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Measurement of the Clinical Effects of a Marine Fish Extract on Periodontal Healing—A Preliminary Clinical Interventional Study

Appl. Sci. 2022, 12(2), 677; https://doi.org/10.3390/app12020677

by Cristina Gabriela Pușcașu^1,*,†

, Aureliana Caraiane^1,†, Elena Dumea^2,†

, Erdogan Elvis Șachir^1,†, Gheorghe Raftu^1,†, Radu Andrei Pușcașu^3,†, Cristina Bartok-Nicolae^1,*,†

, Eduard Radu Cernei^4,*,†, Liliana Sachelarie^5,*,†

and Liliana Loredana Hurjui^4,*,†

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Appl. Sci. 2022, 12(2), 677; https://doi.org/10.3390/app12020677

Submission received: 23 December 2021 / Revised: 7 January 2022 / Accepted: 9 January 2022 / Published: 11 January 2022

(This article belongs to the Special Issue Applied Interdisciplinarity Medical Areas in Maintaining Oral Health)

Round 1

Reviewer 1 Report

Congratulations on the corrections

Author Response

Thank you very much!

I remain most respectfully yours,

Prof.dr. Liliana Sachelarie

Reviewer 2 Report

Dear Authors, some typo errors are present in the manuscript.

In the abstract in the underline 26 you write "pacients". Please check all the manuscript.

In the conclusion section add the limitations of the present study and future prospects

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript initially entitled “The clinical effects of a marine fish extract on the periodontal healing”, co-authored by Cristina Gabriela Pușcașu¹*, AurelianaCaraiane¹, Elena Dumea², Erdogan Elvis Șachir¹, GheorgheRaftu¹, Radu Andrei Pușcașu³, Cristina Bartok-Nicolae¹, Cernei Eduard Radu^4*, Liliana Sachelarie^5*, Liliana Loredana Hurjui⁴*

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

In the abstract in the underline 26 you write "pacients". Please check all the manuscript.

Patients were divided into two groups: group I - after scaling and root planning (SRP) patientsfollowed therapy with marine fish extract, Alflutop®, group II - SRP therapy alone.

We checked all the manuscript.

In the conclusion section add the limitations of the present study and future prospects.

This study is a limited study but provides information that can be used in future research on the therapeutic efficacy of marine fish extract Alflutop®.

Thank you very much for both review reports and for the extremely useful suggestions!

I remain most respectfully yours,

Prof.dr. Liliana Sachelarie

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The English language is poor and should be revised by native English speaker familiar with scientific English. The methods are not adequately described and the number of subject included in the two groups are too different.

line 44: you probably forgot POOR ORAL HYGIENE, the first cause of periodontal inflammation

line 78: please use the current 2017 world workshop classification

line 84: please delete "sitting on dental chair"

line 86: Why you don't use BOP?

line 89: which probe?

113: what do you mean with "evolution of inflammation"?

126: mean age/years old

177: asses

180: please don't use personal pronouns in scientific papers

the conclusions are too strong

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled “The clinical effects of a marine fish extract on the periodontal healing”, co-authored by Cristina Gabriela Puşcaşu * , Aureliana Caraiane , Elena Dumea , Erdogan Elvis Șachir , Gheorghe Raftu , Radu Andrei Puşcaşu , Cristina Bartok-Nicolae * , Radu Eduard Cernei * , Loredana Liliana Hurjui *

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

The English language is poor and should be revised by native English speaker familiar with scientific English.

We have made the revision of the paper’s English language.

The methods are not adequately described and the number of subjects included in the two groups are too different.

The initial study group included 68 subjects, divided in two groups of 34 subjects each (group I-test and group II-control). Group I (test) received Alflutop®, after scaling, root planning, debridement (SRP), and group II (control) received only SRP therapy.

Although the study group was divided in 2 groups (test and control), the number of patients from both groups who finished the study protocol was different.

