Abstract
Objective
To evaluate the effect of fluoride on the cytotoxicity of buccal mucosa cells in the presence of nickel-titanium archwires.
Materials and methods
Seventy-five subjects requiring comprehensive orthodontic treatment were randomly allocated to three groups. Healthy control group included normal subjects who did not receive orthodontic treatment. In the treatment control group, only stainless-steel attachments were placed without archwire. Experimental group included subjects in whom stainless-steel attachments and 0.014″ nickel-titanium archwires were used. Fluoride mouthwash and toothpaste were prescribed to all subjects. Buccal mucosa cell smear was assessed for cell viability, micronucleus scores and caspase-3 reactive cells just before (T0) and 3-months (T1) after the intervention.
Results
The buccal mucosa cell viability score was significantly lower in the experimental and treatment control groups than in the healthy control group subjects (P < 0.001). Micronucleus score was significantly higher in the experimental and treatment control groups compared to the healthy control group subjects (P < 0.001). The caspase-3 reactive cells and micronucleus scores were significantly more in the experimental group subjects compared to treatment control group subjects (P < 0.001). There was a negative but statistically non-significant correlation between cell viability and micronucleus score among three groups of subjects.
Conclusion
The use of fluoride mouthwash and toothpaste in the presence of stainless-steel attachments and nickel-titanium archwire decreased the buccal cell viability by 8.51 % and increased the micronucleus score by 3.03 %. The use of fluoride toothpaste and mouthwash had a significant cytotoxicity effect on buccal mucosa cells in the presence of nickel-titanium archwire during comprehensive orthodontic treatment.
Keywords: Orthodontics, Genotoxicity, Cytotoxicity, Metal leaching, Cell viability, Micronucleus
1. Introduction
Stainless-steel (SS) attachments and nickel-titanium (NiTi) archwires are the critical components of orthodontic appliances. As orthodontic attachments and archwires fulfill their crucial role within the oral cavity over an extended period, there is a possibility for patients to be exposed to corrosion products emanating from these orthodontic components.1 Therefore, the selected alloy's biocompatibility is intrinsically tied to its corrosive qualities, as any corrosion process might affect how the archwire interacts with the oral environment.2
Enamel demineralization is a common complication during comprehensive orthodontic treatment, and sodium fluoride is commonly used for its prevention.3 However, fluoride has been implicated in corrosion processes, particularly by degrading the protective titanium oxide layer on NiTi archwires, potentially leading to increased metal ion leaching.4 The continuous release of metal ions from corroded orthodontic materials can result various health concerns.5 The nickel in the corrosion product has been shown to exhibit cytotoxic effects in the oral cavity.6 Even in non-toxic amounts, nickel exposure can cause DNA damage.7
The existing literature on the effect of fluoride on orthodontic archwires is limited and inconclusive.8, 9, 10, 11 Few studies have reported increased metal ion release and cytotoxic effects following fluoride exposure, whereas others have found no significant impact.8, 9, 10, 11 Also the cytotoxicity and genotoxicity effects of corrosion products on buccal mucosa in an in-vivo setting remain unclear.8, 9, 10, 11 Given these uncertainties, the present study aimed to evaluate the cytotoxic and genotoxic effects of fluoride use in patients undergoing comprehensive orthodontic treatment with NiTi archwires. The study assessed buccal cell viability, micronucleus formation and caspase-3 expression, providing direct in-vivo evidence on the biological impact of fluoride exposure in orthodontic patients.
The existing literature concerning the effect of fluoride on orthodontic archwires is limited, and their results are inconclusive.8, 9, 10, 11 Therefore, the current study aimed to investigate fluoride's influence on buccal mucosa cells' cytotoxicity in the presence of NiTi archwires during comprehensive orthodontic treatment.
2. Materials and methods
2.1. Trial design
Single-center, three-arm parallel randomized controlled trial.
