Effect of root canal irrigants on attachment and differentiation of human dental pulp cells

Abstract

Purpose: Sodium hypochloride (NaOCl) and ethylenediaminetetraacetic acid (EDTA)are commonly used for each antibacterial and cleansing effect in conventional root canaltreatment, but they are questionable candidates for cytotoxicity in regenerativeendodontic treatment. In clinical practice for regenerative endodontics, NaOCl is obligedto be used for the canal disinfection despite the damage to the living cells. EDTA hasbeen suggested as a chelating agent to release growth factor form dentin matrix, withoutscientific evidence analog to actual clinical situation. The aim of this study was toevaluate the effect of dentin application with NaOCl and EDTA on dental pulp cells(DPCs) attachment and differentiation on the dentin surface. I investigated whetherEDTA could release any biochemical components from dentin and change cell behaviors.Materials & Methods: Human dental pulp cells were obtained from 11 normal humanthird molars and investigated the stemness by flow cytometry analysis. Dentin specimenswere cut into disc shape and applied with 5.25% NaOCl (30 minutes) and 17% EDTA (12minute). After procedure to minimize the residual toxicity, pulp cells were seeding onthe conditioning dentin slices. To investigate the growth factor releasing effect, weincluded a group that dental pulp cells were cultured without dentin contact under a cellculture insert, in which EDTA-treated dentin slices were placed. After 3 day culture, cellattachment of each group was measured. After 21 day culture, I investigateddifferentiation gene expression level by quantitative real-time PCR. Then we observedcalcification level by Alizarin Red S staining and scanning electron microscopy (SEM).Results: After residual irrigants were removed after application of NaOCl and EDTA,pulp stem cell attached significantly denser on EDTA-treated dentin surface. The pulpstem cells on EDTA or NaOCl-treated dentin surface showed higher gene expression ofDSPP and DMP-1 than that on untreated dentin or no dentin after 3weeks culture. In thisstudy, dental pulp cells under a cell culture insert, in which EDTA-treated dentin sliceswere placed, did not show any differentiation gene expression.Conclusion: The physical property or topography of dentin surface might be changed byEDTA or NaOCl, which is considered to induce cell differentiation. How to treat thesubstrate (dentin) can be a crucial factor to regulate pulp stem cell behaviors (migration,attachment, proliferation, and differentiation) in regenerative endodontic treatment.