Tissue-friendly dentin treatments as a potential element in revascularization protocol (ex-vivo study)

Abstract

Background: Endodontic treatment aims to eliminate pulp tissue, microorganisms, and toxins while creating an environment conducive to tissue revitalization and regeneration. Sodium hypochlorite, the gold-standard irrigant, is effective but has significant cytotoxic effects, prompting the need for safer alternatives. This study investigates the cytotoxicity, cell proliferation, adhesion to dentin, and osteogenic differentiation of cells exposed to Dual Rinse HEDP, curcumin, and sodium hypochlorite (2.5%) for 1, 5, and 15 min, focusing on their potential application in revitalization and regenerative endodontic protocols.

Methodology: Samples were assigned to groups based on the irrigant used: control, HEDP, curcumin, or sodium hypochlorite (2.5%) for exposure durations of 1, 5, and 15 min. Cytotoxicity was assessed using the MTT assay, with optical density measured at the specified times. Cell proliferation was evaluated via the Trypan blue exclusion test, with viable cells counted using a hemocytometer. Data were presented as mean and standard deviation (SD) values and statistical significance was set at p < 0.05 for all tests. Cell adherence to dentin discs was analyzed using scanning electron microscopy (SEM) after 5-min irrigant exposure. Osteogenic differentiation was assessed through alizarin red staining for calcium deposition and quantitative PCR analysis of BMP-2, TGF-β1, VEGF, and DSPP gene expression.

Results: Cell cytotoxicity differed significantly across groups (p < 0.05), with HEDP showing the best results at 1 and 5 min. After 15 min, Group II had the highest value, followed by Group I. HEDP also recorded the highest cell proliferation, followed by curcumin. HEDP exhibited substantial calcium deposition and significantly upregulated BMP-2, TGF-β1, VEGF, and DSPP gene expression, surpassing other materials. Curcumin moderately promoted calcified nodule formation. Osteogenic media also induced significant gene upregulation.

Conclusions: Dual Rinse HEDP and curcumin are tissue-friendly. Dual rinse HEDP efficiently increases stem cell adherence to dentin discs and their osteogenic differentiation. So, this irrigant has the potential to be used in regeneration protocols.

Keywords: Cell viability; Curcumin; Cytotoxicity; Dentine Treatments; Dual rinse HEDP; Human Periodontal Stem Cells; Revascularization; SEM.

Fig. 1

Showing laboratory flow chart of the study

Fig. 2

Composite figure showing photomicrographs of PDLSCs co-cultured with tested reagents for 1 min. A: PDLSCs + Group I (HEPD), B: PDLSCs + Group II ( curcumin) and C: PDLSCs + Group III NaOCl. (trypan blue × 10). 5 min. A: PDLSCs + Group I (HEPD), B: PDLSCs + Group II ( curcumin) and C: PDLSCs + Group III NaOCl. (trypan blue × 10). 15 min. A: PDLSCs + Group I (HEPD), B: PDLSCs + Group II ( curcumin) and C: PDLSCs + Group III NaOCl. (trypan blue × 10)

Fig. 3

Scanning electron microscopy (SEM) of periodontal ligament stem cells (PDLSCs) cultured onto treated dentin discs. A The dentin surface of the control group appears with open dentinal tubules, and there is no visible attachment of cells. B Stem cells partially cover the dentinal tubules of the NaOCl-treated group. C Cluster of cells partially covering the dentin surface conditioned with curcumin. D Evident attachment of multiple stem cells with clear cytoplasmic extensions after HEDP treatment

Fig. 4

Qualitative results of Alizarin red staining of (A) control, (B) Osteogenic media, (C) NaOCl, (D) Curcumin, (E) HEDP

Fig. 5

Means and standard deviation of Alizarin Red absorbance at 405 nm. Different superscript small letters indicate significant differences

Fig. 6

Bar graphs representing the relative fold changes in the expression of genes (BMP-2, TGF-β1, VEGF, DSPP) in cell cultures under different treatment conditions. Different superscript small letters indicate significant differences