2.2. Sample size calculation
It was done by G Power software version 3.1.9.2. The effect size (f) was interpolated using an exponential model based on the data from a previous study yielding an effect size (f) of 0.4.12 Considering the effect size to be measured (f) as 0.4, power of the study as 80 %, and the margin of error as 5 %, the sample size in each group was calculated as 22. Considering 10 % dropout, the sample size inflated to 25 in each group. Post-hoc power analysis confirmed that the study was well-powered (>80 %) to detect the observed differences.
2.3. Participants, eligibility and settings
The study protocol was reviewed and approved by the Institutional Ethics Committee (IEC/AIIMS BBSR/PG Thesis/2021–22/105) and registered with The Clinical Trial Registry of India (CTRI) with CTRI Reg. No. CTRI/2022/04/042323. Subjects within the age range of 18–25 years having minimum crowding/spacing in maxillary and mandibular arches (≤5 mm), a full complement of teeth except for 3rd molars, and good oral hygiene (gingival sulcus probing depth <3 mm, gingival index score <1) were selected for the study.
Subjects with a history of orthodontic treatment, any known disease affecting salivary glands, oral premalignant disorder, metallic restoration or prosthesis, occupational exposure to metals, on long-term medications, known allergy to nickel, lack of manual dexterity, under any medicines for a systemic illness, recent bacterial or viral infection, who smokes or consumes alcohol, who fails to maintain good oral hygiene during the study period and pregnant subjects were excluded from the study. After an initial screening, eligible subjects were explained the study, and written consents were obtained.
2.4. Randomization
A computer random number generator software (Microsoft Excel, Microsoft, Washington, USA) was used for allocating the subjects into Group 1 (Healthy control; M = 14, F = 11), Group 2 (Treatment control; M = 11, F = 14) and Group 3 (Treatment; M = 10, F = 15). Fig. 1 shows the CONSORT flowchart.
Figure-1.
CONSORT flow diagram showing the patient allocation and their follow-up.
2.5. Blinding
The outcome assessor was blinded during data analyses. It was not possible to blind the subject and the treatment provider.
2.6. Interventions
Group 1 subjects were followed for three months without treatment. The same orthodontist (MB) corrected the malocclusions of Groups 2 and 3 subjects by using the standard edgewise appliance (0.018″ slot, Leone SpA, Sesto Fiorentino, Firenze, Italy). In Group 2 and 3 subjects, pre-welded SS bands (Leone SpA, Sesto Fiorentino, Firenze, Italy) were cemented on maxillary 1st molars and, SS brackets were bonded on all teeth mesial to 1st molars. In Group 2 subjects, the figure of 8 ligations of brackets was done using 0.009″ SS ligature wire, and no archwire was placed. In Group 3 subjects, 0.014″ NiTi archwire was ligated by 0.009″ SS ligature wire.
All subjects were instructed to brush their teeth twice daily with fluoride toothpaste (Colgate Strong Teeth, 1000 ppm fluoride, Colgate Palmolive Co. India.) and rinse their mouth with sodium fluoride mouthwash (Colgate Plax, 225 ppm Fluoride, Colgate Palmolive Co. India) twice daily, once in the morning and evening.
2.7. Collection of buccal mucosa cells
Buccal mucosa cells were collected at the beginning (T0) and end of the 3-month (T1) follow-up period. Before cell collection, each subject rinsed mouth with tap water for 1 min to remove the exfoliated dead cells. Then, cells from the buccal mucosa (both right and left) were collected with the help of a cytobrush (ColMed, India) as per the prescribed protocol.12 Then, the cytobrush was agitated in a 15 ml tube filled with 8 ml of cold phosphate-buffered saline solution.13 A random number was assigned for each sample, and stored immediately in an ice-packed closed container for cell block preparation by the Plasma-Thrombin method.14 (Figure-2)
Figure-2.
Collection of buccal mucosa cell smear using cytobrush (A) and cell suspension in the centrifuge tube (B).
