Abstract
Background:
Activated inflammatory cells produce reactive oxygen species (ROS) to eliminate pathogens. Under normal conditions, the pathogens are taken care of, and tissues are repaired. However, in periodontal disease, persistent inflammation causes increased ROS release and impaired healing. Therefore, removal of overproduced ROS using antioxidants is necessary. Hydrogen water has an antioxidative effect on cells and impedes oxidative stress-related disorders.
Aim:
To study the effect of hydrogen water on cell viability, migration, and its antioxidative potential in fibroblasts obtained from chronic periodontitis patients.
Materials and Methods:
The gingival tissue samples were obtained from 26 subjects (13 periodontally healthy individuals and 13 chronic periodontitis patients) and processed. The human gingival fibroblasts were cultured and the assays were commenced once adequate growth was detected. The effect of hydrogen water on cell viability was checked by neutral red assay, while the migration potential was assessed by transwell migration assay. The antioxidative potential of hydrogen water was evaluated by CUPRAC assay.
Statistical Analysis:
Intergroup comparison was done using Mann–Whitney U-test. Intragroup comparison was done using Wilcoxon signed-rank test.
Results:
Hydrogen water was nontoxic to the fibroblasts at 24 h and 48 h. The intergroup comparison of the cell viability between hydrogen water-treated periodontally healthy gingival fibroblasts (HF) and fibroblasts from patients with chronic periodontitis (CF) showed a statistically significant (P = 0.00) difference at 24 h and 48 h. Hydrogen water also positively influenced the migratory capacity. Hydrogen water-treated fibroblasts obtained from chronic periodontitis patients showed more migration in comparison to the healthy group (P = 0.00). Hydrogen water showed an antioxidative potential. The maximum potential was seen in relation to the fibroblasts obtained from chronic periodontitis patients at 48 h.
Conclusion:
Hydrogen water was nontoxic, increased the migratory capacity, and showed an antioxidative potential on human fibroblasts obtained from periodontally healthy individuals and patients with chronic periodontitis.
Keywords: Antioxidants, cell migration assays, cell survival, chronic periodontitis, fibroblasts
INTRODUCTION
Reactive oxygen species (ROS) include all reactive forms of oxygen, including both the radical and nonradical species.[1] These species are generated by exogenous or endogenous sources. Endogenously, host defense cells produce ROS to eliminate the pathogens. Under normal circumstances, the pathogens are taken care of, and the tissue can be repaired.[2] However, in chronic inflammatory diseases, like periodontal disease,[3] low-grade inflammation causes persistent activation of inflammatory cells, resulting in a high degree of ROS release. Overproduced ROS deplete endogenous antioxidants, increase oxidative damage, and impair healing.
Antioxidants can be used to improve tissue repair in chronic inflammatory conditions.[4] Various antioxidative agents, in different forms, are available. To have an agent which is naturally occurring and can be consumed on daily basis is desirable. One such agent is “hydrogen water.”
Hydrogen water has an antioxidative effect on cells and tissues and plays a beneficial role in preventing oxidative stress-related disorders. It also diminishes physical injury-induced ROS generation and promotes healing.[5]
Taking into account the above applications, we conducted a study to assess the effect of hydrogen water on gingival fibroblasts obtained from periodontally healthy individuals and those with chronic periodontitis. We aimed to assess the effect of hydrogen water with respect to cell viability, migratory potential, and antioxidative potential.
MATERIALS AND METHODS
Subjects for the study were selected from patients visiting the Department of Periodontology and Oral and Maxillofacial Surgery, in the Institute. The study commenced after obtaining approval from the institutional review board. The nature and purpose of the study were explained to the subjects, and written informed consent was obtained. A total of 26 subjects (13 periodontally healthy patients and 13 patients with chronic periodontitis) in the age group of 18–50 years were included in the study. Based on probing pocket depth, clinical attachment level, and gingival index, patients were categorized into two groups.
Group 1: Periodontally healthy individuals
Group 2: Patients with chronic periodontitis.
Generation of hydrogen water was done using a commercially available hydrogen water bottle (Hydrocare Plastic and Tritan Water Bottle with USB Charger, 500 ml-Hydride hydropower generator, Gdh pure). The base of the bottle has electrodes, which helps in production of hydrogen water. Distilled water was infused in the bottle. The process of electrolysis was initiated after the power of the bottle was in an ON mode. The electrolysis of water generated hydrogen (H2) at the cathode, thus producing hydrogen water in the bottle at the end of 5 min.
