The null hypothesis was rejected. Ozonated water induced proliferation of primary dental pulp cells and proliferation rate was time and concentration- dependent.
The role of infection and inflammation is a challenge for regenerative endodontics [34]. The key to the successful regenerative endodontic therapy is the eradication of microorganisms from the root canal system effectively [6, 35]. NaOCl which has a strong antibacterial efficiency, is used in the current regenerative endodontic protocols of AAE and ESE in low concentrations [13, 14]. Although NaOCl has a considerable antibacterial effect it is also highly toxic on SCAPs. Moreover, previous studies have shown that necrotic immature teeth may contain vital pulp cells as well which also could be affected by the toxicity of irrigating agents [36, 37]. Regarding the side-effects of NaOCl, the wide open apex and/or resorbed apex has been described as a condition where ozone can be an adjuvant irrigant to NaOCl [38].
In a previous study, effects of both gaseous ozone and ozonated water on human oral epithelial cells have been discussed. The results of the study have revealed that ozonated water is more biocompatible than gaseous ozone [33]. For this reason, ozonated water was involved instead of gaseous ozone in the present study.
In recent years, more studies have been made on the antimicrobial efficiency of ozonated water. Previous studies have proven the antimicrobial efficacy of OW, however, authors have failed to agree on an exact dose to be used more sufficiently as an irrigating agent [26,27,28,29, 31, 39, 40]. Nagayoshi et al. [27] concluded that the antimicrobial efficacy of ozonated water between the concentrations of 0.5–4 mg/L was highly effective and rapid on killing both gram-positive and gram-negative bacteria. Nogales et al. [31] have considered antimicrobial activity of ozonated water in concentrations of 2, 5 and 8 mg/L. Cardoso et al. [28], evaluated the effect of 24 mg/L OW against E. faecalis and endotoxins in root canals. Considering the proven doses that have antibacterial efficiency in these studies, 2 mg/L, 4 mg/L, 8 mg/L, and 16 mg/L OW have been used in the present study.
Although the antibacterial effect of ozone therapy has proven, cytotoxic or/and proliferative effect of ozonated water is rarely discussed [26, 31,32,33]. Therefore, the present study aimed to assess the biological response of dental primary pulp cells to various doses of ozonated water by using MTT assay.
MTT assay is one of the most commonly used colorimetric assays to evaluate cytotoxicity [41] and it simply measures cytotoxicity based on the mitochondrial activity of cells. It is easy to use, has a high reproducibility, and it is widely used to determine both cell viability and cytotoxicity [42].
An adequate cell line selection is an important issue for cytotoxicity assessment. Permanent cell lines, standard cell lines and primary cells collected from gingiva, periodontium or pulp are the recommended alternatives. Primary cell line were used in this study which has better ability to represent clinical conditions[43].
The results of the present study revealed that ozonated water was biocompatible in each concentration, and also the proliferation rate of dental primary pulp cells was induced. Ozonated water has distinctive properties such as high oxidizing power and increased intracellular metabolic activity. Thus, high proliferation rate of dental primary pulp cells can be associated with these properties. Although the mean percentage value of viable cells decreased for 16 mg/L OW and 8 mg/L OW at 48 h time point, both groups cannot be considered as toxic because the viable cell percentages were above 70 [44].
Nagayoshi et al. [26] observed that 4 mg/L OW is biocompatible although there was not a statistically significant difference when compared to mean OD values of distilled water. Our results are in agreement with the mentioned study but yet there are differences in methodology. While the authors examined only one concentration of ozonated water, present study includes a wide range of ozonated water in four different concentrations. We have standardized the results by converting the OD values into viable cell percentages, however, Nagayoshi et al. [26] declared results in mean OD values. For this reason, we are not able to compare our cell viability results in percentages. In addition, contact time of the cells with irrigating agents was set as 2 min in the previous study but in the current study the contact time was set as 5 min and the MTT was applied in two separate time points. Required irrigation time for regenerative endodontic protocol for NaOCl was taken in consideration while determining 5 min as the contact time with cells and irrigating agents in our study [13]. Nagayoshi et al. [26] did not mention any induced proliferation by ozonated water at contact time but our results showed that there is a highly induced proliferation in every concentration of ozonated water at 0 h time point which is statistically significant when compared to positive control group except Group 3 (p < 0.05). In another point of view, induced proliferation in the experimental groups may be associated with a high proliferation rate of dental primary pulp cells.
The experimental design of Nogales et al. [31] was almost similar to the present study. The authors included 2 mg/L, 5 mg/L, and 8 mg/L OW concentrations in this study. According to this study, there is an increase in mean percentage values of viable cells over time and proliferation did not occur at 0 h time point of all concentrations of ozonated water. Moreover, at 48 h time point, Nogales et al. [31] pointed that the highest proliferation rate was achieved by the 8 mg/l OW which is the highest concentration used in the study and the only cytotoxic concentration at 0 h time point with the mean percentage value of 68.6.
In the present study, 2 mg/L OW gave the most stable results between two time points, which means that there was not a significant change in cell viability over time. When we compare our result with Nogales et al. [31], it can be observed that the 2 mg/L OW is the most stable ozonated water concentration among all time points as well, no dramatically high increase was observed. However, cell viability percentage of 2 mg/L OW in our study is higher than the previous study. This can be attributed to higher proliferation rate of our dental primary pulp cells.
In another study, cytotoxicity of OW with concentrations of 5 mg/L, 10 mg/L, and 20 mg/L were tested by using MTT assay at 5, 10 and 15-min time points of interaction with stem cells from human exfoliated deciduous teeth (hSHEDs). The study revealed that the 20 mg/L OW at 5 and 10-min time points showed the highest proliferation rate whereas the 5 mg/L ozonated water showed the highest proliferation rate at the 15-min time point [32]. The authors evaluated the cytotoxicity by using MTT assay and the cell viability results were given in percentages. Although hSHEDs which have high capacity of proliferation were preferred in their study, the proliferation rate was not as high as the proliferation rate in our results. This may be due to the cells’ going through a cryopreservation procedure before the experiment or to the contact time which differs from our methodology.
Considering the results of this study 2 mg/L OW is recommendable as a possible adjuvant irrigating agent for the regenerative endodontic procedures. Due to the biocompatibility of OW, in addition to a sufficient disinfection, it provides an environment which supports the tissue engineering strategies and achievement of a success in pulp repair or pulp regeneration. However, further studies are needed to evaluate the biological response and odontoblastic differentiation mechanism of SCAPs to ozonated water. Future studies should also focus on interactions between ozonated water and common irrigating solutions such as NaOCl, ethylenediamine tetraacetic acid and citric acid. In addition, for the evaluation of recovery ability of the cells, a long-term experimental period can be planned, different cytotoxicity evaluation methods can be preferred and number of the patients can be increased to have a cell proof for further studies.