From the test group, only 25 patients could finalize the research. The rest of 9 patients did not finalize the protocol (2 patients did not present in time and could not finish all 10 infiltrations, 7 patients did not present for the final assessment one month after therapy). Among the test group, after SRP therapy, a number of 14 patients did not come at the final examination, one month after therapy. So, the final study groups are: group I (test) with 25 cases, and control group II with 20 patients.

line 44: you probably forgot POOR ORAL HYGIENE, the first cause of periodontal inflammation

Periodontal inflammation can be influenced by poor oral hygiene, but also by the immune response, the type of subgingival microbiota and other possible systemic diseases.

line 78: please use the current 2017 world workshop classification

All patients diagnosed with periodontitis, no comorbidities or known previous allergies to any drug, were considered eligible and included in the study group after signed informed consent for the research

line 84: please delete "sitting on dental chair"

A previously standardized dentist performed the oral examinations of the patients, with Williams-periodontal probe and a flat dental mirror.

line 86: Why you don't use BOP?

PBI was preferred to be used because the most affected sites are approximal areas, in terms of alveolar bone loss and inflammation, according to the 2017 European workshop staging and grading classification of periodontitis.

line 89: which probe?

Williams periodontal probe was used for clinical measurements.

113: what do you mean with "evolution of inflammation"?

Evolution of inflammation indices

126: mean age/years old

177: asses, 180: please don't use personal pronouns in scientific papers

The present preliminary study aimed to evaluate the clinical effects of treatment with SRP and Alflutop®, as compared with clinical effects of SRP alone.

the conclusions are too strong

This clinical investigation suggests that the marine fish extract (Alflutop®) could have certain therapeutic efficiency also in periodontal healing, in terms of reduction of clinical signs of inflammation and tooth mobility.

Thank you very much for the review report and for the useful suggestions!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Reviewer 2 Report

Great confusion between the initial and post-test study groups.

Just like the numbers post the final numbers that do not match the final table

These numbers must be corrected for publication.

The initial study has 68 participants, divided into 2 groups of 34(G-I=34, GII=34)

After the tests:

GI= 34 initial, final has= 35 finished the test(?) and 8 didn't finish(?), as if the total were 34? And now there are 43??

GII = initial 34, in the end there were 20 (the absence of 14 is not mentioned)

20 patients did not appear in a total of 68, would give an n=48, however the sum gives 55??

In the table, GI has 27 patients and GII has 28, for a total of 56??

Author Response

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

Great confusion between the initial and post-test study groups.

Just like the numbers post the final numbers that do not match the final table

These numbers must be corrected for publication.

The initial study has 68 participants, divided into 2 groups of 34(G-I=34, GII=34)

After the tests:

GI= 34 initial, final has= 35 finished the test(?) and 8 didn't finish(?), as if the total were 34? And now there are 43??

GII = initial 34, in the end there were 20 (the absence of 14 is not mentioned)

20 patients did not appear in a total of 68, would give an n=48, however the sum gives 55??

The numbering errors were corrected as follows:

45 patients with periodontitis were divided, by accessibility to Alflutop® therapy (Figure 1), into group I with 25 cases, mean aged 50.4±10.6 year old (limits: 34-67 years), and group II with 20 controls, mean aged 44±19 year-old (limits: 25-81 years).

In the table, GI has 27 patients and GII has 28, for a total of 56

The numbers in Table 1 were corrected as well.

Group	Sex	N	Minimum	Maximum	Mean	Std. Deviation
Alflutop	Female/Age	14	42	67	51.57	8.31
Alflutop	Male/Age	11	34	64	49	13.42
Control	Female /Age	14	25	45	33.36	8.88
Control	Male/Age	6	54	81	69.33	9.20

Table 1Age (mean) by group and by sex of the final study groups

Thank you very much for the review report and for the useful suggestions!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Reviewer 3 Report

applsci-1480352-peer-review-

Puscasu et al: The clinical effects of a marine fish extract on the periodontal healing

I do recommend a change in the title “..on clinical periodontal healing or the clinical measures of periodontal healing: as there is no histological investigation! The study design also must be indicated in the title, like case-control study or clinical interventional study.

Introduction:

Second sentence: “ reattachment of the periodontal ligaments at a higher level on the cementum” ? In this case if there is attachment gain it is not reattachment. In this case it is a new attachment. If we take the original definition true. I could not check what was in the original paper as it is in Rumanian.