Patients with probing pocket depth <3 mm, no bleeding on probing, and no clinical attachment loss were included in Group 1. Patients with the presence of bleeding on probing, probing pocket depth >3 mm, and clinical attachment level ≥5 mm were included in Group 2. Pregnant and lactating women, patients who received any medications 3 months before sampling, patients with cancerous or precancerous lesion, patients with medically compromised conditions, and patients with tobacco habitués were excluded from the study.
For Group 1, the gingival tissue was harvested from the patients undergoing crown lengthening procedure, while for Group 2, the gingival tissue specimen was harvested from the tooth indicated for extraction due to periodontitis. The tissue was then transported in Dulbecco’s modified Eagle’s media and the specimens were processed.
The tissue samples were minced and transferred to a 24 well microtiter plate containing Dulbecco’s modified eagle medium (DMEM) supplemented with fetal calf serum, penicillin, streptomycin, fungizone, and nonessential amino acids and incubated at 37°C in a humidified atmosphere, containing 5% carbon dioxide. Media replacement was done every 2 days. The assays were commenced once adequate growth of fibroblasts was detected under the microscope.
The effect of hydrogen water on cell viability was checked by neutral red assay. Human gingival fibroblasts were treated with DMEM + hydrogen water (50-50) for a total of 48 h. Medium replacement was done after the first 24 h and then 2 h before the assay. 150 µL of neutral red working solution was added. The cells were then incubated for 2 h. Postincubation, the working solution was removed and cells were washed with phosphate buffer saline. Desorb solution (150 µL) was added next and the cells were incubated again for 30 min. The optical density (OD) values were recorded after 24 h and 48 h using an enzyme-linked immunosorbent assay Reader at 492 nm and 630 nm. The assay was also done for the control groups, where positive control consisted of human gingival fibroblasts treated only with DMEM, while the negative control group had fibroblasts treated with DMEM + distilled water (50-50). The mean cell viability was then calculated.
Mean cell viability (percentage) = {Mean of OD (Fibroblasts treated with hydrogen water)/Mean of OD (Fibroblasts treated with only DMEM)} ×100.
The influence of hydrogen water on the migratory capacity was checked by transwell migration assay. Fibroblasts growing in the microtiter plates, were mechanically detached. The detached cells were then transferred to transwell migration assay kit (HIMEDIA Cell culture insert). 800 microliters of media including the detached fibroblast cells was added to the upper chamber of the kit. 2.4 ml of hydrogen water/distilled water was added below the membrane in the wells for the test group and the negative control group, respectively. The assembly was kept overnight in an incubator at 37°C. The migrated cells in the lower chamber were then fixed with 70% ethanol for 10 min. The fixed cells were dried and crystal violet stain was added. The migrated stained cells were then counted under the microscope.
CUPRAC assay was used to assess the antioxidative potential of hydrogen water. The assay was done using fibroblast cell lysate. The culture well plate was labeled with different concentrations of standard, sample, and blank. 100 µL deionized water was added to each well. Then, 10 µL sample (hydrogen water-treated fibroblast lysate) was added to the sample well, the standard well contained 10 µL of standard, and the blank well had only deionized water. The chromogenic substrate was then added to each well and the assembly was inoculated for 10 min in dark at room temperature. The absorbance was measured at 450 nm wavelength using a microplate reader at 24 h and 48 h.
Statistical procedure
The data were subjected to statistical analysis using Statistical Package for the Social Sciences (SPSS v 21.0, IBM, Armonk, NY, USA). Normality of numerical data was checked using Shapiro–Wilk test and it was found that the data did not follow a normal curve; hence, nonparametric tests were used. Intergroup comparison was done using Mann–Whitney U-test. For all the statistical tests, P < 0.05 was considered to be statistically significant, keeping α error at 5% and β error at 20%, to achieve a study power of 80%.
RESULTS
The mean cell viability of the treated fibroblasts is depicted in Table 1. The hydrogen water-treated fibroblasts obtained from periodontally healthy patients had a mean cell viability of 87% after 24 h and 88.6% after 48 h. The gingival fibroblasts which were obtained from patients with chronic periodontitis, after treatment with hydrogen water, showed the mean viability of 80% after 24 h and 73% after 48 h. The fibroblasts treated with distilled water showed condensation and shrinkage, indicating the cell death.
Table 1.
