The Effects of Irrigants on the Survival of Human Stem Cells of the Apical Papilla, Including Endocyn Mark B. Scott, II, DMD,* Gregory S. Zilinski, DDS,* † Timothy C. Kirkpatrick, DDS, † Van T. Himel, DDS,* Kent A. Sabey, DDS,* and Thomas E. Lallier, PhD ‡ Abstract Introduction: Endocyn, a pH-neutral solution of hypo- chlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human peri- odontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants. Methods: To determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse- transcriptase polymerase chain reaction. Results: Endo- cyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concen- trations of >20% inhibited SCAP proliferation, whereas concentrations of $10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did signifi- cantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin. Conclusion: Endocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used end- odontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phospha- tase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn. (J Endod 2017;-:1–6) Key Words Cell survival, chlorohexidine, hypochlorous acid, stem cells, sodium hypochlorite I t is well established that microorganisms are the principal cause of end- odontic disease (1–3), thus the paramount goal of endodontic treatment is their elimination. Regenerative endodontics is indicated for management of immature permanent teeth with pulpal necrosis. This procedure is performed with the goal of allowing and promoting further root development. This is accomplished by first disinfecting the canal space, and then allowing the ingress of stem cells into that space. Those stems cells of the apical papilla (SCAP) typically reside in close proximity to the root end and have exhibited faster proliferation, and greater differentiation into odontoblast-like cells than other dental stem cells (4, 5). SCAP survival during the disinfection process is essential to the regenerative process (6). If these stem cells are exposed to standard irrigants or medicaments, it may render them nonviable, less functional, or unable to migrate appropriately to the needed site (7, 8). This creates a conundrum regarding one’s ability to adequately disinfect the canal space while promoting the survival of native cells, allowing regeneration to proceed. Sodium hypochlorite (NaOCl) has proven to be an effective irrigant because of its ability to effectively disrupt microbial biofilms (9) and dissolve necrotic tissue (10). However, a significant disadvantage of using NaOCl is its toxicity to periodontal ligament (PDL) cells (11) and SCAPs, (7) and the significant pain and morbidity involved when it is extruded beyond the confines of the tooth (12). Chlorhexidine (CHX) is another endodontic irrigant that efficiently elim- inates microorganisms but also negatively impacts PDL (11) and gingival fibroblasts (13). An optimal irrigant for regenerative procedures would disinfect the canal space while not harming human cells in the vicinity (6). It has been shown that 17% EDTA promotes SCAP survival and attachment. In contrast, 2% CHX is detrimental to SCAP sur- vival, and 6% NaOCl + EDTA decreased cell viability when compared with EDTA alone (7). Unfortunately, the use of EDTA alone is not sufficient to consistently produce a dis- infected canal (14). Other intracanal medicaments, such as triple- and double- antibiotic pastes, were both detrimental to SCAP survival, whereas Ca(OH) 2 promoted SCAP survival and proliferation (8, 15). Thus, many root canal medicaments have been demonstrated to be detrimental to SCAP and are therefore not suitable for regenerative endodontics. The ideal endodontic irrigant would eliminate microorganisms but have insignificant or no toxicity to healthy host cells (16). From the *Departments of Endodontics and ‡ Cell Biology and Anatomy, Louisiana State University, School of Dentistry, New Orleans, Louisiana; and † Department of Endodontics, Keesler Air Force Base, Biloxi, Mississippi. Address requests for reprints to Thomas E. Lallier, Louisiana State University, Department of Cell Biology and Anatomy, 1100 Florida Avenue, New Orleans, LA 70119. E-mail address: tlalli@lsuhsc.edu 0099-2399/$ - see front matter Copyright ª 2017 American Association of Endodontists. https://doi.org/10.1016/j.joen.2017.09.001 Significance Regenerative pulp therapy requires host stem cell survival in the root canal. Most endodontic irrigants are highly cytotoxic. Endocyn is less cytotoxic to stem cells of the apical papilla and is better suited for irrigation of teeth with open apexes. Basic Research—Biology JOE — Volume -, Number -, - 2017 Effects of Irrigant Endocyn on SCAP Survival 1