2.8. Cellblock and slide preparation
Centrifugation of cell suspension was done at 2000 rpm for 5 min. The supernatant was discarded. For approximately 1 ml of sediment, 4 drops of 3.2 % Tri-sodium citrate plasma were added. Four drops of thrombin were added. The contents were mixed by tapping gently on the counter. This mixture was allowed to sit for a few minutes until a soft ball formed. This mass was removed and placed in an appropriately labeled cassette, which was kept in fixative and submitted for processing as a tissue.
The slides were prepared as per the process described by Suvarna et al.15 The sample was processed in a carousel-type automatic tissue processor. After tissue processing, the sample was sent to a modular embedding station where the processed specimen was correctly oriented and embedded in paraffin wax that provided external support during microtomy. The array block prepared was placed on a rotary microtome to get sections of tissue at 4–5 μm. The sections were placed on a positively charged glass slide in the same orientation and sent for Hematoxylin and Eosin (H&E) stain and immunohistochemistry (IHC) stain (Caspase-3 antibody).
2.9. Staining procedure
The H&E staining was performed as described by Suvarna et al.15 The deparaffinization of slides was done in an incubator at 700C for 10-min. Then, rehydration was carried out, which involved sequential immersion in absolute alcohol for 2-min and 95 % alcohol for another 2-min. Then, the tissue was rinsed in water for 3-min to prime the sample for subsequent staining with hematoxylin. Hematoxylin stain was applied for 3-min, and then a 4-min water rinse was done. Differentiation was achieved through a brief dip in 1 % acid alcohol, accentuating the contrast between stained and unstained areas. Subsequently, thorough washing in tap water was done to arrest the differentiation process. The tissue sample was then subjected to "bluing" in an alkaline solution (ammonia water) and a 10-min tap water wash. Eosin was then applied for two brief dips to impart color to the cytoplasm. The final steps involved dehydrating the sample through ascending alcohol grades, followed by clearing and mounting for microscopic examination.
The IHC staining procedure was performed as described by Suvarna et al. 15 The deparaffinization of slides was carried out in an incubator at 700C for 10-min and dipped in subsequent grades of xylene and absolute alcohol for 3-min each. Slides were rehydrated by a 3-min dip each in 90 % and 70 % alcohol, followed by rinsing in distilled water. Slides were transferred to citrate buffer, which was kept in a pressure cooker to reach at 10.3 kPa (15psi) and then immediately transferred to cold water for 20-min. After washing, a hydrophobic slide marker was used to encircle, and a peroxide blocker was added, which was incubated for 10-min. These slides were washed with wash buffer, and a primary antibody (Caspase-3) was added. The slides were incubated for 45-min and washed 3 times with wash buffer. Secondary antibody was added and kept for incubation for 30-min. Following this, slides were washed with wash buffer, and freshly diluted 3,3′-diaminobenzidine (DAB) chromogen was added and incubated for 10-min. Slides were then washed with wash buffer, and counter-staining with hematoxylin was done.
2.10. Evaluation of cell viability and Caspase-3 apoptotic marker
Cells with intact cytoplasm that remained relatively flat on the slide and did not clump and overlap with adjacent cells were screened (Figure-3). The presence of caspase-3 reactive cells in the smear was assessed according to the number of immunoreactive (brown) cells (Figure-4). The cell morphology was studied with an Olympus BX43 light microscope (Olympus Life Science, Tokyo, Japan) and an attached Olympus U-CMAD3 (DP22) video camera (Olympus Life Science, Tokyo, Japan) equipped with 1920 × 1440 resolution and 3.69 × 3.69 μm pixel size sensor. Olympus cellSens Entry software (Olympus Life Science, Tokyo, Japan) was used for recording of the images.
Figure-3.
Hematoxylin and Eosin-stained section from cell block in high power view (80X) showing the (A) morphology of a viable squamous cell, (B) cell with micronuclei and (C) other characteristics like karyorrhexis (i) and karyolysis (ii).
