Cytotoxicity evaluation of calcium hypochlorite and other commonly used root canal irrigants against human gingival fibroblast cells: An in vitro evaluation

Background. The conventional endodontic therapy primarily focuses on biomechanical preparation, which is achieved by the application of various intracanal irrigants and intracanal medicaments. One of the most commonly used intracanal irrigants – sodium hypochlorite (NaOCl) – has already been proven to have an antimicrobial effect as well as the ability to dissolve tissues in the areas where files cannot reach. One of the recently used irrigants having a promising effect is calcium hypochlorite (Ca(OCl)2), which has been shown to be relatively more stable than NaOCl and has much more chlorine ions.


Introduction
The typical aim in the endodontic therapy is to perform the optimal disinfection of the root canal system, after which a suitable filling material is placed in this disin fected space, preventing any further ingress of microorga nisms leading to further complications. 1 It is well known that microorganisms are a critical factor in the develop ment of pulp and periapical diseases, and have an influ ence on the prognosis of the endodontic therapy in the long run. 2,3 Currently, along with mechanical instrumen tation, various adjuvants, such as intracanal irrigants and intracanal medicaments are used, which helps to signifi cantly reduce the amount of microorganisms. Due to the complexity of the root canal anatomy, intracanal irrigants are of great importance in the endodontic procedure, since they have the ability to get into the sites where me chanical instrumentation fails to reach. 4 Various intracanal irrigants have been used in the con ventional endodontic therapy in the past decades in order to help the clinician to completely eradicate microbes, necrotic tissues and debris from the root canal system, the most common being chlorhexidine (CHX) and so dium hypochlorite (NaOCl), which have been shown to have an antimicrobial effect, the ability to inactivate endo toxins as well as the ability to remove some part of the smear layer and necrotic tissue remnants. 5 Sodium hypochlorite, one of the most common intracanal irrigants, also shows antimicrobial activity and is able to dissolve pulpal tissue remnants in spaces such as fins and ramifications, where instrumentation cannot reach. 6 The capability of NaOCl to dissolve pulpal tissue results from its alkaline nature as well as from the release of HOCl − and OCl − ions. 7 Sodium hypochlorite is known to show certain draw backs, such as the lack of substantivity, the ability to dis solve only the organic portions of the smear layer and a cytotoxic effect when extruded into the periapical re gion. One of the ways to overcome these drawbacks is to use it in combination with ethylenediaminetetraacetic acid (EDTA), which reduces the pH of NaOCl in a time dependent manner as well as the amount of the available free chlorine, making NaOCl more stable and preventing the weakening of the dentinal matrix. 8 One of the recently introduced intracanal irrigants of a promising potential is calcium hypochlorite (Ca(OCl) 2 ), which has shown an anti microbial effect and at the same time a much higher chlorine release in comparison with NaOCl. 9 These irrig ants have been reported to affect various cells present in the periapical complex. Although numerous assessments of their cytotoxicity against multiple cell lines have been performed, there is no data available in the dental litera ture on the cytotoxic effect of these irrigants on human gingival fibroblast (hGF) cells. The current study aimed to evaluate the individual cytotoxic activity of various root canal irrigants, and the combined cytotoxicity of NaOCl and Ca(OCl) 2 with EDTA against hGF cells.

Material and methods
This in vitro study had received prior approval of the insti tutional review board (No. SRB/SDC/ENDO1805/20/04).

Preparation of the cell line
This in vitro study was conducted under aseptic con ditions in a research laboratory. The hGF cell lines were received from the National Centre for Cell Science, Pune, India. The hGF cells were seeded in the αminimal essen tial medium (αMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 2.5 μg/mL streptomycin, 2.5 μg/mL amphotericin B, and 50 μg/mL ascorbic acid, which was replaced twice a week, and incubated in 5% CO 2 at 37°C for humidification.