The chemical composition of “Alflutop” can go to “materials and methods” part. The study design also must be precisely stated in this part. The authors must describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection, as well. Relevant dates are fully missing. The rationale for the choice of cases and controls is not presented. It should have been randomization, in this case. How did the authors randomize patients? Diagnostic criteria were not given for periodontitis. Was it based on previous guidelines or new guidelines? What was the level of the attachment loss? It is important as deeper pockets normally can get more clinical attachment than a normal depth crevice just by the custom mechanical treatment.

Gingival index: is it the Löe-Silness index (1963)? In that case score 1 is colour change of the gingiva+ no BOP, score 3 means severe bleeding on probing, not spontaneous bleeding!

What was the reason (theory behind) that Alflutop was administered “mostly” to the canine –premolar area? Upper or lower arch? If it was not “mostly” than where?

Statistical methods are not described at all. Please, clearly describe the methods used in the study. Please, present the reason why they were used, please, also describe the efforts to address potential source of bias. E.g: it seems that the in the control group the age has a different mean and variance.

Results: report number of individuals at each stage of the study. Some numbers are given but make it more clear. A flow chart would be advantageous to use! The reason for non-participation must also be given at the different steps.

Please, also make comparisons on the initial stage variables to final stage variables of the periodontal condition in the same group. Practically, the improvement in periodontal condition must be significantly different to prove the effectivity of Aflutopf. Both for case and control groups. The reduction of mobility is secondary to inflammation reduction. The lack of side-effects must go to the result section.

Discussion part: start with your own main findings, then compare results. Discuss limitations of the study, as well.

Comments for author File: Comments.pdf

Author Response

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

I do recommend a change in the title “..on clinical periodontal healing or the clinical measures of periodontal healing: as there is no histological investigation! The study design also must be indicated in the title, like case-control study or clinical interventional study.

Thank you very much for the title change suggestions.

Measurement of the clinical effects of a marine fish extract on periodontal healing - a preliminary clinical interventionalstudy

Introduction:

Second sentence: “ reattachment of the periodontal ligaments at a higher level on the cementum”? In this case if there is attachment gain it is not reattachment. In this case it is a new attachment. If we take the original definition true. I could not check what was in the original paper as it is in Rumanian.

This kind of therapy is known as periodontal biostimulation and it is used after SRP (Scaling and Root Planing) therapy and periodontal surgery, in patients with chronic periodontitis, to help the healing process and clinical attachment gain.

A clinical interventional study was conducted in Constanța, on adults, selected randomly among the ones who required periodontal therapy, in the Periodontology Department of Dentistry Faculty, Ovidius University of Constanța. The recruitment of the patients was done during Summer of 2021. Patients included in the control group were selected among the subjects that declared that are not willing to came at 10 appointments for Alflutop injections, but they agreed with the SRP therapy alone and the second evaluation after one month. The patients from the test group agreed that after SRP treatment to be scheduled for the 10 appointments for Alflutop therapy.

Diagnostic criteria were not given for periodontitis. Was it based on previous guidelines or new guidelines? What was the level of the attachment loss? It is important as deeper pockets normally can get more clinical attachment than a normal depth crevice just by the custom mechanical treatment.

A periodontitis case was defined according to the new classification proposed by consensus in the last World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions in 2017 [18]. So, a periodontitis case was considered when: interproximal clinical attachment loss (CAL) in ≥2 non-adjacent teeth, or ≥2 teeth with buccal or palatal site affected by CAL ≥3 mm and PD >3 mm (in the same site) are detected and PD ≥4 mm.

Gingival index: is it the Löe-Silness index (1963)? In that case score 1 is colour change of the gingiva+ no BOP, score 3 means severe bleeding on probing, not spontaneous bleeding!

GI scores were [2]: 0-normal aspect, 1-slight change in color and texture of the gingiva, no bleeding, 2- inflammation and bleeding on probing, 3-advanced inflammation, severe bleeding on probing.

What was the reason (theory behind) that Alflutop was administered “mostly” to the canine –premolar area? Upper or lower arch? If it was not “mostly” than where?

If the periodontal disease is localized, the infiltrations are done in the mobile mucosa of affected sites. In generalized forms, at the canine-premolar mobile mucosa.-I have included this explanation also into the text:

After SRP, cases received infiltrations with Alflutop® solution, into mobile mucosa of the affected sites, or, in case of generalized periodontitis into mobile mucosa of canine-premolar area, 2-3 times weekly, until all 10 vials finished (approximately 3- 4 weeks).

