EDTA Treatment for Sodium Hypochlorite-treated Dentin Recovers Disturbed Attachment and Induces Differentiation of Mouse Dental Papilla Cells

Introduction: The disturbance of cellular attachment to dentin by sodium hypochlorite (NaOCl) may hamper pulp tissue regeneration. The aims of this study were to examine the recovering effect of EDTA on the attachment/differentiation of stemlike cells and to address the mechanisms of EDTA-induced recovery under the hypothesis that attachment to the exposed dentin matrix and the subsequent activation of integrin/phosphatidylinositol 3-kinase (PI3K) signaling play a crucial role.

Methods: Mouse dental papilla (MDP) cells were cultured on bovine dentin disks treated with NaOCl (0%, 1.5%, or 6%) followed by EDTA (0%, 3%, or 17%). Cell attachment was evaluated by cell density, viability, and scanning and transmission electron microscopy. Odonto-/osteoblastic gene expression in attached MDP cells was analyzed with or without a pan-PI3K inhibitor (LY294002) using real-time polymerase chain reaction.

Results: NaOCl treatment (1.5%, 10 minutes) significantly diminished attached MDP cells (P < .00001), but EDTA treatment (3% and 17%, ≥10 minutes) of NaOCl-pretreated dentin induced a significant increase in attached cells (P < .05). Ultrastructurally, MDP cells on EDTA-treated dentin showed attachment to exposed collagen fibers. MDP cells cultured on EDTA-treated disks (with or without 1.5% NaOCl pretreatment) showed significant up-regulation of alkaline phosphatase, dentin matrix protein 1, and dentin sialophosphoprotein messenger RNAs (P < .05). Alkaline phosphatase expression was down-regulated by LY294002 (P < .05).

Conclusions: Under the present experimental conditions, 10 minutes of EDTA treatment was sufficient to recover attachment/differentiation of MDP cells on 1.5% NaOCl-pretreated dentin. EDTA-induced exposure of collagen fibers and subsequent activation of integrin/PI3K signaling may contribute, at least partly, to the recovery.