Figure-4.
Caspase-3 reactive cell.
2.11. Evaluation of micronucleated cells
Thousand intact epithelial cells were examined, and the average percentage of micronucleated cells was counted. The findings were compared with those of controls to assess the difference in cytotoxicity of buccal mucosa cells. The criteria for the identification of micronucleus as suggested by Tolbert et al.16 and Countryman et al.17 with some additions as described by Bolognesi et al.18 were followed.
2.12. Statistical analysis
The data were analyzed using the statistical software package IBM SPSS Statistics for Windows version 26.0 software (IBM Corporation, Armonk, New York, USA). Descriptive statistics were applied and data distribution was assessed using the Shapiro-Wilk test. Descriptive statistics and Shapiro-Wilk test were applied. A paired t-test was used within the group comparison. One-way ANOVA and Bonferroni (post-hoc) tests were used for between the group comparison. The P-value of 0.05 was considered as a level of significance.
3. Results
3.1. Participants details
Participants' details are described in table-1. The mean age of the subjects at T0 was comparable among three groups (P = 0.898). The mean gingival index score among the three groups of subjects was comparable at T0 (P = 0.930) and T1 (P = 0.870). The mean probing depth was comparable among the three groups at T0 (P = 0.727) and T1 (P = 0.995).
Table 1.
Characteristics of the subjects at different time points of observation.
| Variable | Groups |
Significance (P-value) | ||
|---|---|---|---|---|
| Group 1 (Mean ± SD) | Group 2 (Mean ± SD) | Group 3 (Mean ± SD) | ||
| Age at T0 (yr) | 21.76 ± 2.50 | 20.92 ± 2.12 | 21.16 ± 2.15 | 0.898NS |
| Gingival index score at T0 | 0.32 ± 0.14 | 0.37 ± 0.16 | 0.35 ± 0.18 | 0.930NS |
| Gingival index score at T1 | 0.36 ± 0.17 | 0.41 ± 0.13 | 0.42 ± 0.14 | 0.870 NS |
| Gingival probing depth at T0 (mm) | 1.57 ± 0.51 | 1.78 ± 0.19 | 1.63 ± 0.13 | 0.727NS |
| Gingival probing depth at T1 (mm) | 1.96 ± 0.87 | 1.98 ± 0.09 | 1.94 ± 0.17 | 0.995 NS |
T0: Beginning of follow up; T1: After 3-months of follow up.
SD: Standard Deviation, NS: Non-significance.
3.2. Analysis of cell viability
Cell viability at different time points of observation is mentioned in table-2. In Group 1 subjects, the cell viability at T0 and T1 was comparable (P = 0.496). In Group 2 subjects, the cell viability decreased significantly by 4.08 ± 1.43 % from T0 to T1 (P < 0.001). For Group 3 subjects, the cell viability at T0 was 79.30 ± 4.16 % and 70.78 ± 4.04 % at T1, and the difference (8.51 ± 1.85 %) was statistically significant (P < 0.001). The cell viability was significantly less in group 3 compared to group 1 and 2 subjects (P < 0.001).
Table 2.
Buccal mucosa cell viability, micronucleus and caspase-3 reactive cell scores at various time points of observation.