Data are presented as mean ± standard deviation (SD) or percentage for categorical variables. Two proportion z-testallow the comparison of two proportions,to see if they are the same, being used to evaluate the evolution of the GI, PBI, and degree of pathologic tooth mobility in both control and test group, before and after therapy.The null hypothesis (H₀) for thetestis that theproportionsare the same. Wilcoxon signed rank test and Mann Whitney U test were used to compare the difference between probing depth (max) before and after therapy in both test and controls. Levene’s test for equality of variances was used to assess the age and sex distribution among the subjects in both groups.The subject was the unit of analysis for all statistical comparisons with the level of statistical significance set at p<0,05.

Sample selection bias, confirmation bias, publishing bias could influence the results. To address potential publishing and confirmation bias, the non-significant improvements of the clinical parameters were reported together with relevant correlations. The loss of participants could influence the study results, as the control group lost many participants then the test group. The performance bias was addressed providing same treatment protocol in both groups (SRP), followed by additional 10 infiltrations with the active ingredient in the test group, the patients that could not present for all 10 injections, being excluded from final evaluation. Also, smokers were excluded from the study participants.

Please, also make comparisons on the initial stage variables to final stage variables of the periodontal condition in the same group. Practically, the improvement in periodontal condition must be significantly different to prove the effectivity of Alflutop®. Both for case and control groups. The reduction of mobility is secondary to inflammation reduction. The lack of side-effects must go to the result section.

Indeed, the clinical improvement of the periodontal condition was not significantly of all clinical parameters in test group, therefore the conclusion is not very strong.

No side effects of Alflutop® were reported, not even pain at the infiltration sites.

Discussion part: start with your own main findings, then compare results. Discuss limitations of the study, as well.

The strength of the study might be that it includes both men and women, with different age. Also, no similar study was published before, this could reflect its originality, but also, a limitation because the comparison of the study results with similar research is impossible to de done. Other limitations of the study include the randomly selection of the participants, among the patients addressed for periodontal examination. Due to COVID 19 pandemic variations, the addressability of patients to dental care decreased, and an invitation for study participation could have a very low response rate. Another limitation of the study was the level of oral hygiene, which varied between subjects during study. The low number of study participants is another limitation, this could be the consequence of current pandemic, because 23 of the initially enrolled patients did notfinalize the protocol, even study protocol did not require a long-term assessment.

Thank you very, very much for the review report and for the extremely useful suggestions!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Reviewer 4 Report

The introduction provides a good, generalized background of the topic that quickly gives the reader an appreciation of the wide range of problems about the periodontal healing.However, to make the introduction more substantial, the author may wish to provide several references to substantiate the claim made in the introduction (that is, provide references to other groups who do or have done research in this area). Materials and methods- My biggest concern is the number of participants.

Could you include in the title of this paper that it is a preliminary study? " A preliminary study of the clinical effects of a marine fish extract on the periodontal healing".

Discussion- this paragraph should be rearranged. you should explain and highlight better what you have achieved.

Author Response

According to the reviewer’s recommendations, all the suggestions were carefully considered, as follows:

The introduction provides a good, generalized background of the topic that quickly gives the reader an appreciation of the wide range of problems about the periodontal healing. However, to make the introduction more substantial, the author may wish to provide several references to substantiate the claim made in the introduction (that is, provide references to other groups who do or have done research in this area).

Previous research used the marine fish extract (Alflutop) in dentistry in endodontics, in chronic periapical periodontitis, after cleaning and shaping the root. The results showedregenerative properties of Alflutop used in endodontic temporary root canal treatment.

Materials and methods- My biggest concern is the number of participants.

The numbering errors were corrected as follows:

From the test group, only 25 patients could finalize the research. The rest of 9 patients did not finalize the protocol (2 patients did not present in time and could not finish all 10 infiltrations, 7 patients did not present for the final assessment one month after therapy). Among the test group, after SRP therapy, a number of 14 patients did not come at the final examination, one month after therapy. So, the final study groups are group I (test) with 25 cases, and control group II with 20 patients

Could you include in the title of this paper that it is a preliminary study?

