Effects of Calcium Hypochlorite and Octenidine Hydrochloride on L929 And Human Periodontal Ligament Cells

Brazilian Dental Journal (2019) 30(3): 213-219 http://dx.doi.org/10.1590/0103-6440201902280 ISSN 0103-6440 Effects of Calcium Hypochlorite and Octenidine Hydrochloride on L929 And Human Periodontal Ligament Cells Hernán Coaguila-Llerena1 , Elisandra Márcia Rodrigues1 , Mário Tanomaru-Filho1 , Juliane Maria Guerreiro-Tanomaru1 , Gisele Faria1 The aim of this study was to assess cytotoxicity and cell migration of calcium hypochlorite [Ca(OCl)2] and octenidine hydrochloride - OCT (Octenisept®, Schülke & Mayr, Norderstedt, Germany) in L929 and human periodontal ligament (hPDL) cells. The cells were exposed to different doses of different solutions: 2.5% and 5% Ca(OCl)2, 0.1% OCT, 2.5% NaOCl and 2% CHX for 10 min. Cell viability was assessed by methyl-thiazol-tetrazolium (MTT) and neutral red (NR) assays, and cell migration was determined by wound-healing assay. Statistical analysis was performed by two-way ANOVA and Bonferroni tests (α=0.05). The MTT and NR assays revealed that 0.1% OCT was less cytotoxic in hPDL cells (p<0.05), followed by 2% CHX and 2.5% Ca(OCl)2 (p<0.05). There was no significant difference between 2.5% NaOCl and 5% Ca(OCl)2 (p>0.05), but these solutions showed greater cytotoxicity than the others. The result was the same for L929 cells, except that there was no significant difference between 2% CHX and 2.5% Ca(OCl)2 (p>0.05). Wound-healing assay in L929 and hPDL cells showed that cell migration of 0.1% OCT, 2% CHX and 2.5% Ca(OCl)2 groups was higher than 5% Ca(OCl)2 and 2.5% NaOCl groups at 24 h (p<0.05). In conclusion, 0.1% OCT had lower cytotoxicity in tested cell lines than CHX, Ca(OCl)2 and NaOCl. Cell migration was higher for 0.1% OCT, 2% CHX and 2.5% Ca(OCl)2. Therefore, in terms of cytotoxicity, OCT and Ca(OCl)2 have the potential to be used as root canal irrigants. Introduction Current scientific evidence indicates sodium hypochlorite (NaOCl) as the most widely used irrigant solution due to its potent antimicrobial activity (1) and organic dissolution capacity (2). However, NaOCl is cytotoxic at high concentrations (3), and has a pronounced negative effect on the survival and differentiation of stem cells of the apical papilla, factors which may hinder periapical repair and pulpal regeneration (4). CHX is a potent antiseptic used in endodontic treatment due to its antimicrobial efficacy (1) and substantivity (5). However, CHX cannot dissolve organic tissues (6). Currently, there is no root canal irrigant considered ideal, and alternative solutions continue to be studied. Calcium hypochlorite [Ca(OCl)2] has been studied as a root canal irrigant (7,8). It has tissue dissolution capacity (2) and higher chlorine content than NaOCl at the same concentration (9). The preparation of a Ca(OCl)2 solution may be more accurate than that of NaOCl, because Ca(OCl)2 powder can be weighed and incorporated into water prior to use. On the other hand, a NaOCl solution is prepared by diluting a more concentrated and therefore unstable solution, thus making it difficult to obtain an accurate concentration of NaOCl (9). When used as an irrigant solution during biomechanical preparation of Enterococcus 1Department of Restorative Dentistry, Araraquara School of Dentistry, UNESP – Universidade Estadual Paulista, Araraquara, SP, Brazil Correspondence: Gisele Faria, Rua Humaitá, 1680, 14.801903 Araraquara, SP, Brasil. Tel: +55-16-3301-6398. e-mail: Key Words: calcium