Innovative Alveolar Socket Preservation Procedure Using Demineralized Tooth Dentin as Graft Biomaterial Covered with Three Reabsorbable Membranes: Human Histological Case Series Evaluation

Round 1

Reviewer 1 Report

The study is very well structured. Still there are some issues that need to be addressed before the publication of the study.

1. The authors state that “……after the healing period, (follow the patients requirement) bone biopsies using 4x18 mm 133 graduated trephine cylindrical drills (Meisinger, Neuss, Germany) were performed, 134 matching the center of the original extraction socket in the area….” How was this matching of the center of the original extraction socket achieved? Was it through visual distinctions between the graft and surrounding area, a prefabricated jig, or the use x-ray? Please further elaborate in the methods and materials..

2. In the “Histology” section, please mention what staining protocol was utilized for the histological samples.

3. Was any patient related outcomes (pain, edema, …) compared between the two groups or not? If there was no comparison between these two groups please discuss this at the end of the discussion by adding a limitations recommendations section and recommend that future studies measure patient reported outcomes as well.

4. In the 190, 191 line: “In a recent literature review the different market graft materials histomorphometric results were compared without to show the best biomaterial to obtain the ideal solution.” Does not make any sense, please revise this sentence.

5. The sentence “Therefore, in the literature, actually no scientific or clinical evidence regarding the ARR procedure were showed.” Should also be edited.

6. The statement “the encouraging histological and histomorphometrical results of the bone biopsies were showed in a several publications.” Only cites one study. There should be a citation to several studies or the cited study should be a systematic review.

7. Overall, the study and specially the Discussion section need an English language overhaul.

Author Response

thank you for your suggestions

1. Line 126 added Four months after surgery, through a periodontal probe measurement with the mesial tooth, into the center of the original extraction socket, specific bone biopsies were obtained using graduated trephine cylindrical drills (4 x 18 mm Meisinger, Neuss, Germany).
2. Line 171 added and colored with hematoxylin and eosin
3. Was eliminated it because no critical evaluation system was used. First version Line 255 : we delete the part

4. Line 235 modified In a recent histomophometrical literature review the different market graft materials were compared without showing the best biomaterial
5. The sentence was cancelled
6. added the references 16-17-18
7. The article is proofed from a professional mother tongue (Joan L. Oliver) as referred to the editor

Reviewer 2 Report

Firstly congratulation to the authors for conducting this interesting clinical study. Its not common to secure ethics approval for such human studies particularly when post-operative biopsy is needed for research purpose only. Therefore this study findings is considered "rare" and valuable.

This manuscript can be further improved along the following comments:

1. Materials and Methods

At Line 75: Please describe the age range of the patients who participate in this study.

At line 78: the term "alveolar socket reconstruction' was used. Need to use consistent terminology either "alveolar socket reconstruction' or 'alveolar socket preservation' throughout the manuscript.

Surgical Protocol:

It is important to describe which tooth was selected for the study in since healing of teeth at different sites and in the upper and lower jaw is different. Need to mention the site of biopsy (which tooth socket) in the histology too.

Author must mention how residual tooth socket walls were measured immediately after the tooth extraction.

Please mention the type of chemical and its concentration use for decontamination of the oral cavity and extracted tooth.

The dentine was extracted from the tooth and was demineralized. Please mention the type of chemical (acid ?) and at which concentration and the time taken to immerse the dentine tissue during demineralization procedure. 

Was there any post-operative dentin grafting radiographs done to compare  the grafted sites at 4 months? There could have been radiograph taken after the bone biopsy and implant insertion and this could be a valuable image of peri-implant dentin graft in the socket, and compare it among the patients.

The authors describe the healing process in the maxilla but the clinical photographs do not clearly show the maxillary dental arch. Please select other clinical photos that are much clearer if available.

STATISTICAL ANALYSIS:

At line 158, data statistical analysis was carried out to obtain average values. It is preferable to calculate mean values, with standard deviations.

RESULTS:

The Methodology did not include CBCT in the planning but the Results describe CBCT measurements. Please correct this discrepancy. 