Intergroup comparison of the mean cell viability percentage for hydrogen water-treated fibroblasts obtained from periodontally healthy individuals and patients with chronic periodontitis after 24 h and 48 h
| Group | Mean cell viability (%)* | |
|---|---|---|
|
| ||
| 24 h | 48 h | |
| Hydrogen water-treated periodontally HF | 87 | 88.6 |
| Hydrogen water-treated fibroblasts from chronic periodontitis patients (CF) | 80 | 73 |
*Considering the positive control has 100% viability. HF-Healthy fibroblasts; CF – Fibroblasts from patients with chronic periodontitis
Table 2 depicts the intergroup comparison of the cell viability between hydrogen water-treated periodontally healthy gingival fibroblasts (HF) and fibroblasts from patients with chronic periodontitis (CF). A statistically significant (P < 0.05) difference was seen in the values between the two groups at 24 h and 48 h. The absorbance values (nm) were more in the CF group (0.292 nm) as compared to the HF group (0.254 nm) at 24 h, while higher values were seen in HF group (0.435 nm) as compared to CF group (0.276 nm) at 48 h.
Table 2.
Intergroup comparison of cell viability between hydrogen water-treated periodontally healthy fibroblasts and chronic periodontitis fibroblasts after 24 h and 48 h
| Groups | n | Mean | SD | Median | Mann-Whitney-U value | Z | P* | |
|---|---|---|---|---|---|---|---|---|
| Absorbance (nm) at 24 h | Hydrogen water-treated periodontally HF | 13 | 0.256 | 0.013 | 0.254 | 0.000 | −4.337 | 0.000 |
| Hydrogen water-treated fibroblasts from chronic periodontitis patients (CF) | 13 | 0.290 | 0.005 | 0.292 | ||||
| Absorbance (nm) at 48 h | Hydrogen water-treated periodontally HF | 13 | 0.434 | 0.008 | 0.435 | 0.000 | −4.345 | 0.000 |
| Hydrogen water-treated fibroblasts from chronic periodontitis patients (CF) | 13 | 0.275 | 0.004 | 0.276 |
*Statistically highly significant difference (P<0.01). n – Number of samples; Z – Number of standard deviations from mean; P – Level of significance; SD – Standard deviation; nm – Nanometers; HF – Healthy fibroblasts; CF – Fibroblasts from patients with chronic periodontitis
Table 3 represents migration potential of hydrogen water-treated fibroblasts. There was a statistically significant difference in the values between the groups (P < 0.05) with the highest migration potential value in the positive control group followed by hydrogen water-treated chronic periodontitis group fibroblasts, negative control and least for hydrogen water-treated periodontally healthy fibroblasts.
Table 3.
Intergroup comparison of migration potential of hydrogen water-treated fibroblasts
| Groups | n | Mean | SD | Median | Mean rank | χ 2 | P* |
|---|---|---|---|---|---|---|---|
| Positive control (media+serum) | 10 | 72.70 | 12.81 | 70 | 35.50 | 36.848 | 0.000 |
| Hydrogen water-treated periodontally HF | 10 | 1.30 | 0.67 | 1 | 5.50 | ||
| Hydrogen water-treated fibroblasts from chronic periodontitis patients (CF) | 10 | 33.30 | 3.33 | 34 | 25.50 | ||
| Negative control (distilled water) | 10 | 4.10 | 0.73 | 4 | 15.50 |
*One-way ANOVA; Statistically highly significant difference (P<0.01). n – Number of samples; SD – Standard deviation; P – Level of significance; HF – Healthy fibroblasts; CF – Fibroblasts from patients with chronic periodontitis; χ2 – Chi square value
The intergroup comparison of the antioxidative potential of hydrogen water is represented in Table 4. Maximum antioxidative potential of hydrogen water was seen in relation to the fibroblasts obtained from chronic periodontitis patients at 48 h.
Table 4.
Intergroup comparison of antioxidative potential of hydrogen water
| Hydrogen water-treated periodontally HF | Hydrogen water-treated fibroblasts from chronic periodontitis patients (CF) | |||
|---|---|---|---|---|
|
|
|
|||
| 24 h | 48 h | 24 h | 48 h | |
| OD values (nm) | 0.278 | 0.327 | 0.286 | 0.697 |
| Results (µM) | 58.0 | 68.31 | 59.74 | 205.8 |
OD-Optical density; nm-Nanometers; µM-Micromolar; HF-Healthy fibroblasts; CF – Fibroblasts from patients with chronic periodontitis
DISCUSSION
Periodontal disease is characterized by high production of ROS, leading to oxidative stress.[3] Endogenous antioxidants by themselves cannot attenuate the excessively produced ROS. Therefore, in this condition, there is a need for administering exogenous antioxidants so as to improve tissue repair. Numerous antioxidants are available. Hydrogen water is one such naturally available agent.