Thank you very much for the good tile change suggestion,

Measurement of the clinical effects of a marine fish extract on periodontal healing - a preliminary clinical interventional study

Discussion- this paragraph should be rearranged. you should explain and highlight better what you have achieved.

I have rearranged the Discussion paragraph according to Your suggestions, thank you very much!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Reviewer 5 Report

Authors has undertaken an interesting subject. Clinical protocol is well designed.

There is some information which should be explained.

The number of patients is not correct in correct: in line 115 it is 35, in line125 it is 55 and in table 1 it is 15+12 which gives 27.

The authors report that the final measurements were compared with the patients' initial state. These results could be compared with a chlorhexidine preparation, then the effectiveness of the new gingivitis treatment could be evaluated.

A control group would also need to be added in which the inflammation was rinsed with the solvent alone without the addition of the extract. Then the reduction of inflammation would be confirmed by the effect of the extract.

The graphs presented in the paper are unreadable. It is not clear if the data presented refer to the percentage of tooth mobility reduction or if they refer to the percentage of tooth condition improvement.

Comments for author File: Comments.docx

Author Response

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

Authors has undertaken an interesting subject. Clinical protocol is well designed.

There is some information which should be explained.

The number of patients is not correct in correct: in line 115 it is 35, in line125 it is 55 and in table 1 it is 15+12 which gives 27.

The numbering errors were corrected as follows:

From the test group, only 25 patients could finalize the research. The rest of 9 patients did not finalize the protocol (2 patients did not present in time and could not finish all 10 infiltrations, 7 patients did not present for the final assessment one month after therapy). Among the test group, after SRP therapy, a number of 14 patients did not come at the final examination, one month after therapy. So, the final study groups are group I (test) with 25 cases, and control group II with 20 patients.

Indeed, the use of an antiseptic product in conjunction with SRP can have beneficial effects on the gingival inflammation. According to new EFP guidelines (Mariano Sanz,David Herrera,Moritz Kebschull,Iain Chapple,Søren Jepsen,Tord Berglundh,Anton Sculean, Treatment of stage I–III periodontitis—The EFP S3 level clinical practice guideline, https://doi.org/10.1111/jcpe.13290), antiseptics can be used as adjunctives in the treatment of periodontitis (grade 0 recomandation), also adverse effects should be taken into account. The recomandation is open, this supporting our idea to evaluate the clinical effects of other compounds, such natural products, which could have clinical benefits on the periodontal healing.

The idea of an additional control group who received the solvent alone was taken into consideration, but the Alflutop product is available commercially in pharmacy shops in sterile individual vials, so it is impossible to separate the solvent. Also, the application of a sterile saline solution (a non active product) by 4 injections (infiltration) was not approved from ethical reasons, the subjects from control group should not be subjected to additional pain (caused by the 4 injectionsx10 appointments).

The graphs are illustrating the percentage of tooth mobility reduction in both groups, after therapy (may be it could be useful to include the tables as well)

Tooth mobility (max) Index
Alflutop
Before					After
Index	Frequency		Percent		Index	Frequency		Percent
0	0		0.00%		0	6		24.00%
1	8		32.00%		1	19		76.00%
2	17		68.00%		2	0		0.00%
3	0		0.00%		3	0		0.00%
Total	25		100.00%		Total	25		100.00%

Tooth mobility (max) Index
Control
Before					After
Index	Frequency		Percent		Index	Frequency		Percent
0	11		55.00%		0	11		55.00%
1	6		30.00%		1	6		30.00%
2	1		5.00%		2	3		15.00%
3	2		10.00%		3	0		0.00%
Total	20		100.00%		Total	20		100.00%

		Index
		0		1			2
Alflutop	Total	25		25			25
	N1	6		19			0
	p1	0.2400		0.7600			0.0000
	%p1	24.00		76.00			0.00
Control	Total	20		20			20
	N2	11		6			3
	p2	0.5500		0.3000			0.1500
	%p2	55.00		30.00			15.00
	%(p1-p2)	-31.00		46.00			-15.00
	OSP	0.3778		0.5556			0.0667
	z	-2.131		3.086			-2.004
	abs(z)	2.131		3.086			2.004
	P(z<zcr)	0.983		0.999			0.977
	p	0.033		0.002			0.045
	Decizie	DA		DA			DA

