In Vitro Bioactivity and Cytotoxicity Assessment of Two Root Canal Sealers

Abstract

The development of bioactive materials in endodontics has advanced tissue regeneration by enhancing the biological responses of periradicular tissues. Recently, calcium silicate-based sealers have gained attention for their superior biological properties, including biocompatibility, osteoconductivity, and cementogenic potential. This study aimed to evaluate the cytotoxicity, biocompatibility, and bioactivity of EndoSequence BC Sealer (ES BC) and AH Plus Bioceramic Sealer (AHP BC) using human periodontal ligament stromal cells (hPDLSCs). Biocompatibility was assessed using MTT, Live/Dead, and wound healing assays. ES BC and AHP BC demonstrated significantly higher cell viability and proliferation compared to AH Plus used as a control. Gene expression analysis via real-time quantitative PCR demonstrated that ES BC, especially in set form, significantly upregulated osteogenic markers-alkaline phosphatase (2.49 ± 0.10, p < 0.01), runt-related transcription factor 2 (2.33 ± 0.13), and collagen type I alpha 1 chain (2.85 ± 0.40, p < 0.001)-more than cementogenic markers (cementum protein 1, cementum attachment protein, and cementum protein 23). This differential response may reflect the fibroblast-dominant nature of hPDLSCs, which contain limited cementoblast-like cells. This study supports the superior biocompatibility and regenerative capacity of ES BC and AHP BC compared to AH Plus. While in vitro models provide foundational insights, advanced ex vivo approaches are crucial for translating findings to clinical practice.

Keywords: PDL cells; bioceramic sealer; biocompatibility; cementogenesis; osteogenesis; periodontal ligament stem cells.

Figure 1

Cell proliferation assessed by MTT assay at various sealer extract dilutions (1:1 (undiluted) to 1:64) after exposure periods of 1 and 3 days. Sealers tested were AH Plus (AHP), EndoSequence Bioceramic Sealer (ES BC), and AH Plus Bioceramic Sealer (AHP BC). Data represent mean ± standard deviation (n = 3). Statistical significance was determined by post hoc Tukey tests; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns: non-significant.

Figure 2

Live/Dead assay illustrating the viability of PDLSCs exposed to extracts of EndoSequence Bioceramic Sealer (ES BC) and AH Plus Bioceramic Sealer (AHP BC), in both fresh and fully set conditions after 1 day and 3 days. Green fluorescence indicates live cells, while red fluorescence indicates dead cells. Scale bars represent 100 µm.

Figure 3

Cytoskeleton staining of human periodontal ligament stem cells (PDLSCs) following 72 h of culture in the presence of fully set EndoSequence Bioceramic Sealer (ES BC) and AH Plus Bioceramic Sealer (AHP BC) extracts compared with the control group. Actin filaments are visualized in red (phalloidin staining) and nuclei in blue (DAPI staining). Scale bars represent 100 µm.

Figure 4

Results from the cell migration (wound healing) assay with human periodontal ligament stem cells (hPDLSCs) exposed to 1:1, 1:2, and 1:4 eluates of EndoSequence Bioceramic Sealer (ES BC) and AH Plus Bioceramic Sealer (AHP BC) after 6, 24, 48, and 72 h. (A,B) Representative light microscopy images showing the wound closure process, with a scale bar of 200 µm and magnification of ×20. (C,D) Graphical representation of wound area closure, presented as the percentage of remaining open wound areas relative to the negative control. Panels (A,C) correspond to fresh sealer samples, whereas panels (B,D) depict fully set samples. Data represent mean ± standard deviation (n = 3). Statistical significance was determined by post hoc Tukey tests; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 5

RT-qPCR results showing gene expression of osteogenic and cementogenic markers (RUNX2, ALPL, COL1A1, CAP, CP23, and CEMP1) in human periodontal ligament stem cells (hPDLSCs) cultured with fresh or set EndoSequence Bioceramic Sealer (ES BC) and AH Plus Bioceramic Sealer (AHP BC) undiluted extracts for 7 and 14 days. Gene expression levels are presented relative to the negative and positive control groups. Statistical significance was determined by post hoc Tukey tests; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Expanded gene names: RUNX2: runt-related transcription factor 2, ALPL: alkaline phosphatase, COL1A1: collagen type I alpha 1 chain, CAP: cementum attachment protein, CP23: cementum protein 23, CEMP1: cementum protein 1.