Huang et al.[6] studied the about the use of hydrogen as a therapeutic agent. Hydrogen was shown to have antioxidative, anti-inflammatory, and protective effects on cells and organs. The authors suggested different delivery modes for therapeutic hydrogen namely direct inhalation, drinking hydrogen-rich water, and injection with hydrogen saturated saline. In the present study, hydrogen was delivered using water as a medium. The results of the cell viability assay showed the hydrogen water-treated human gingival fibroblasts had a mean cell viability of >50% at 24 h and 48 h (in cell viability assay, IC50 denotes the half-maximal inhibitory concentration of a substance, indicating its nontoxicity above 50%). Hence, an inference can be drawn that hydrogen water is nontoxic to the human gingival fibroblasts.
In a study by Kato et al.,[7] the defensive effects of hydrogen-rich water were demonstrated on keratinocytes. The results of their study indicated that the percentage of apoptotic keratinocytes was increased after ultraviolet A (UVA)-irradiation, but the preliminary use of hydrogen water to every irradiation, suppressed the cell death and retained the cell viability. Thus, application of hydrogen water could significantly attenuate UVA-induced cell death over regular water.
Migration is a key property of live cells and is critical in the normal development, immune response and disease processes such as inflammation. Impaired healing is a common pathological change in chronic inflammatory diseases including periodontitis. Overproduced ROS delay healing and tissue regeneration. Hence, in the present study, a migration assay was done to evaluate the migration potential of hydrogen water-treated human gingival fibroblasts. Xiao and Miwa.[5] studied if hydrogen water could achieve cyto-protection from oxidative stress injury in gingival fibroblasts. The results of their study suggested that hydrogen water could reduce over produced ROS, reduce intra-cellular ROS production and protect fibroblasts in comparison to control. Hydrogen water also showed protective effects on endogenous antioxidants, which in turn can scavenge the overproduced ROS. The authors concluded hydrogen water could delay cellular senescence, promote cell migration, and promote healing.
Yoneda et al.[8] suggested that hydrogen water has an antioxidative potential. Drinking of hydrogen water lowered the serum and gingival levels of 8-OHdG, which is an indicator of oxidative stress. The results of our present study also suggested an antioxidative potential of hydrogen water. We found; the effects were more pronounced in relation to the fibroblasts obtained from patients with chronic periodontitis at 48 h. Chronic periodontitis, being an inflammatory disease, is associated with high production of ROS thereby leading to oxidative stress. We believe that hydrogen water being a potent antioxidative agent was more effective in scavenging the overproduced ROS in the chronic periodontitis group, thereby showing a striking difference in OD values in comparison to other groups.
Azuma et al.[9] studied the effects of drinking hydrogen water on nonsurgical periodontal treatment. The results showed an increase in the serum level of the total antioxidant capacity in the patients consuming hydrogen water. The participants in the hydrogen water group also showed greater improvements in probing pocket depth and clinical attachment level as compared to the control group at 2,4 and 8 weeks.
The mechanism by which Hydrogen (H) shows antioxidative activity and anticytotoxic effect was given by Ohsawa et al.[10] Hydrogen selectively reduces the hydroxyl radical which is the most cytotoxic of the ROS and changes it into harmless water and effectively protects the cells.
To the best of our knowledge, no studies have reported any unwanted/adverse effects of consumption of hydrogen water. Evidence suggests use of hydrogen water will help combat the oxidative stress and thereby limit the progression of the disease.[5,10,11]
Recent studies also indicate hydrogen water has an antibacterial activity.[12] This remarkable finding of something as natural as water, but having an antibacterial and antioxidative potential, has certainly opened our minds to further research on hydrogen water and its applications. In addition, a more comprehensive understanding of pharmacokinetics and biology will certainly help us to harness the protective potential of hydrogen water prior to its clinical application.
CONCLUSION
In the light of the results of the present in vitro study, it was observed, that hydrogen water was nontoxic to the human gingival fibroblasts at 24 h and 48 h. An increased migratory potential of the human gingival fibroblasts was seen as a response to hydrogen water when compared to distilled water. Hydrogen water also showed an antioxidative potential.
Measuring the amount of hydrogen generated in the hydrogen water bottle after electrolysis, could have further strengthened our study, this perhaps can be considered one of the limitations. Financial constraints prevented us to conduct tests that would reveal the exact mechanism through which hydrogen-rich water exhibits its antioxidative potential.
The adjunctive use of hydrogen water as an irrigant, and mouth rinse especially in patients with chronic periodontitis, needs to be further explored. The encouraging results of this basic research prompt us to undertake further prospective clinical studies for the application of hydrogen water as an adjunct in the treatment of patients with chronic periodontitis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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