F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.145643.1 Study Protocol Articles Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C–reactive protein levels after surgical removal of impacted mandibular third molar: a randomized, double-blind, controlled clinical trial.

[version 1; peer review: 1 approved with reservations]

 Singhai Anuroop Conceptualization Methodology Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-8806-1283 a 1 2 Kambala Rajanikanth Supervision Validation https://orcid.org/0000-0001-7695-3770 1 Bhola Nitin Validation 1 Oral and Maxillofacial Surgery, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442001, India Oral Surgery, General Dentistry Program, Batterjee Medical College, Jeddah, Makkah, 21442, Saudi Arabia anuroopsinghai@gmail.com

No competing interests were disclosed.

 16 2 2024 2024 13 106 6 2 2024 Copyright: © 2024 Singhai A et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction

Surgical removal of an impacted third molar is one of the most common oral surgical procedures performed in dental offices. The postoperative phase is often associated with severe inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) are usually prescribed to manage postoperative discomfort. NSAIDs have been associated with gastrointestinal bleeding, renal function disturbances, and platelet count reductions. Thus, the present study demonstrates the utility of aescin in managing postoperative discomfort after the surgical removal of impacted mandibular third molars.

This study aimed to correlate salivary PGE2 levels and serum C-reactive protein levels with subjective and objective symptoms after surgical extraction of the mandibular third molar and their relationship with drug therapy.

 Methods

The planned study is a single-center, double-blind, randomized, parallel, prospective clinical trial. Each patient will be prescribed either diclofenac sodium 150 mg/day or aescin (escin) 120 mg/day to be taken orally in divided doses for five days after surgically removing the impacted mandibular third molar.

Pain will be assessed using a visual analog scale. Facial swelling and mouth opening will be recorded using a metric scale with standardized reference points. ELISA (enzyme-linked immunosorbent assay (ELISA) will be employed to measure salivary Prostaglandin E2 and serum C–reactive protein levels. All parameters will be recorded preoperatively (T0) on the second postoperative day (T1) and fifth postoperative day (T2).

 Conclusion

The proposed study is expected to show a favorable response to the administration of aescin for the management of postoperative discomfort compared to diclofenac sodium after third molar surgery.

The proposed study is expected to positively manipulate the levels of salivary Prostaglandin E2 and serum C–reactive protein, which are reliable inflammatory markers.

The outcome of this study may provide an efficacious and safe alternative to conventional nonsteroidal anti-inflammatory drugs for managing postoperative discomfort following third molar surgery.

 Aescin Diclofenac pain swelling C-reactive protein prostaglandin E2 third molar None The author(s) declared that no grants were involved in supporting this work. Introduction Background and rationale

The backbone of most oral surgical practices worldwide is the extraction of impacted third molars. It is the most common oral surgical procedure 1 performed in dental offics. However, the postoperative phase is often concomitant with severe pain, reduced mouth opening, and marked facial inflammation. Most patients are prescribed corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs) to manage their postoperative discomfort. NSAIDS have been associated with gastrointestinal bleeding, disturbance in renal function, and a reduction in platelet count. 2 Because of these limitations, many phytotherapeutic alternatives have been suggested. Aescin (Escin) has generated interest among researchers over the years. It is a practical part of Aesculus hippocastanum and was first isolated in 1953. It combines triterpene saponins and has demonstrated anti-inflammatory, antiedematous, and venotonic properties in various preparations. 3 Thus, the present study demonstrates the utility of aescin in managing postoperative discomfort after the surgical removal of impacted mandibular third molars.

Another concern over the years has been the selection of NSAIDs, which predominantly depends on subjective factors, including the surgeon’s preference and personal experience. The need for an hour is to use a measurable marker of inflammation that can be quantitatively controlled by drug administration. Prostaglandin E2 (PGE2) is a reliable marker of inflammation, which has been isolated from various body fluids, including saliva. 4 Owing to inflammation, C-reactive protein (CRP) emerges in the blood. Various studies have used CRP concentrations to monitor the healing process after drug therapy because of its sensitivity. 5 This study aimed to correlate salivary PGE2 levels and serum C-reactive protein levels with subjective and objective symptoms after surgical extraction of the mandibular third molar and their relationship with drug therapy.