Preparation of calcium hypochlorite and other irrigants
The CaOH 2 powder of 65% purity (Merck, Darmstadt, Germany) was weighed on a precision balance and mixed with distilled and sterilized water. When the solutions were completely dissolved, they were filtered twice to remove debris, and then stored in bottles. Other chemicals used in the study were as follows: NaOCl (Molychem, Mumbai, India); EDTA (Thermo Fisher Scientific, Waltham, USA); and CHX (SigmaAldrich, St. Louis, USA).

Cytotoxicity test with the sulforhodamine B assay
The cytotoxicity test was performed using a modifi cation of the method developed by Vichai and Kirti kara. 10 The assessment was carried out within a time frame of 6 h and 24 h, with each concentration being tested in triplicate. The microtiter plates were incubat ed with Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, Grand Island, USA) supplemented with 10% FBS, 100 μL/well of the culture medium, for 24 h at 37°C with 5% CO 2 . After 24 h of incuba tion, cell adhesion and growth were observed. The cel lular concentration used was 1.4 × 104 cells/mL. The medium was then discarded, and the treatment group samples, controls and DMEM without FBS were added to each well. After 24 h of incubation, the cells were fixed with trichloroacetic acid (TCA) (100 μL/well) and placed under refrigeration for 1 h at 4°C. Trichloro acetic acid was then removed, and the plates were washed in lowflow water 3 times and dried. After that, the plates were stained for 20 min at room temperature with sulforhodamine B (SRB) (SigmaAldrich) 0.4% (50 μL/well). The plates were washed with 1% acetic acid solution and dried at room temperature. The un bound dye was removed after washing; the dye bound to the protein was solubilized in the basic medium to determine the optical density in a plate reader set at 570 nm. The colorimetric evaluation gives an estimate of total protein mass. The cellular concentration used was 1.4 × 104 cells/ml. The percentage of living cells for each concentration of the tested substances was as sessed with the following equation (Equation 1): (1) where: AbsT -average absorbance of the tested substances; AbsC -average absorbance of the control.

Statistical analysis
The statistical analysis was carried out using IBM SPSS for Windows, v. 21.0 (IBM Corp., Armonk, USA) The oneway analysis of variance (ANOVA) was applied to as sess differences in absorbance at 570 nm at different time intervals between the treatment groups, posthoc Tukey's test was used for the intergroup comparison of absor bance between the test groups; the independent t test was done to assess cell viability at different time points between the groups. The level of significance was set at p < 0.05.

