Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation.

Intracanal disinfection is a crucial step in regenerative endodontic procedures. Most published cases suggest the use of sodium hypochlorite (NaOCl) as the primary irrigant. However, the effect of clinically used concentrations of NaOCl on the survival and differentiation of stem cells is largely unknown. In this study, we tested the effect of various concentrations of NaOCl on the stem cells of the apical papilla (SCAPs) survival and dentin sialophosphoprotein (DSPP) expression. Standardized root canals were created in extracted human teeth and irrigated with NaOCl (0.5%, 1.5%, 3%, or 6%) followed by 17% EDTA or sterile saline. SCAPs in a hyaluronic acid-based scaffold were seeded into the canals and cultured for 7 days. Next, viable cells were quantified using a luminescence assay, and DSPP expression was evaluated using quantitative real-time polymerase chain reaction. There was a significant reduction in survival and DSPP expression in the group treated with 6% NaOCl compared with the untreated control group. Comparable survival was observed in the groups treated with the lower concentrations of NaOCl, but greater DSPP expression was observed in the 1.5% NaOCl group. In addition, 17% EDTA resulted in increased survival and DSPP expression partially reversing the deleterious effects of NaOCl. Collectively, the results suggest that dentin conditioning with high concentrations of NaOCl has a profound negative effect on the survival and differentiation of SCAPs. However, this effect can be prevented with the use of 1.5% NaOCl followed by 17% EDTA. The inclusion of this irrigation regimen might be beneficial in regenerative endodontic procedures.

Martin DE ，De Almeida JF ，Henry MA ，Khaing ZZ ，Schmidt CE ，Teixeira FB ，Diogenes A ... -  《-》

Dentin Conditioning with Bioactive Molecule Releasing Nanoparticle System Enhances Adherence, Viability, and Differentiation of Stem Cells from Apical Papilla.

Temporal-controlled bioactive molecule (BM) releasing systems allow the delivery of appropriate concentration of BM to enhance the interaction of stem cells to dentin matrix and subsequent odontogenic differentiation in regenerative endodontics. The goal of this study was to evaluate the effect of dentin conditioning with 2 variants of dexamethasone (Dex) releasing chitosan nanoparticles (CSnp), (1) Dex-CSnpI (slow releasing) and (2) Dex-CSnpII (rapid releasing), on adherence, viability, and differentiation of stem cells from apical papilla (SCAP) on root dentin exposed to endodontic irrigants. Slab-shaped dentin specimens were prepared parallel to the root canal and treated with 5.25% sodium hypochlorite (NaOCl) for 10 minutes and/or 17% EDTA for 2 minutes. Dentin was then conditioned accordingly by (1) no nanoparticle treatment, (2) CSnp, (3) Dex-CSnpI, and (4) Dex-CSnpII. The effect of nanoparticle conditioning on SCAP viability was determined by cell count and a circularity index. SCAP adherence and viability on dentin were assessed by fluorescence and scanning electron microscopy and odontogenic differentiation by immunofluorescence. SCAP on dentin treated with NaOCl alone or NaOCl as the last irrigant showed the least adherence, minimal cytoplasmic extensions, and higher circularity. SCAP adherence and viability on Dex-CSnpI and Dex-CSnpII conditioned dentin were increased and had a well-developed cytoplasmic matrix and significantly lower circularity (P < .05). SCAP cultured in Dex-CSnpII group expressed higher levels for DSPP and DMP-1 than in CSnp or Dex-CSnpI groups. Dex-CSnpI and Dex-CSnpII conditioning of dentin enhanced SCAP adherence and viability. Temporal-controlled release of Dex from Dex-CSnpII enhanced odontogenic differentiation of SCAP. This study highlighted the ability of dentin conditioning with temporal-controlled BM releasing nanoparticles to improve the local environment in regenerative endodontics.

Shrestha S ，Torneck CD ，Kishen A 《-》

Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips.