 Background and rationale: choice of comparators

Diclofenac was chosen for comparison because it has dose equivalence to aescin and is widely used to manage postoperative sequelae associated with third molar surgery.

 Protocol Aim of the study

To compare the efficacy of aescin (escin) and diclofenac in managing postoperative sequelae following surgical removal of impacted mandibular third molars and their effects on salivary Prostaglandin E2 and serum C-reactive protein levels.

 Objectives

The objectives of the present study are as follows:

To compare the efficacy of aescin and diclofenac sodium in managing postoperative sequelae following surgical removal of impacted mandibular third molars.

To compare the efficacy of aescin and diclofenac sodium in manipulating salivary Prostaglandin E2 (PGE2) and serum C-reactive protein (CRP) levels in patients undergoing surgical removal of the impacted mandibular third molar.

To assess patient satisfaction regarding therapy.

To evaluate the need for rescue analgesics in the postoperative period.

Trial design

A single-center, double-blind, randomized, parallel, prospective clinical trial will be conducted at the Sharad Pawar Dental College, Datta Meghe Institute of Higher Education and Research (DMIHER), Sawangi, Wardha.

Two groups will be created:

Group A – Diclofenac sodium

Group B – Aescin (escin)

The randomization process will be performed using a computer-generated subject allocation list.

Trial participants and outcome assessors will be blinded.

 Study setting

A total of 50 patients presenting to the outpatient department of oral and maxillofacial surgery, Sharad Pawar Dental College and Hospital, Sawangi, Wardha, India, with impacted mandibular third molars with a definitive indication for extraction will be enrolled in the study.

 Eligibility criteria

Inclusion criteria

Patients giving informed written consent.

Age group 18-40 yrs.

ASA I and healthy ASA II physical status.

Patients presenting with impacted mandibular third molars.

Slightly or moderately difficult impacted mandibular third molars, according to the Pederson difficulty index.

Exclusion criteria

Presence of comorbid conditions.

Patients not reporting for follow-up as per study protocol.

Presence of systemic infections.

Presence of acute infection.

Known allergies to drugs under study.

Methods Interventions: description

A clinical trial will be conducted with approval from the institutional ethics committee. The Consolidated Standards of Reporting Trials (CONSORT) statement guidelines will be used for this study.

Patients will be prescribed either diclofenac sodium 150 mg/day or aescin (escin) 120 mg/day 6 to be taken orally in divided doses for five days. Each patient will be subjected to one of the drugs after the procedure.

Saliva collection will be done using a sterile polystyrene tube. A kit specific to PGE2 will be used to measure PGE2 levels in each specimen. 4

Venous blood will be subjected to a C-reactive protein (CRP) test to measure C-reactive protein 7 levels in blood serum.

Saliva and serum samples will be processed in Central Research Laboratory.

 Surgical procedure

All patients will be administered 1 g of oral amoxicillin 1 h before the procedure. The standard surgical protocol for the removal of impacted mandibular third molars under local anesthesia will be followed.

 Interventions: modifications

Drug therapy will be discontinued in cases of adverse reactions reported by the patient.

 Interventions: adherence

Patients will be recalled on the second and fifth postoperative days. The number of tablets remaining will be calculated to assess compliance with the trial protocol.

 Interventions: concomitant care

Patients will be required to adhere to the standard postoperative instructions following extraction.

Tramadol 50 mg will be prescribed as a rescue analgesic for the postoperative period.

 Outcomes

Postoperative sequelae after surgical removal of the impacted mandibular third molar will be estimated by assessing pain, facial swelling, and mouth opening.

Pain: The visual analog scale (VAS) 8 will be used to assess pain on a scale ranging from 0 to 10. The worst imaginable pain will be graded as 10, and the absence of pain will be graded as 0.