Results
The experiments were carried out in triplicate. The re sults of the assessment of cytotoxicity against hGF cells are shown in a dosedependent and timedependent man ner, with a total timeframe of 6 h and 24 h. They are pre sented as mean (M) ± standard deviation (SD). Table 1 shows the absorbance values for different root canal irrigants at different concentrations at 6 h and 24 h. Table 2 presents the percentage of cell viability for different root ca nal irrigants at different concentrations at 6 h and 24 h. The findings of the study suggest that CHX, NaOCl and Ca(OCl) 2 had an effect on hGF cells (p = 0.001). Figure 1 shows the dosedependent effect of various root canal irrigants on hGF cells at 6 h. The intergroup analysis of the rate of absorbance at the 0.025% concentration showed statistically significant differences in various combinations: NaOCl vs Ca(OCl) 2 (p = 0.002); NaOCl vs negative control (NC) (p = 0.001); Ca(OCl) 2 vs CHX (p = 0.013); and CHX vs NC (p = 0.001).   Figure 2 shows the dosedependent effect of various root canal irrigants on hGF cells at 24 h. The compari son of the absorbance of various irrigants at 0.025% showed statistically significant differences for the fol lowing: NaOCl vs Ca(OCl) 2 (p = 0.004); NaOCl vs NC (p = 0.003); Ca(OCl) 2 vs CHX (p = 0.004), Ca(OCl) 2 vs NC (p = 0.003), Ca(OCl) 2 vs NC (p = 0.008), and CHX vs NC (p = 0.005). At the 0.050% concentration, the com parison of absorbance showed statistically significant differences for NaOCl vs Ca(OCl) 2 (p = 0.002), NaOCl vs NC (p = 0.001), Ca(OCl) 2 vs CHX (p = 0.001), and CHX vs NC (p = 0.001). At the 0.10% concentration, the intergroup comparison of absorbance showed sta tistically significant differences for NaOCl vs Ca(OCl) 2 (p = 0.001), NaOCl vs NC (p = 0.001), Ca(OCl) 2 vs CHX (p = 0.001), and CHX vs NC (p = 0.001). In the case of the 0.20% concentration, the intergroup absorbance rate was observed to be statistically significantly differ ent for NaOCl vs Ca(OCl) 2 (p = 0.008), NaOCl vs NC (p = 0.001), Ca(OCl) 2 vs CHX (p = 0.008), Ca(OCl) 2 vs NC (p = 0.006), and CHX vs NC (p = 0.001). Table 3 shows the percentage of cell viability for hGF cells post reaction with NaOCl/EDTA and Ca(OCl) 2 /EDTA at different concentrations at 6 h and 24 h. Table 4 presents the final concentrations of NaOCl and Ca(OCl) 2 after serial dilution with EDTA at 6 h and 24 h. Figure 3 depicts the combined cyto toxicity against hGF cells at 6 h of the combinations of NaOCl/EDTA and Ca(OCl) 2 /EDTA. The comparison of cell viability done at 6 h showed statistically significant difference between NaOCl/EDTA and Ca(OCl) 2 /EDTA at 0.025% (p = 0.001), 0.050% (p = 0.001), 0.10% (p = 0.001), and 0.20% (p = 0.001). Figure 4 shows the combined cyto toxicity against hGF cells at 24 h for the combinations of NaOCl/EDTA and Ca(OCl) 2 /EDTA. The comparison of cell viability done at 24 h showed statistically significant difference between NaOCl/EDTA and Ca(OCl) 2 /EDTA at 0.025% (p = 0.019), 0.050% (p = 0.007), 0.10% (p = 0.005), and 0.20% (p = 0.029).