hypochlorite, cytotoxicity, fibroblasts, octenidine, root canal treatment. faecalis-infected teeth, 2.5% Ca(OCl)2 showed antibacterial efficacy similar to 2.5% NaOCl (7). Regarding cytotoxic effects, Blattes et al. (8) found no difference between Ca(OCl)2 and NaOCl in 3T3 embryonic mouse fibroblast cells. Octenisept® - OCT (Schülke & Mayr, Norderstedt, Germany) contains 0.1% octenidine hydrochloride (an antimicrobial agent) and 2% phenoxyethanol, a derivative of ethanol, which serves as a preservative (10). OCT is used primarily for antisepsis of burns and wounds, and as a mouthwash (10). As a root canal irrigant, OCT has showed similar efficacy to 2.5% and 5.25% NaOCl, and to 2% CHX against Candida albicans (11) and E. faecalis (12). Regarding cytotoxic effects, another 0.1% octenidine hydrochloridebased antiseptic, Octenidol®, has presented cytotoxicity lower than 0.2% CHX in human gingival fibroblasts and nasal epithelial cells (13). Therefore, OCT and Ca(OCl)2 have the potential to be used as alternative root canal irrigants to NaOCl and CHX. However, further studies comparing the cytotoxicity of OCT and Ca(OCl)2 with that of other root canal irrigants in different cell lines are needed to indicate these solutions for endodontic treatment. The aim of this study was to assess effects of OCT and Ca(OCI)2, in comparison with NaOCl and CHX, on viability and proliferation/migration of human periodontal ligament (hPDL) cells and L929 fibroblasts. The Braz Dent J 30(3) 2019 null hypothesis was that there would be no difference in effects of solutions on viability and proliferation/migration of the cells tested. Material and Methods H. Coaguila-Llerena et al. Preparation of Irrigant Solutions The solutions evaluated were 2.5% and 5% Ca(OCl)2 (Sigma-Aldrich, St. Louis, MO, USA), 0.1% OCT (Octenisept®, Schulke & Mayr), 2.5% NaOCl (AraQuímica, Araraquara, SP, Brazil) and 2% CHX (Reactive Manipulation Pharmacy, Araraquara, SP, Brazil). Ca(OCl)2 solution was prepared immediately prior to use by diluting Ca(OCl)2 powder in distilled water, and 2.5% NaOCl was prepared by diluting 9% NaOCl solution in distilled water. The available chlorine content in NaOCl and in Ca(OCl)2 solutions was determined by the physicochemical spectrophotometric method (14). The Ca(OCl)2 concentrations of 2.5% and 5%, as well as 0.1% OCT, 2.5% NaOCl and 2% CHX, were considered grade 1 dilutions, and were serially diluted in saline solution (0.9% sodium chloride) using a 1.5 dilution factor (15). The cells were incubated with solutions in the following dilutions: 1/111, 1/166, 1/250, 1/375, 1/562, 1/844, 1/1266 and 1/1898, which corresponded to doses/concentrations of 0.9%, 0.6%, 0.4%, 0.26%, 0.18%, 0.12%, 0.08% and 0.05%, respectively. Cell Culture and Treatment Protocol with the Irrigant Solutions Permanent cell lines of L929 mouse fibroblasts (American Type Culture Collection) and human hPDL cells were used. All procedures conformed to the applicable ethical guidelines and regulations of the dental school’s Research Ethics Committee which approved the project, with written informed consent obtained from all subjects. Human third molars with no evidence of carious lesions or periodontal disease were obtained from healthy patients aged 16-25 year, who were being treated at the dental school’s surgery clinic. After extraction, the teeth were immediately stored in Dulbecco’s Modified Eagle’s Medium - DMEM (Sigma-Aldrich). The periodontal ligament was removed from the middle third of the root surface with a #15 scalpel blade, a (...truncated)