Facial swelling will be measured using a metric tape and standard reference points. 6 The following reference points will be used for the measurement:

Soft tissue pogonion - Tragus

The angle of the mandible - Corner of the eye

Corner of mouth - Tragus

Mouth opening: The maximum interincisal distance will be recorded using a metric scale with standardized ruler and a digital Vernier caliper. 9

Salivary Prostaglandin E2 and serum C-reactive protein levels will be measured by ELISA (enzyme-linked immunosorbent assay (ELISA) preoperatively (T0) on the second postoperative day (T1) and fifth postoperative day (T2).

Patient satisfaction regarding therapy: Study subjects will classify their experience from 1 to 5, where 1 = poor, 2 = fair, 3 = good, 4 = very good, and 5 = excellent.

The need for rescue analgesics will be evaluated by counting the number of tramadol capsules or tablets required by the patient until the fifth postoperative day (T2).

Participant timeline

Pain, facial swelling, mouth opening, serum C-reactive protein, and salivary prostaglandin E2 levels will be recorded preoperatively (T0) on the second postoperative day (T1) and fifth postoperative day (T2).

Patient satisfaction regarding therapy and the need for rescue analgesics will be recorded on the fifth postoperative day.

 Sample size

The study will be a non-inferiority trial with a continuous outcome.

The sample size was established based on data obtained from a previous study conducted by Salgia et al. 10 Serum C-reactive protein level was chosen as the primary outcome variable.

Formula using mean difference n 1 = n 2 = 2 Z α + Z β 2 σ 2 δ 2 Z α = 1.96

α = Type I error at 5% at both sides two tailed

Z β = 0.84 = Power at 80%

Primary Variable = CRP (C-reactive protein) levels

The mean difference after treatment CRP (mean ± SD) for the diclofenac group was 16.305 ± 3.977 (as per the reference article). 9

Considering 20% clinically relevant margin for aescin = ( 16.305 ∗ 20 ) / 100 = 3.26

Sample size: N = n 1 = n 2 = 2 1.96 + 0.84 2 3.977 2 3.261 2 = 23

Assuming 10% dropout = 2

Total sample size required = 23 + 2 = 25 each group.

Thus, a total sample size of 50 subjects presenting with impacted mandibular third molars will be enrolled in the study.

 Recruitment

All patients presenting to the outpatient unit of the Oral and Maxillofacial Department and fulfilling the enrolment criteria of the study will be recruited. The principal investigator coordinated with each patient to achieve the desired sample size.

 Methods: Assignment of interventions (for controlled trials) Allocation: sequence generation

Study participants will be randomized using the SCIENCER application. As a specific age range was chosen for the study, further stratification and subset creation will not be performed.

 Allocation concealment mechanism

All the patients will be sequentially numbered for the purpose of allocation concealment.

 Allocation: implementation

The principal investigator will then enrol the participants. The research supervisor allocate the sequence and assign the participants to the intervention.

 Blinding

Trial participants and the principal investigator will be blinded.

 Blinding (masking): emergency unblinding

Unblinding will be performed if a patient reports adverse side effects to the emergency unit during the follow-up period.

 Data collection plan

Demographic, clinical, and laboratory data for each patient will be collected using the JOTFORM application.

 Data collection plan: retention

Telephonic reminders will be administered to each patient, according to the follow-up protocol. The research supervisor will directly dispense medication to the participants to promote retention.

 Data management

Demographic, clinical, and laboratory data will be segregated in the JOTFORM application based on patient codes. The filtered data will be downloaded and stored in an Excel spreadsheet for statistical analysis.

 Statistical analysis plan

Statistics: outcomes

Statistics: additional analyses

Statistics: analysis population and missing data

Primary variable

Inferential statistics will be used to compare the two groups for measurement scores, resulting in their mean change in a primary variable (visual analog scale, facial swelling, mouth opening, salivary prostaglandin E2, serum C-reactive protein) between baseline and end visits.