At Line 167, the observation mentioned was between 20 weeks and 34 weeks; but the text mentioned follow-up at 4 months;

and at Line 133, "after the healing period, (follow the patients requirement) bone biopsies were performed. Does this mean that the bone biopsies was performed at different time point for each patient depending on their individual need?

At Line 168 : Please describe the methods of measuring bone width (in the Methodology Section) in this study.

Table 1 : i) Are patients 'C' and 'C'  and 'B' and 'B' the same patients having two separate operations?

              ii) What is the unit use in the values describe in Table 1 ?

              iii) A 'mean' value will need a standard deviation 

It is not clear what does 'Vertical bone variation' and 'Horizontal bone variation' mean? What does the word 'variation' signify?

DISCUSSION:

Based on the authors research question, whether flapless or using 3 layers of membrane will give different results, there is no discussion on this important point in this study.

At Line 224, the authors mentioned the following results : "While the test group has a high density but loses 15% percentage of volume, the control group maintains the volume but has a minor density". Please discuss this interesting and important finding.

Author Response

Thank you for your suggestions

(in the attached file there are some images)

the english has been totally checked and corrected by a professional 

At Line 75: Please describe the age range of the patients who participate in this study.

Line 89 added - from 50 to 73

At line 78: the term "alveolar socket reconstruction' was used. Need to use consistent terminology either "alveolar socket reconstruction' or 'alveolar socket preservation' throughout the manuscript.

Line 93 erased reconstruction and added preservation

It is important to describe which tooth was selected for the study in since healing of teeth at different sites and in the upper and lower jaw is different. Need to mention the site of biopsy (which tooth socket) in the histology too.

line 106 added : needing the extraction of a mandibular molar

Author must mention how residual tooth socket walls were measured immediately after the tooth extraction.

Line 112 added :dimensions were recorded using CBCT measurement and again  after healing period (4 months)

Please mention the type of chemical and its concentration use for decontamination of the oral cavity and extracted tooth.

Line 108 added : Two weeks before the oral surgery treatment, all patients received a professional oral           hygiene session, and chlorhexidine 0.2% mouth rinses, twice a day for two weeks, were prescribed.

The dentine was extracted from the tooth and was demineralized. Please mention the type of chemical (acid ?) and at which concentration and the time taken to immerse the dentine tissue during demineralization procedure. 

Line 122 added : The liquids used are constituted by hydrochloric acid,  hydrogen peroxide and demineralized water as a wash.

(The others informations , time, temperature etc are covered by a patent and are not available)

Was there any post-operative dentin grafting radiographs done to compare  the grafted sites at 4 months? There could have been radiograph taken after the bone biopsy and implant insertion and this could be a valuable image of peri-implant dentin graft in the socket, and compare it among the patients.

We don’t add the xray images because the article is full of images and 20 xray are too much

but we send you some cbct images to better understand

The authors describe the healing process in the maxilla but the clinical photographs do not clearly show the maxillary dental arch. Please select other clinical photos that are much clearer if available.

This study was conceptualized to produce Histological and Histomorfometrical informations about the regenerated sites. That is the reason why we don’t made clinical photographs showing the maxillary dental arch

The Methodology did not include CBCT in the planning but the Results describe CBCT measurements. Please correct this discrepancy. 

At the line 85 (before the revision) was described the cbct measurement

Line 112 added : dimensions were recorded using CBCT measurement and again  after healing period (4 months)

At Line 167, the observation mentioned was between 20 weeks and 34 weeks; but the text mentioned follow-up at 4 months;

Line 200 cancelled In a range between 20 and 34 weeks added :4 months after the

and at Line 133, "after the healing period, (follow the patients requirement) bone biopsies were performed. Does this mean that the bone biopsies was performed at different time point for each patient depending on their individual need?

The healing period was 4 months for all patients

At Line 168 : Please describe the methods of measuring bone width (in the Methodology Section) in this study.

Line 112 added : dimensions were recorded using CBCT measurement and again  after healing period (4 months)

Table 1 : i) Are patients 'C' and 'C'  and 'B' and 'B' the same patients having two separate operations?

The B and C are the surname initials. I agree that in this way it is not clear 

I changed the table with incremental letters

What is the unit use in the values describe in Table 1 ?