| Groups | Percentage of buccal cell viability |
Between group comparison of change (P-value) |
|||||
|---|---|---|---|---|---|---|---|
| T0 | T1 | Change (T0 to T1) | Within group comparison of change (T0 Vs. T1) (P-value) | 1 Vs. 2 | 1 Vs. 3 | 2 Vs. 3 | |
| Group 1 | 80.57 ± 2.70 | 80.40 ± 2.28 | 0.17 ± 1.22 | 0.496NS | <0.001 | <0.001 | <0.001 |
| Group 2 | 80.41 ± 5.64 | 76.34 ± 5.07 | 4.08 ± 1.43 | <0.001 | |||
| Group 3 | 79.30 ± 4.16 | 70.78 ± 4.04 | 8.51 ± 1.85 | <0.001 | |||
| Groups | Percentage of micronucleus score |
Between group comparison of change (P-value) |
|||||
|---|---|---|---|---|---|---|---|
| T0 | T1 | Change (T0 to T1) | Within group comparison of change (T0 Vs. T1) (P-value) | 1 Vs. 2 | 1 Vs. 3 | 2 Vs. 3 | |
| Group 1 | 2.38 ± 0.91 | 2.37 ± 0.86 | 0.008 ± 0.82 | 0.962NS | <0.001 | <0.001 | <0.001 |
| Group 2 | 2.34 ± 0.38 | 3.92 ± 0.80 | 1.58 ± 0.70 | <0.001 | |||
| Group 3 | 3.26 ± 1.39 | 6.28 ± 2.18 | 3.03 ± 1.61 | <0.001 | |||
| Groups | Percentage of caspase-3 reactive score |
Between group comparison of change (P-value) |
|||||
|---|---|---|---|---|---|---|---|
| T0 | T1 | Change (T0 to T1) | Within group comparison of change (T0 Vs. T1) (P-value) | 1 Vs. 2 | 1 Vs. 3 | 2 Vs. 3 | |
| Group 1 | 0.38 ± 0.32 | 0.37 ± 0.26 | 0.005 ± 0.43 | 0.984NS | <0.001 | <0.001 | <0.001 |
| Group 2 | 0.34 ± 0.34 | 1.92 ± 0.43 | 1.57 ± 0.08 | <0.001 | |||
| Group 3 | 0.29 ± 0.21 | 4.54 ± 0.64 | 4.25 ± 0.43 | <0.001 | |||
T0: Beginning of follow up; T1: After 3 months of follow up.
NS: Non-significance.
3.3. Analysis of micronucleated cells
Micronucleated cell details are described in table-2. In Group 1 subjects, cells with micronucleus at T0 and T1 was comparable (P = 0.962). In Group 2 subjects, the score at T0 was 2.34 ± 0.38 %, and it increased significantly to 3.92 ± 0.80 % at T1 (P < 0.001). In Group 3 subjects, the score increased significantly from T0 (3.26 ± 1.39 %) to T1 (6.28 ± 2.18 %) (P < 0.001). There was a significant increase in the micronucleated cells in Group 2 and 3 compared to Group 1 (P < 0.001). In group 3, the frequency was significantly more compared to group 2 (P < 0.001).
3.4. Analysis of caspase-3 reactive cells
The detailed analysis of Caspase-3 reactive cells is highlighted in table-2. In Group 1 subjects, the caspase positive cell score at T0 was 0.38 ± 0.32 %, and 0.37 ± 0.26 % at T1 (P = 0.984). In Group 2 subjects, the score was 0.34 ± 0.34 % at T0, and it increased significantly to 1.92 ± 0.43 % at T1 (P < 0.001). In Group 3 subjects, the score increased significantly from 0.29 ± 0.21 % at T0 to 4.54 ± 0.64 % at T1 (P < 0.001). There was a significant increase in the Caspase-3 reactive cells in Group 2 and 3 subjects compared to Group 1 ((P < 0.001).
4. Discussion
Orthodontic treatment often involves prolonged exposure of patients to stainless steel and nickel-titanium alloys materials. The oral cavity is exposed to various chemicals, which leads to the corrosion of these materials. Thus, the potential impact of these corrosion products on the cellular health and genetic stability demands a thorough investigation. The use of fluoride toothpaste and mouthwash for the prevention of enamel white spot lesions exacerbates the corrosion process, leading to a further leaching of toxic ions from orthodontic materials.19
This present three-arm parallel clinical trial helps in estimating the cytotoxic and genotoxic effects of fluoride exposure in the presence of NiTi archwires during comprehensive orthodontic treatment. role of nickel-titanium archwire in the cytotoxicity of buccal mucosa cells. This study design facilitates the comparison of two experimental groups against the control, as well as a head-to-head comparison between the groups. The epithelium of buccal mucosa is non-keratinized and in immediate contact with the archwire and brackets, making it more prone to absorption of leached ions. Many factors influence the absorption corrosion products. The permeability of buccal mucosa decreases with age.20 Hence to avoid any age-related bias, young adults in the age range of 18–25 years were included. Although comprehensive orthodontic treatment takes about two years to complete, but a short-term exposure is sufficient to detect cellular changes.11 Thus a three-month follow-up period was considered in the present study.