Baseline variables will be tested for significance in the mean using ANOVA or Kruskal-Wallis test for more than two assessment periods. A post-hoc (Tukey’s or Duncan’s) test will be used to find a significant difference between the two groups for pair-wise comparison.

Outcome variables

Outcome variables will be tested for intra-difference in measurement at pre- and post-visits using a paired t-test to determine the mean’s significance. The unpaired t-test for comparison of two groups and ANOVA for comparison of three groups will be used for intergroup differences.

Statistical analysis will be carried out using R-software free version.

 Methods: monitoring Data monitoring: formal committee

Data monitoring will be carried out under the aegis of DSCC at DMIHER, Sawangi, and Wardha.

 Data monitoring: interim analysis

The interim analysis and review will be carried out by a member of the institutional ethics committee. Trial termination rights were reserved by the Institutional Ethical Committee.

 Harms

Telephonic conversations will be conducted prior to follow-up visits to inquire about any untoward events. In case of any adverse events, patients will be managed at the institutional hospital, and the necessary care will be rendered free of cost.

All adverse events will be reported to the Institutional Ethical Committee.

 Auditing

An interim audit of the trial will be conducted by the research supervisor. A final audit will be carried out by nominated members of the DSCC at the DMIHER, Sawangi, and Wardha.

 Ethics and dissemination

Research ethics approval

Ethical approval has been obtained from the Institutional Review Board of the Datta Meghe Institute of Medical Sciences (now renamed as Datta Meghe Institute of Higher Education and Research) Sawangi, Wardha. (Reference number: DMIMS (DU)/IEC/2022/842 on 05/04/2022).

 Protocol amendments

Amendments to the protocol will be carried out only after approval from the Institutional Ethical Committee and DSCC at DMIHER, Sawangi, Wardha.

 Consent or assent

The Principal investigator will be required to answer any queries by the patient regarding the trial, and will be responsible for obtaining informed consent.

 Consent or assent: ancillary studies

No biological specimen will be stored for ancillary studies and hence, ancillary study consent is not applicable.

 Confidentiality

Access to information from individual participants will be restricted to the principal investigator and research supervisor.

 Declaration of interests

There are no financial or competing interests for the principal investigators for the overall trial or each study site.

 Data access

The principal investigator and research supervisor will be custodian of the final trial data. The IEC and DSCC have access to the final trial dataset.

 Ancillary and post-trial care

The trial will be conducted in accordance with good clinical practice and a strong adverse event-monitoring protocol. The safety of the procedures and drugs used in this study has been established. However, in case of any harm, compensation will be carried out according to institutional policy.

 Dissemination policy: trial results

This clinical trial will be published as an original research article in an indexed journal. Open access release of the trial results will be performed to ensure optimum exposure of the scientific community.

 Dissemination policy: authorship

The final trial data will be published as postdoctoral research (PHD thesis). The authorship associated with this trial will be limited to the principal investigator and research supervisor, with acknowledgements to individuals involved at various stages of the trial.

 Dissemination policy: reproducible research

The PHD thesis will be made public after approval from DSCC.

 Study status

The study is currently in the stage of a pilot project.

 Discussion

There is growing interest in medical fraternity to find safer alternatives to NSAIDs. Despite advances in pharmaceutical technology, the pharmacokinetic properties of NSAIDs, such as diclofenac, have improved, however the dose-dependent gastrointestinal severe, renal, and cardiovascular adverse effects remain a cause of concern with the use of NSAIDs.

Previous studies have indicated that phytotherapeutic drugs have a favorable impact on inflammatory parameters and are safe for managing pain and discomfort following surgical removal of the third molar. Aescin (escin) is a phytotherapeutic drug that has been recognized for its anti-inflammatory properties. Previous studies have also suggested the glucocorticoid-like activity of aescin along with its gastroprotective action. However, further research is needed to examine the effectiveness of aescin in managing postoperative pain, facial swelling, and mouth opening following surgical removal of the third molar.

