Cytocompatibility and bioactive potential of AH Plus Bioceramic Sealer: An in vitro study

Abstract

Aim: To assess the cytocompatibility and bioactive potential of the new calcium silicate cement-based sealer AH Plus Bioceramic Sealer (AHPbcs) on human periodontal ligament stem cells (hPDLSCs) compared with the epoxy resin-based sealer AH Plus (AHP) and the calcium silicate cement-based sealer Endosequence BC Sealer (ESbcs).

Methodology: Standardized sample discs and 1:1, 1:2 and 1:4 eluates of the tested materials were prepared. The following assays were performed: surface element distribution via SEM-EDX, cell attachment and morphology via SEM, cell viability via a MTT assay, cell migration/proliferation via a wound-healing assay, osteo/cemento/odontogenic marker expression via RT-qPCR and cell mineralized nodule formation via Alizarin Red S staining. HPDLSCs were isolated from extracted third molars. Comparisons were made with hPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media. Statistical significance was established at p < .05.

Results: A higher peak of Ca² + was detected from ESbcs compared with AHPbcs and AHP in SEM-EDX. Both AHPbcs and ESbcs showed significantly positive results in the cytocompatibility assays (cell viability, migration/proliferation, attachment and morphology) compared with a negative control group, whilst AHP showed significant negative results. Both AHPbcs and ESbcs exhibited an upregulation of at least one osteo/odonto/cementogenic marker compared with the negative and positive control groups. Both ESbcs and AHPbcs showed a significantly higher calcified nodule formation than the negative and positive control groups, indicative of their biomineralization potential and were also significantly higher than AHP group.

Conclusion: AH Plus Bioceramic Sealer exhibited a significantly higher cytocompatibility and bioactive potential than AH Plus and a similar cytocompatibility to that of Endosequence BC Sealer. Endosequence BC Sealer exhibited a significantly higher mineralization potential than the other tested sealers. The results from this in vitro study act as supporting evidence for the use of AH Plus Bioceramic Sealer in root canal treatment.

Keywords: AH Plus Bioceramic Sealer; AH Plus sealer; Endosequence BC sealer; bioactivity; biocompatibility.

FIGURE 1

Results from the SEM‐EDS analysis for the tested sealers (ESbcs [column a], AHPbcs [column b], AHP [column c]). The first row illustrates SEM images of each material (scale bar: 100 μm). The second row shows the EDS elemental spectra. The third row lists the elements present per sealer by weight and atomic weight.

FIGURE 2

Results from MTT assay for the different eluates (1:1, 1:2, 1:4) of the tested sealers (ESbcs, AHPbcs and AHP) after 24, 48 and 72 h of culture with hPDLSCs. Data are presented absorbance values (570 nm) at the different measurement time‐points, compared with the negative control group. ***p < .001 (One‐way anova analysis).

FIGURE 3

Results from the wound healing assay for the different eluates (1:1, 1:2, 1:4) of the tested sealers (ESbcs, AHPbcs and AHP) after 24, 48 and 72 h of culture with hPDLSCs. Graphical results are presented as percentages of open wound areas at the different measurement time points, compared with the negative control group. ***p < .001 (One‐way anova analysis). Images: Scale bar 100 μm.

FIGURE 4

Results from the SEM visualization after 72 h of culture of hPDLSCs seeded onto the surface of the tested material sample discs (ESbcs, AHPbcs and AHP). Magnifications: 100×, 300× and 1500×. Scale bars: 500 μm, 100 μm and 30 μm.

FIGURE 5

Results from the analysis of hPDLSCs osteo/odonto/cementogenic marker expression via RT‐qPCR after 3, 7, 14 and 21 days of culture with DMEM (negative control), ESbcs, AHPbcs, or Osteodiff (postive control). *p < .05; **p < .01; ***p < .001 (Two‐way anova analysis). Asterisks above the bars indicate a significant difference with the negative control group. Asterisks above the lines indicate a significant difference between the groups which the line is connecting.

FIGURE 6

Results from the Alizarin Red S staining of hPDLSCs after 21 days of culture with DMEM (negative control), ESbcs, AHPbcs, AHP or Osteodiff (positive control). *p < .05; **p < .01; ***p < .001 (Two‐way anova analysis). Asterisks above the bars indicate a significant difference with the negative control group. Asterisks above the lines indicate a significant difference between the groups which the line is connecting.

FIGURE 7

Preferred Reporting Items for Laboratory studies in Endodontology (PRILE 2021)‐based flowchart.