Activation of the caspase-3 pathway is a hallmark of apoptosis. The expression of caspase-3 is time and concentration dependent.21 Caspase-3 expression precedes DNA fragmentation in apoptotic cells, making it a specific marker for assessment of cell damage. The activation of caspase-3 lies at the intersection of several apoptotic pathways, which gives it a broad range over other apoptotic markers.22
Cell viability refers to the number of cells with the ability to remain alive and functional within a given condition. The assessment of change in cell viability is a predictor of cytotoxic effects of the intervention used. In the present study, the cell viability was around 80 % in individuals prior to the beginning of orthodontic treatment. Faccioni et al. also observed 73.43 ± 12.29 % cell viability among healthy control subjects without orthodontic treatment.23
Our study revealed that the use of fluoride products had no adverse effects on cell viability in normal individuals. However, when fluoride products are used in the presence of stainless-steel attachments, the viability of cells decreases by 4.08 %. This could be due to the cytotoxic effect of various corrosion products released by the interaction between fluoride and stainless-steel. However, when fluoride products are used in the presence of both stainless-steel attachments and nickel-titanium archwire, the cell death was almost two times more compared to subjects with only stainless-steel attachments. Sodium fluoride (NaF) combines with hydrogen ions (H+) in the oral cavity forms hydrofluoric acid (HF), a potent inorganic acid that erodes the oxidative layers shielding alloy surfaces and starts the corrosion process.24 Hafez et al., reported a significant reduction in buccal mucosa cell viability and increase in cellular nickel and chromium levels in subjects undergoing orthodontic treatment with stainless-steel brackets and archwire.25
Micronuclei are the product of abnormal mitosis. This study revealed that the frequency of having micronuclei among normal individuals was nearly 2–3 % which was similar to finding of study done by Natarajan et al.26 The use of fluoride products for 3-months had no effect on the micronuclei formation in normal subjects. However, the use of fluoride products in the presence of stainless-steel attachments resulted in 1.5 % more micronuclei formation. In contrast, the use of fluoride products in the presence of stainless-steel attachments and NiTi archwire resulted in 4 times more micronuclei formation. This indicates that although the use of stainless-steel attachments itself has genotoxic potential, but this effect is four-fold more when used along with NiTi archwires. Chitra et al. found 5.55 micronucleus/200 cells when fluoride products are used in presence of stainless-steel brackets and NiTi archwires.27 In contrast, Apiwantanakul et al. found a significant decrease in cell viability but no change in micronucleus score in subjects with stainless-steel brackets and NiTi archwire after 3-months of professional fluoride application.11 However, subjects with sodium fluoride gel and sodium fluoride varnish had no significant change in cell viability and micronucleus score.11 This disparity could be due to one-time exposure to fluoride at the beginning of the study, unlike continuous exposure to low-dose fluoride in our study.
Cell damage leading to apoptosis usually works through extrinsic or intrinsic signaling pathways. Both pathways work by the activation of the caspase-3 inflammatory marker. Nickel has been observed to induce apoptosis of buccal mucosa cells by regulating caspase-3 activation.28 The present study revealed that the use of fluoride products had no adverse effect on oral mucosa cells in the absence of metal attachments. However, in the presence of stainless-steel attachments and nickel-titanium archwire, the use of fluoride toothpaste and mouthwash for 3-months substantial increased the cell apoptosis.