Nowak et al. (2021) conducted a study of 60 patients to assess the impact of A-PRF application during third molar surgery and its effect on C-reactive protein concentrations and wound healing. Patients who received A-PRF after the procedure had a faster rate of decreased CRP levels. They also suggested that A-PRF application significantly reduced the symptoms of regional inflammation and incidence of dry socket. 5

In a detailed review of escin, Gallelli (2019) suggested that it has potent anti-inflammatory, antiedematous, venotonic, and endothelial protective properties. They described the basic mechanism of action of escin. Oral escin and its topical gel have been demonstrated to increase venous tone and reduce edema, resulting in quantifiable clinical progress in patients with blunt injury and chronic venous insufficiency. 3

Isola et al. (2018) conducted a clinical trial of eighty-two patients needing mandibular third molar extraction surgery. The study participants were randomly allocated into three groups. Group 1, placebo; Group 2, ibuprofen; Group 3, a phytotherapeutic drug (baicalin 190 mg, bromelain 50 mg, and escin 30 mg). All medications were prescribed twice daily for five days after tooth extraction. A visual analog scale was used to assess pain. A metric scale was used to record alterations in facial contours and mouth opening in millimeters. The outcome of this clinical trial implies that phytotherapeutic drug therapy effectively manages postoperative pain and discomfort. 8

Graziani et al. (2017) enrolled 40 patients in a case-control study demonstrating systemic inflammation following third molar removal. Patients who underwent third molar removal had more significant systemic inflammation, oxidative stress, and triglyceride levels than controls, and the first postoperative week had increased WBC counts and serum C-reactive protein and fibrinogen levels. The baseline values of the markers were observed three months after extraction. Higher systemic inflammation is associated with impacted third molars, and their elimination may signify a novel method for investigating acute inflammation and defining favorable systemic outcomes in humans. 7

Mehra et al. (2013) conducted a double-blind, randomized clinical trial of 80 patients to assess the effect of four different pharmacological regimens on prostaglandin E2 (PGE2) levels in urine and saliva. Saliva and urine samples were collected at planned intervals. After removing the impacted lower third molars, the findings were compared with clinical progression. Patients who received ibuprofen fared significantly better in terms of most parameters. They further concluded that PGE2 is measurable in saliva and urine, and its rise and fall can be detected in both saliva and urine. They further suggested that PGE2 correlates with subjective and objective clinical symptomatology and that administration of anti-inflammatory drugs manipulates its levels in the body. 4

The current scientific literature must be more extensive in demonstrating the correlation of the drug’s efficacy with reliable inflammation markers and associated clinical symptomatology.

Considering this, the present study will be a randomized, double-blind, non-inferiority trial to compare the efficacy of aescin and diclofenac sodium in the management of postoperative pain, swelling, and mouth opening, as well as their effects on salivary Prostaglandin E2 and serum C-reactive protein levels after surgical removal of the impacted mandibular third molar. The trial will be conducted as per SPIRIT guidelines. 11

Trial registration

www.ctri.nic.in

Trial Registration Number is: CTRI/2023/09/058004

 Trial registration: data set

Not applicable

 Protocol version

v1 dated 26/07/2023

Identifier: Dr. Rajanikanth Kambala

 Roles and responsibilities: sponsor and funder

Study planning, data collection, research management, analysis, preparation of report and publication

 Roles and responsibilities: committees

The study will be carried out under the aegis of the Institutional Ethical Committee and the DSCC at DMIHER, Sawangi, Wardha, Maharashtra, India.

 Data availability

No data is associated with this article.