Line 210 added : The volume was measured before surgery with CBCT and after 4 months and the images were superimposed. During the measurement (millimeters) before surgery the hope dimensions (vertical and horizontal) were signed and compared with the same dimensions (vertical and horizontal) superimposing the image after 4 months (Fig.3). The differences in percentages are shown in table 1

Fig.3 the superimposition of these two CBCT sections allows to understand the regeneration of 0.81 mm greater than expected

 A 'mean' value will need a standard deviation

These value are absolute values not mean value

It is not clear what does 'Vertical bone variation' and 'Horizontal bone variation' mean? What does the word 'variation' signify?

Line 210 added : The volume was measured before surgery with CBCT and after 4 months and the images were superimposed. During the measurement (millimeters) before surgery the hope dimensions (vertical and horizontal) were signed and compared with the same dimensions (vertical and horizontal) superimposing the image after 4 months (Fig.3). The differences in percentages are shown in table 1

Fig.3 the superimposition of these two CBCT sections allows to understand the regeneration of 0.81 mm greater than expected

Based on the authors research question, whether flapless or using 3 layers of membrane will give different results, there is no discussion on this important point in this study.

Line  278 added : This article compares this new technique with a traditional technique using a patient’s tooth as biomaterial. It is also possible to compare the results obtained by Grassi (15), who used the same new  technique but with a market xenograft biomaterial. It is interesting to observe that: the mean value among eight patients showed by Grassi is very different to that found in this research. The residual graft was 31,8+-8,3 and the vital bone was 25,4+- 8,7. In our study the obtained results, as showed before, are better. The residual graft is  5,24 (GA) and 5,29 (GB) and the  Vital bone is  57,53 (GA) and 42.41 (GB) . Probably the difference is due  to the autologous graft material wich is very similar to the bone.

At Line 224, the authors mentioned the following results : "While the test group has a high density but loses 15% percentage of volume, the control group maintains the volume but has a minor density". Please discuss this interesting and important finding.

Line 275 added It was possible to draw these conclusions as the volume, measured with the superimposition, was connected with the histological and histomorphometrical results

Reviewer 3 Report

This study histologically and clinically evaluated a new flapless socket-sparing procedure with dentin graft materials extracted from demineralized teeth covered by three resorbable membranes. His main contribution is in the field of surgery, periodontics and oral implantology, to publicize a new flapless socket preservation technique.

The manuscript is clear, relevant, and presented in a well-structured manner. In addition, it is scientifically sound and presents an appropriate design to test the hypothesis. Nearly 50% of the cited references are from the last 5 years. The results are reproducible depending on the details given in the methods. The tables and images are appropriate, correctly display the data, and are easy to interpret and understand. Data are interpreted appropriately and consistently throughout the manuscript. A correct statistical analysis is carried out and the conclusions are consistent with the evidence and the arguments presented.

However, it presents the following points to take into account for an improvement of the study:

- It is mentioned that the study took the Helsinki Declaration of 2008 as an ethical consideration, when the latest version is from 2013.

- No mention is made of how the distribution of patients was made for each group

- It is mentioned that a box containing disposable liquid solutions was used to guarantee the decontamination of the grafts, if perhaps the authors could mention these substances in the study, in order to have a little more clarity on the complete methodology.

- Although in the images it can be seen that work was done in posterior sectors, it would be good if the text mentions whether this was the case or whether anterior teeth were also worked on.

Author Response

Thank you for your suggestions

It is mentioned that the study took the Helsinki Declaration of 2008 as an ethical consideration, when the latest version is from 2013.

Line 94 cancelled 2008 and added 2013

No mention is made of how the distribution of patients was made for each group

Patients were randomly distributed between the two study groups by drawing lots from outside the study group with this  scheme

5 Pz group A , 5 Pz group B

It is mentioned that a box containing disposable liquid solutions was used to guarantee the decontamination of the grafts, if perhaps the authors could mention these substances in the study, in order to have a little more clarity on the complete methodology.