Discussion
Root canal irrigants have a pivotal role in the success of the endodontic therapy. It is well known that they parti cipate in removing necrotic tissue and debris from fins and ramifications, where mechanical instrumentation cannot reach. Still, there is a risk that during the endodontic pro cedure, intracanal irrigants will be extruded periapically. 11 This can cause damage to cells and also impair wound heal ing. 12 Another potential effect of the extrusion of irrigants is that they may impair the survival of stem cells, which are required for successful tissue regeneration. 13 Human gingival fibroblast cells are reported to have a much higher reparative potential than human periodon tal ligament fibroblasts (hPDLF). 14 Though hPDLF cells could simulate periodontal ligament fibroblasts better, they are known to produce a higher amount of collagen than hGF cells, and could potentially influence the results of cytotoxicity for the materials tested. 15 Apart from hav ing great regenerative properties as compared to other fi broblasts, hGF cells also have the multilineage capability to differentiate into different types of cells, such as osteoblasts and periodontal ligament cells, which can be potentially used for periodontal tissue engineering. 16 The pre sence of lipopolysaccharides in endodontic pathogens has an immunosuppressive effect on the human pulp, leading to pulpal pain and periapical inflammation. 17 Unlike other cells present in the periodontium, hGF cells do not express lipopolysaccharide tolerance and have been shown to release inflammatory cytokines for up to 7 days, which makes them the least susceptibile to microbial action. 18 As compared to other standard cell lines, hGF cells are diploid host cells, continuous in nature, with the special ized properties retained; they are more likely to demon strate the cytotoxic effect exerted by dental materials on cells than other commercially available cell lines, which are aneuploid in nature, showing a heteroploid chromosome pattern and responding differently to different materials. 19 The use of commercially available standardized cell lines has an advantage that they are easily accessed, but can demonstrate greater toxicity levels and show an altered effect of the use of dental materials as compared to specific lineage cell lines, which are more desirable, as they bridge the gap between in vitro and in vivo biocompatibility. 20 Various studies have already been done in the field of endodontics, using hGF cells. Barnhart et al. assessed the cytotoxicity of various intracanal irrigants with the CyQUANT ® assay and found potassium iodide and calcium hydroxide to be the least cytotoxic against hGF cells. 21 Lee et al. also evaluated the cytotoxicity of various dental adhesives against hGF cells and found that all of the tested adhesives had a cytotoxic effect on hGF cells. 22 In the present study, the SRB assay was used for the cytotoxicity assessment, as it has been shown to have a higher sensitivity; the cell viability count is not affected by any compounds released from the specimen, is inde pendent of any cellular metabolic action, which makes the assay much easier to conduct in comparison with other assay methods. 23 Calcium hypochlorite is another intracanal irrigant in troduced in endodontics which has been shown to have a good antimicrobial activity. [24][25][26] Though it is very simi lar to NaOCl in terms of chemical action, the presence of more chlorine (Cl − ) in Ca(OCl) 2 results in more pro nounced antimicrobial action and a greater tissue dissolv ing capability as compared to NaOCl. 27 The current study showed the least cytotoxic effect in the case of CHX. It is known that CHX has a sustained antimicrobial activity as well as low toxicity, but its major disadvantage is its in ability to dissolve organic pulp tissues. 28 The present study showed that Ca(OCl) 2 maintained the viability of cells when tested within a timeframe of 6 h and 24 h. In contrast, NaOCl was proven to decrease the levels of viable hGF cells after 24 h even further. In an in vitro study, CoaguilaLlerena et al. found that differences in cyto toxicity against L929 and hPDLF cells between Ca(OCl) 2 and 2% CHX were statistically insignificant; also, the cel lular proliferation capacity was maintained when Ca(OCl) 2 at a concentration of 2.5% was used. 29 In another study, Ferraz Blattes et al. conducted an in vitro cytotoxicity as sessment against 3T3 fibroblasts and an analysis of the inflammatory reaction in rats; the authors reported that Ca(OCl) 2 showed lower levels of inflammation, better healing response as well as lower cytotoxicity as com pared to NaOCl. 30 The combined use of NaOCl with EDTA is the recom mended protocol to be followed according to the American Association of Endodontists (AAE) guidelines and was the basis for evaluating the combined cytotoxic effect in our study. It is known that NaOCl/EDTA acts on the organic/ inorganic portion of the smear layer present in dentin and shows a synergistic effect by chelating Ca 2+ ions, causing col lagen deproteinization, which results in a superficial change in dentin, preventing further microbial colonization. 31 The present study also showed that cytotoxicity was reduced to a greater extent with the addition of EDTA. Our results are consistent with those obtained by Vouzara et al., who proved that the application of the combination of NaOCl and EDTA reduced the cytotoxic effect against MRC5 cells. 32 The re sults obtained in our study could be due to an antagoniz ing effect of EDTA on NaOCl, which plays a critical role in reducing its pH in a timedependent manner as well as de creasing exponentially the amount of the released chlorine gas. 33 Due to the chemically similar nature, the same action could be justified in the case of Ca(OCl) 2 as well.

Limitations
One of the limitations of the present study is not using an in vivo model to assess the cytotoxic reaction, since an in vitro model is an ideally conditioned environment and may not necessarily transmit the achieved results in vivo. Another limitation is probably the use of lower con centrations of irrigants, which were diluted further to dif ferent levels for assessment standardization and may not necessarily show the same result when used in their clini cally recommended concentrations.

Conclusions
Based on the present study, it can be concluded that Ca(OCl) 2 showed less cytotoxicity in a timedependent and dosedependent manner than NaOCl, but was more cytotoxic against hGF cells in comparison with chlorhexi dine gluconate. The combined use of Ca(OCl) 2 and NaOCl with EDTA showed a more significant reduction of cyto toxicity against hGF cells. In contrast, the sustainability of hGF cells after 24 h was much greater in the case of using Ca(OCl) 2 as compared to NaOCl.