Extended data

Zenodo: Extended data sheet for “Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C–reactive protein levels after surgical removal of impacted mandibular third molars: a randomized, double-blind, controlled clinical trial.”. Zenodo. https://doi.org/10.5281/zenodo.10605612. 11

This project contains the following extended data:

Model Consent form

Biological specimen

Patient Case Record form

Reporting guidelines

Zenodo: SPIRIT checklist for “Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C–reactive protein levels after surgical removal of impacted mandibular third molars: a randomized, double-blind, controlled clinical trial.” https://doi.org/10.5281/zenodo.10605565. 12

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 References Brkovic B Andric M Calasan D : Efficacy and safety of 1% ropivacaine for postoperative analgesia after lower third molar surgery: a prospective, randomized, double-blind clinical study. Clin. Oral Investig. 2017;21:779–785. 27114091 10.1007/s00784-016-1831-2 Piecuch JF : What strategies are helpful in the operative management of third molars? J. Oral Maxillofac. Surg. 2012;70(9 Suppl 1):S25–S32. 22916697 10.1016/j.joms.2012.04.027 Gallelli L : Escin: a review of its anti-edematous, anti-inflammatory, and venotonic properties. Drug Des. Devel. Ther. 2019;13:3425–3437. 31631970 10.2147/DDDT.S207720 PMC6776292 Mehra P Reebye U Nadershah M : Efficacy of anti-inflammatory drugs in third molar surgery: a randomized clinical trial. Int. J. Oral Maxillofac. Surg. 2013;42(7):835–842. 10.1016/j.ijom.2013.02.017 Nowak JM Surma S Romańczyk M : Assessment of the Effect of A-PRF Application during the Surgical Extraction of Third Molars on Healing and the Concentration of C-Reactive Protein. Pharmaceutics. 2021;13(9):1471. 34575547 10.3390/pharmaceutics13091471 PMC8469162 Landim FS Filho JRL Nascimento J : Effectiveness of nicotine patch for the control of pain, oedema, and trismus following third molar surgery: a randomized clinical trial. Int. J. Oral Maxillofac. Surg. 2020;49(11):1508–1517. 32381373 10.1016/j.ijom.2019.08.013 Graziani F D’Aiuto F Gennai S : Systemic Inflammation after Third Molar Removal: A Case-Control Study. J. Dent. Res. 2017;96(13):1505–1512. 28759304 10.1177/0022034517722775 Isola G Matarese M Ramaglia L : Efficacy of a drug composed of herbal extracts on postoperative discomfort after surgical removal of impacted mandibular third molar: a randomized, triple-blind, controlled clinical trial. Clin. Oral Investig. 2019;23(5):2443–2453. 30311061 10.1007/s00784-018-2690-9 Ali AA Belgasem KA : Maximum Mouth Opening among Libyan Population. J. Oral Health Dent. Sci. 2021;5:102–109. Salgia G Kulkarni DG Shetty L : C-reactive protein estimation: A quantitative analysis for three nonsteroidal anti-inflammatory drugs: A randomized control trial. Indian J. Dent. Res. 2015;26:43–47. 25961614 10.4103/0970-9290.156797 Singhai A : Extended data sheet for “Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C–reactive protein levels after surgical removal of impacted mandibular third molars: a randomized, double-blind, controlled clinical trial.”. Zenodo. 2024. 10.5281/zenodo.10605612 Singhai A : SPIRIT checklist for “Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C–reactive protein levels after surgical removal of impacted mandibular third molar: a randomized, double-blind, controlled clinical trial.”. Zenodo. 2023. 10.5281/zenodo.10605565 10.5256/f1000research.159617.r272570 Reviewer response for version 1 Isola Gaetano 1 Referee https://orcid.org/0000-0003-4267-6992 University of Catania, Catania, Italy

Competing interests: No competing interests were disclosed.

 22 5 2024 Copyright: © 2024 Isola G 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

In the manuscript entitled: " Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C–reactive protein levels after surgical removal of impacted mandibular third molar: a randomized, double-blind, controlled clinical trial" the authors aimed to assess salivary PGE2 levels and serum Creactive protein levels with subjective and objective symptoms after surgical extraction of the mandibular third molar and their relationship with drug therapy.

The proposed study is expected to show a favorable response to the administration of aescin for the management of postoperative discomfort compared to diclofenac sodium after third molar surgery.