Intracanal disinfection is a crucial step in regenerative endodontic procedures. However, this novel endodontic treatment lacks standardization, and numerous treatment protocols have been reported without knowledge of the effect of disinfection protocols on the survival of stem cells. The aim of this study was to test the hypothesis that different root canal irrigation protocols alter survival of stem cells from the apical papilla (SCAP). SCAP were isolated from immature human third molars, and a subpopulation of STRO-1 expressing cells was selected and expanded in vitro. Standardized human root segments (n = 5/group) were irrigated with 1 of 4 protocols: (1) 17% ethylenediaminetetraacetic acid (EDTA), (2) 6% NaOCl/17% EDTA/6% NaOCl, (3) 17% EDTA/2% chlorhexidine (CHX), or (4) 6% NaOCl/17% EDTA/6% NaOCl/isopropyl alcohol/2% CHX. Subsequently, STRO-1-enriched SCAP were mixed with platelet-rich-plasma, seeded into the root tips, and cultured for 21 days. Roots were then decalcified, processed for immunohistochemistry, and stained for vimentin and TO-PRO-3. The proportion of viable (vimentin-positive) cells was calculated on the basis of the total cell counts (TO-PRO-3) for each group. Irrigation with 17% EDTA best supported cell survival (89% viability; P < .001 versus all other groups), followed by irrigation with 6% NaOCl/17% EDTA/6% NaOCl (74%; P < .001 versus the 2 groups containing 2% CHX). Conversely, protocols that included 2% CHX lacked any viable cells. Collectively, the results suggest that irrigants alone greatly affect the survivability of STRO-1-enriched SCAP within the root canal environment and that inclusion of EDTA in irrigation protocols might be beneficial in regenerative procedures.

Trevino EG ，Patwardhan AN ，Henry MA ，Perry G ，Dybdal-Hargreaves N ，Hargreaves KM ，Diogenes A ... -  《-》

Dentin conditioning codetermines cell fate in regenerative endodontics.

Recent successes in dental pulp engineering indicate that regenerative treatment strategies in endodontics are feasible. Clinically, revascularization procedures render completion of root formation in immature teeth. The generation of a pulp-like tissue after seeding of dental pulp stem cells into dentin discs or cylinders and transplantation in vivo is possible. In this experimental setup, which mimics the situation in the root canal, the pretreatment of dentin might influence cellular behavior at the cell-dentin interface. Thus, the objective of this study was to investigate whether dentin conditioning can determine cell fate. Dental pulp stem cells (DPSCs) were seeded into a growth factor-laden peptide hydrogel, transferred into dentin cylinders, and transplanted subcutaneously into immunocompromised mice. Before cell seeding, dentin cylinders were either pretreated with sodium hypochloride (NaOCl) or conditioned with EDTA. The constructs were explanted after 6 weeks and subjected to histological and immunohistochemical analysis. In dentin treated with NaOCl, resorption lacunae were found at the cell-dentin interface created by multinucleated cells with clastic activity. After conditioning with EDTA, DPSCs adjacent to the dentin formed an intimate association with the surface, differentiated into odontoblasts-like cells that expressed dentin sialoprotein, and extended cellular processes into the dentinal tubules. A vascularized soft connective tissue similar to dental pulp was observed inside the dentin cylinder. Dentin conditioning considerably influences DPSC fate when seeded in close proximity to dentin. This information might be critical for optimized strategic planning for future regenerative endodontic treatment.

Galler KM ，D'Souza RN ，Federlin M ，Cavender AC ，Hartgerink JD ，Hecker S ，Schmalz G ... -  《-》

Effect of EDTA on attachment and differentiation of dental pulp stem cells.

In regenerative endodontics, it is believed that EDTA induces odontoblast differentiation by releasing growth factors from the dentin matrix. The aim of this study was to evaluate the effect of EDTA on the attachment and differentiation of dental pulp stem cells (DPSCs). We also investigated whether the behavioral changes of DPSCs could be caused by biochemical components released from EDTA-treated dentin. Cells were obtained from human third molars, and the stem-like nature of the cells was investigated by flow cytometric analysis. DPSCs were seeded on EDTA-treated and untreated dentin slices. After 3 days of culture, cell attachment was evaluated by cell density, fibronectin 1 gene expression level using quantitative real-time polymerase chain reaction, and scanning electron microscopy. After 21 days of culture, the expression of differentiation genes was investigated by quantitative real-time polymerase chain reaction, and calcification was observed using alizarin red S staining. To investigate the EDTA-induced growth factor release, DPSCs were cultured with or without direct contact with the EDTA-treated dentin surface. After 3 days of culture, both the cell density and fibronectin expression level were significantly higher in the EDTA-treated dentin group. After 3 weeks, the DPSCs on the EDTA-treated dentin surfaces showed higher expression levels of dentin sialophosphoprotein and dentin matrix protein 1, whereas the DPSCs cultured without direct contact with the EDTA-treated dentin surfaces did not exhibit these findings. Our results showed that EDTA induced cell attachment and odontoblastic/osteoblastic differentiation, which was observed only in the group in which the DPSCs were placed in direct contact with the EDTA-treated dentin surfaces. These findings suggest that EDTA is beneficial for achieving successful outcomes in regenerative endodontics.

Pang NS ，Lee SJ ，Kim E ，Shin DM ，Cho SW ，Park W ，Zhang X ，Jung IY ... -  《-》