Effect of Enzymatic-based chemomechanical caries removal agent on proliferation and osteogenic differentiation of dental pulp stem cells

TL;DR

This study shows that an enzymatic caries removal method is safe and effective for dental pulp stem cells, supporting their growth and bone-like development.

Contribution

The study introduces evidence that enzymatic-based caries removal is biocompatible and promotes stem cell osteogenic differentiation.

Findings

Low-concentration enzymatic CMCR and indirect exposure via dentin discs showed minimal cytotoxicity and high cell viability.

Enzymatic CMCR exposure significantly upregulated DSPP gene expression, indicating enhanced osteogenic differentiation.

The method supports wound healing and proliferation of dental pulp stem cells over time.

Abstract

Chemomechanical caries removal (CMCR) offers a non-invasive alternative to conventional drilling techniques; however, the potential cytotoxic and genotoxic effects of CMCR agents on the dental pulp complex remain underexplored. This study aimed to evaluate the effects of enzymatic-based CMCR on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs).

MTT assay, scratch assay, and gene expression analysis were conducted to assess cell proliferation (viability percentage), migration (wound healing percentage), and osteogenic differentiation (RT-qPCR). The viability of DPSCs after direct exposure to enzymatic-based CMCR (0.5%, 1%, and 2% in DMEM) was measured at 24 and 48 h post-exposure. Indirect exposure was also tested using sound mineralized (0.5 mm and 1 mm) and demineralized (0.5 mm) dentin discs. Scratch assay was performed for control, direct, and indirect groups at 0, 24, and 48 h, followed by fixation and crystal violet staining at 48 h. Dentin sialophosphoprotein (DSPP) gene expression was quantified using RT-qPCR after 48 h.

Two-way ANOVA of proliferation and migration assays revealed that both indirect (0.5 mm demineralized dentin) and direct (0.5% DMEM) exposure groups showed the lowest cytotoxic effect after 24 h (82.75 ± 3.37 and 68.11 ± 4.99, respectively). After 48 h, a recovery trend in DPSC viability was observed, with both groups exhibiting the highest proliferation (93.60 ± 4.67 and 78.25 ± 4.88, respectively). These groups also showed the highest wound closure percentages (78.78 ± 0.89 for direct and 74.19 ± 1.03 for indirect exposure). One-way ANOVA of RT-qPCR results demonstrated significant upregulation of DSPP gene expression in the indirect (0.5 mm demineralized dentin) group (5.35 ± 2.01, p < 0.05).

Enzymatic-based CMCR exhibits favorable biocompatibility, particularly when applied directly on DPSCs at low concentrations and when used indirectly through dentin discs. These findings suggest that this enzymatic caries removal approach is potentially safe, effective, and minimally invasive, making it suitable for conservative restorative dentistry.

The online version contains supplementary material available at 10.1186/s12903-025-07495-w.

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