks above the lines indicate a significant difference between the groups connected by the line