Thank you very much for the review report and for the useful suggestions!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thanks for the corrections sent

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled first “The clinical effects of a marine fish extract on the periodontal healing”, co-authored by Cristina Gabriela Puşcaşu * , Aureliana Caraiane , Elena Dumea , Erdogan Elvis Șachir , Gheorghe Raftu , Radu Andrei Puşcaşu , Cristina Bartok-Nicolae * , Radu Eduard Cernei * , Loredana Liliana Hurjui *

Thank you very much for all your suggestions and good advice!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Reviewer 3 Report

Dear Authors,

Though the study improved a lot, but I still have some concerns. I still do not see the method for patient randomization and examiner blinding. I have also asked about the origination of the method used for “Aflutopf” administration (reference-as one of them is on tumours not periodontal disease, the other one is in Rumanian- or was it just your thought? In case of the least, please, explain why!). In the results the improvement of pathologic mobility was pointed out. But there is no data on the factors affecting initial mobility. As the mobility is affected by the height of the periodontal support, periodontal inflammation, and occlusal load, it would have been advantageous to show the staging of periodontal disease (2017 classification was referenced). As mobility reduction is not only relying on inflammation reduction.

Lines 196-198 are better to go to discussion, as it is an explanation of data. I still miss the flow chart which was advised in my previous review. Missing patients on final examinations must be characterized by their initial values, just to see that what type of patients did not turn up at final examination: those who were in better condition initially, or those who were in worse condition at the beginning both cases and controls. Practically this could be a limitation of the study.

In Figure 1. values are presented in %. Is it BOP or GI? As Gi is not presented as percentage!

Comments for author File: Comments.docx

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript initiallyentitled “The clinical effects of a marine fish extract on the periodontal healing”, co-authored by Cristina Gabriela Puşcaşu * , Aureliana Caraiane , Elena Dumea , Erdogan Elvis Șachir , Gheorghe Raftu , Radu Andrei Puşcaşu , Cristina Bartok-Nicolae * , Radu Eduard Cernei * , Loredana Liliana Hurjui *

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

Though the study improved a lot, but I still have some concerns. I still do not see the method for patient randomization and examiner blinding.

The initial study group included 68 subjects, divided in two groups of 34 subjects each (group I-test and group II-control). Because of the small number of sample size, the simple randomization method was used, by computer-generated numbers assigned initially for every subject, then, the numbers were associated randomly, either within test or control groups. Group I (test) received Alflutop®, after scaling, root planning, debridement (SRP), and group II (control) received only SRP therapy.

Periodontal status was assessed for both cases and controls before and after one month of periodontal therapy, by a single-blinded examiner, who did not discuss with the patients about the type of periodontal treatment they received.

I have also asked about the origination of the method used for “Aflutopf” administration (reference-as one of them is on tumours not periodontal disease, the other one is in Rumanian- or was it just your thought? In case of the least, please, explain why!).

I have included in lines 231-234 the following:

Periodontal disease is similar to osteoarticular diseases, being characterized by the loss of tooth supporting tissues (alveolar bone and periodontal ligaments) induced by an immune response activated by periodontal pathogens. This is the reason why we have used Alflutop infiltrations as adjuvant for periodontal treatment.

Also, I have found indications of Alflutop use in English language, on the above website, but I am not sure if I should add this link at the reference list

https://gerovital4you.com/products/alflutop-biotehnos-1ml-vials

Alflutop is characterized by analgesic and anti-inflammatory action. Alflutop has chondroprotective influence, it effectively regulates cartilage metabolism. Chondroprotective drugs are based on the property of reducing the activity of specific enzymes, including hyaluronidases. These enzymes play a major role in stabilizing the biosynthesis of type II collagen and hyaluronic acid and are involved in the destruction of the extracellular matrix.
The drug reduces the destruction of macromolecular structure of the base material of the connective tissue, reduces the permeability of capillaries. Furthermore, Alflutop stimulate tissue repair process of the articular cartilage, and interstitial tissue. Proteoglycans, which are a component of the drug, cause the trophic effect. With a replacement effect, they greatly increase the performance of MRI-hydrophilic cartilage, its height and uniformity of bone tissue.
Due to this the medicament reduces pain force at rest and in motion. Significantly reduced pain when walking on a flat road, sometimes even while walking up the stairs. Markedly reduced the severity of contracture and local swelling. Thus Alflutop facilitates overall motility of affected joints, increases the volume of its movements.
Besides the drug reduces the globulins seromucoid, C-reactive protein, fibrinogen, and the number of leukocytes into the synovial fluid.
After treatment total duration of 6 months in the synovial fluid levels of hyaluronic acid is increased in two times. But the anti-inflammatory effect is noticeable already at 8 days of therapy.