While statistically significant, the observed reductions in buccal cell viability, increase in micronucleus formation and caspase-3 reactive cells suggest a potential biological impact. These findings indicate that fluoride exposure, in the presence of NiTi archwires, may accelerate cytotoxic and genotoxic effects in the oral mucosa.
5. Limitations and generalizability
Short duration of study may limit the ability to identify the long-term cytotoxic effects. Extended follow-up periods and more frequent assessments could provide a more comprehensive and dynamic understanding of sustained impact and fluctuations on cellular health. The study aimed to evaluate fluoride's effect on buccal mucosa cytotoxicity in orthodontic patients. However, a fluoride-free control group was not included, as fluoride use is a standard preventive measure in orthodontic care. Future studies with an additional non-fluoride group may provide more definitive insights into fluoride's independent role in cytotoxicity. Future investigations should consider incorporating genetic profiling to identify potential susceptibility or resilience factors. The use of techniques like inductively coupled plasma mass spectrometry could help correlate the changes with cellular metal concentrations, localizing the potential cause of these changes.
6. Conclusions
-
1.
The use of fluoride mouthwash and toothpaste had no cytotoxicity effect in individuals without any metal attachments in the oral cavity.
-
2.
The use of fluoride mouthwash and toothpaste decreased the buccal cell viability by 4 % and increased micronuclei formation by 1.58 % in subjects with stainless-steel orthodontic attachments. However, in the presence of stainless-steel attachments and nickel-titanium archwire, the buccal cell viability decreased by 8.51 % and increased micronuclei formation by 3.03 %.
-
3.
The caspase-3 marker score in the buccal mucosa cells was nearly 3 times higher when fluoride mouthwash and toothpaste were used in the presence of stainless-steel attachments and nickel-titanium arch wires compared to only stainless-steel attachments.
Consent declaration
The authors hereby declare that informed consent was obtained from all individual participants included in this study. Each participant was adequately informed about the nature, purpose, procedures, potential risks, and benefits of the research prior to inclusion. The participants were provided with an information sheet and consent form in a language they could comprehend, and sufficient time was given to ask questions and consider their participation.
Participation in the study was entirely voluntary, and all participants retained the right to withdraw at any stage without any consequence to their medical care. Confidentiality and anonymity of the participants have been strictly maintained throughout the research process in accordance with the Declaration of Helsinki and the ethical guidelines.
The study protocol was reviewed and approved by the Institutional Ethics Committee (IEC/AIIMS BBSR/PG Thesis/2021–22/105) and registered with The Clinical Trial Registry of India (CTRI) with CTRI Reg. No. CTRI/2022/04/042323.
Ethical clearance
An informed written consent was obtained from each participant prior to their enrolment in the study. All participants were thoroughly informed about the nature, purpose, methodology, potential risks, and benefits of the research, and they were given the autonomy to withdraw from the study at any stage without any prejudice to their standard care.
The study protocol was reviewed and approved by the Institutional Ethics Committee of AIIMS Bhubaneswar (Approval No. IEC/AIIMS BBSR/PG Thesis/2021–22/105). Furthermore, the study was prospectively registered with the Clinical Trial Registry of India (CTRI) under the registration number CTRI/2022/04/042323.
All procedures performed in the study were in accordance with the ethical standards of the institutional and national research committees and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Declaration of funding
The authors declare that no funding was received from any external sources for the conduct, authorship, or publication of this research.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
The authors declare that no acknowledgements are applicable for this study.
Contributor Information
Mayur Bhola, Email: mb.bhola@gmail.com.
Ashok Kumar Jena, Email: dent_ashok@aiimsbhubaneswar.edu.in.
Madhusmita Sethy, Email: pathol_madhusmita@aiimsbhubaneswar.edu.in.
Jitendra Sharan, Email: dent_jitendra@aiimsbhubaneswar.edu.in.
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