The proposed study is expected to positively manipulate the levels of salivary Prostaglandin E2 and serum C–reactive protein, which are reliable inflammatory markers.

Major comments:

In general, the idea and innovation of this study regards the analysis of perioperative effects of third molar and biomarkers is interesting and novel because the role these aspects in medicine are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information.

The study was well conducted by the authors; However, there are some concerns to revise that are described below.

The introduction section resumes the existing knowledge regarding the important factor linked with the impact mediators involved together with biomarkers in dentistry.

However, as the importance of the topic, the reviewer recommends to update the literature through read, discuss some recent interesting articles, that helps the authors to better introduce and discuss the role of CRP in periodontal treatment (similar to third molar) and on NSAIDs approaches (Isola G, et al., 2024 [Ref 1]) , (Isola G, et al., 2019 [Ref 2])

The authors should be better specified, at the end of the introduction section, the rationale of the study and the aim of the protocol. In the central section, should better clarify inclusions and exclusions criteria of the selected sample.

Please better state the results obtained in the abstract.

The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship regarding the periodontitis and risk of oxidative stress evolution and periodontal treatment that could improve the quality of life in oral diseases patients with impacted third molar. The conclusion should reinforce in light of the discussions.

In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol.

Minor Comments:

Abstract:

Better formulate the abstract section by better describing the aim of the study

Introduction:

Please refer to major comments

Discussion

Please add a specific sentence that clarifies the results obtained in the first part of the discussion

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Partly

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Yes

Reviewer Expertise:

Oral surgery, periodontitis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References : Effects of minimally invasive non-surgical therapy on C-reactive protein, lipoprotein-associated phospholipase A2, and clinical outcomes in periodontitis patients: A 1-year randomized, controlled clinical trial. J Periodontol .2024; 10.1002/JPER.23-0518 38696664 10.1002/JPER.23-0518 : Analysis of the Effectiveness of Lornoxicam and Flurbiprofen on Management of Pain and Sequelae Following Third Molar Surgery: A Randomized, Controlled, Clinical Trial. J Clin Med .2019;8(3) : 10.3390/jcm8030325 30866586 10.3390/jcm8030325 Singhai Anuroop Oral and Maxillofacial Surgery, Sharad Pawar Dental College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442001, India, Wardha, Maharashtra, India

Competing interests: None

 30 5 2024

Dear Dr. Isola

Thank you for taking the time to review our manuscript submitted to F1000research. We greatly appreciate your valuable feedback and constructive comments, which have undoubtedly enhanced the quality of our work.

We have carefully considered each of your suggestions and have made the necessary revisions to address the concerns raised. Below, we outline the changes made in response to your comments:

Introduction Section:

We have provided a more comprehensive rationale for the study and explicitly stated the aim of the protocol at the end of the introduction section. This aims to offer readers a clear understanding of the objectives and purpose of our research.

Methods Section:

In the central section, we have meticulously clarified the inclusion and exclusion criteria of the selected sample. This ensures transparency and assists readers in understanding the criteria used for participant selection.

Abstract:

We have revised the abstract however as this is a study protocol in preliminary stage and data collection is still going on. So the results will be published after the completion of study.

Discussion Section:

We have reorganized the discussion section to further elucidate the relationship between periodontitis, periodontal therapy and inflammatory markers. Additionally, we have incorporated relevant literature to support our conclusions effectively.

Conclusion:

The conclusion has been strengthened to reinforce key insights gleaned from the discussion. By aligning the conclusion with the broader implications discussed, we aim to provide readers with a more robust and conclusive summary of our study approach and relevance.

We are grateful for your acknowledgment of our clinical expertise and the positive assessment of our adopted protocol. Your encouraging words serve as motivation to continually strive for excellence in our research endeavors.

Once again, we extend our sincere gratitude for your insightful review and invaluable input. We believe that the revisions made have significantly improved the manuscript and hope that it now meets the standards for publication in F1000research.

Thank you for your continued support and consideration.