Alflutop indications for use:
Alflutop prescribed for the following diseases and conditions:

Osteochondrosis;
Osteoarthritis, primary and secondary, various sites, including arthritis of small joints, coxarthrosis and gonarthrosis;
Traumatic dysostosis;
Spondylosis;
Periarthritis;
Endochondral ossification and Chondral violations;
Paradontopatiya;
Fibromyalgia.

Alflutop is also administered in the recovery period after surgery, carried on the joints.

In the results the improvement of pathologic mobility was pointed out. But there is no data on the factors affecting initial mobility. As the mobility is affected by the height of the periodontal support, periodontal inflammation, and occlusal load, it would have been advantageous to show the staging of periodontal disease (2017 classification was referenced). As mobility reduction is not only relying on inflammation reduction.

Lines 196-198 are better to go to discussion, as it is an explanation of data.

Lines 196-198 were added to discussion session:

Additional therapy with Alflutop® can contribute also to a reduction of inflammation not only in gingival tissues, but also at the level of the periodontal ligament fibers, suggested by the decrease of pathologic tooth mobility, supporting the hypothesis of anti-inflammatory action in both gingival tissues and periodontal ligament fibers.

I still miss the flow chart which was advised in my previous review.

Missing patients on final examinations must be characterized by their initial values, just to see that what type of patients did not turn up at final examination: those who were in better condition initially, or those who were in worse condition at the beginning both cases and controls. Practically this could be a limitation of the study.

The low number of study participants is one final limitation, this could be the consequence of current pandemic, because 23 of the initially enrolled patients did not finalize the protocol, even study protocol did not require a long-term commitment. Those 9 patients from test group and 14 patients from control had similar initial periodontal condition with the rest of patients, but their periodontal condition after treatment could not be recorded, which might influence the final findings.

In this respect, further research is needed on product compliance and acceptance by different populations and age groups.

In Figure 1. values are presented in %. Is it BOP or GI? As Gi is not presented as percentage!

Figure 1 represent the percentage of cases which recorded reduction of GI scores

Figure 1. Evolution of Gingival Index in cases compared to controls (percentage of cases exhibiting GI reduction in both control and test group).

A two proportion z-test allows to compare two proportions to see if they are the same.

The null hypothesis (H₀) for the test is that the proportions are the same.

The alternate hypothesis (H₁) is that the proportions are not the same.

The results are the following: -14 patients from test group (56%) and 13 patients from control group (65%) had GI 0, at the reevaluation.No statistical correlation was found between these proportions (p = 0.540 >α = 0.05).

-11 patients from test group (44%) and 4 patients from controls (20%) gad GI 1. No statistical correlation was found between these proportions (p = 0.090 >α = 0.05).

Thank you very much for both review reports and for the extremely useful suggestions!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Reviewer 4 Report

The authors has revised according the suggestions.

I would suggest to cite this paper in section introduction:Chitosan use in dentistry: A systematic review of recent clinical studies DOI10.3390/md17070417.

I think that this systematic review could be improved the paper.

Author Response

According to the reviewer’s recommendations, the suggestions were carefully considered, as follows:

I would suggest to cite this paper in section introduction:Chitosan use in dentistry: A systematic review of recent clinical studies DOI10.3390/md17070417.

I think that this systematic review could be improved the paper.

Thank you very much for the useful suggestion, I have included in the discussion section this systematic review (lines 253-254). It is of great help for the present study, indeed!

Thank you very much for all your suggestions and good advice!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

Author Response File: Author Response.docx

Reviewer 5 Report

The authors corrected all elements of the paper suggested for improvement. The paper may be considered for further publication.

Author Response

Thank you very much for all Your suggestions and good advice!

I remain most respectfully yours,

Dr. Cristina Gabriela Puşcaşu

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