Line 103 added : the liquids used were constituted by  hydrochloric acid, hydrogen peroxide and demineralized water as a wash (The others informations , time, temperature etc are covered by a patent and are not available)

Although in the images it can be seen that work was done in posterior sectors, it would be good if the text mentions whether this was the case or whether anterior teeth were also worked on.

line 108 added : needing the extraction of a mandibular molar

Reviewer 4 Report

Please re-write the background section in abstract. Be more descriptive and put the aim of the study within the background. Please rephrase the aim of the study to make it clear.

Please number the tables in order of occurrence. If you quote table 2 in the introduction, please set it as number one and move it closer to the place of citation. Otherwise, delete the sentence. Where is table 2?

The aim, nor conclusions does not say anything about demineralized dentin.

After extraction socket with four bone walls, pregnant subjects, patients with a history of allergies, tobacco use (within the last six months), diabetes, cancer, human immunodeficiency virus (HIV), bone or metabolic diseases, immunosuppressive agents, use of systemic corticosteroids, intramuscular/intravenous bisphosphonates, radio or chemotherapy were used as a exclusion criteria. pre-surgical cbct measurement evaluation was performed in all patients before extraction. – there is a syntax error, please re-write the whole phrase. Inclusion and exclusion criteria should be presented before mentioning the final number of the patients included. Was any of interested patient exclude because of these criteria? Please re-write this paragraph.

What is bone behaviour? Isn't this a translation error from Italian?

Please widen the topic of histomorphometric results in your discussion. There are at least dozen of the articles about demineralized dentin as scaffold which are present in PubMed and weren’t mentioned in Your discussion.

Please discuss the limitations of your pilot study.

In conclusions You mentioned the difference in morbidity in two groups. However, You did not mention how possible morbidity was measured in results section. Please re-write the conclusions and present them in form of bullet points.

Please adjust the reference style to cope with journal instructions for authors. (MDPI style)

There are a lot of syntax error within the text. Many looks like artificially translated. Please revise the syntax carefully. Also in the discussion, I feel the lack of preservation of the train of thought. 

The manuscript looks like as if it has been transferred by the authors without due care and respect to the journal.

Author Response

thank you for your suggestions

please see the attachment

the English language has been totally checked and corrected by a professional  proof reader for several medical research journals

Please re-write the background section in abstract. Be more descriptive and put the aim of the study within the background. Please rephrase the aim of the study to make it clear.

The background and the aim are concerning  to the flapless alveolar preservation and  the choice of comparing the preservation by means of three barriers was because there is a scientific work (reference 15) that talked about major improvements with three membranes. The authors wanted to compare this new technique with a traditional technique using a patient’s tooth as biomaterial instead of market biomaterial.

Please number the tables in order of occurrence. If you quote table 2 in the introduction, please set it as number one and move it closer to the place of citation. Otherwise, delete the sentence. Where is table 2?

I’m sorry was an misprint of previous version

Line 56 deleted

The aim, nor conclusions does not say anything about demineralized dentin.

Our test was not for dentin but with dentin. We use dentin as a material, as has already been demonstrated with articles made by us. If you demand we can send you all our dentin articles

After extraction socket with four bone walls, pregnant subjects, patients with a history of allergies, tobacco use (within the last six months), diabetes, cancer, human immunodeficiency virus (HIV), bone or metabolic diseases, immunosuppressive agents, use of systemic corticosteroids, intramuscular/intravenous bisphosphonates, radio or chemotherapy were used as a exclusion criteria. pre-surgical cbct measurement evaluation was performed in all patients before extraction. – there is a syntax error, please re-write the whole phrase. Inclusion and exclusion criteria should be presented before mentioning the final number of the patients included. Was any of interested patient exclude because of these criteria? Please re-write this paragraph.

Line 97 added : The exclusion criteria were: pregnant subjects, patients with a history of allergies, tobacco use (within the last six months), diabetes, cancer, human immunodeficiency virus (HIV), bone or metabolic diseases, immunosuppressive agents, use of systemic corticosteroids, intramuscular/intravenous bisphosphonates, radio or chemotherapy. Before the  extraction, pre-surgical CBCT (cone-beam computed tomography) measurement evaluation was performed in all patients,again  after healing period (4 months).

What is bone behaviour? Isn't this a translation error from Italian?

I found bone behavior at line 183. That means The behavior of regenerated tissue is different in the two groups, as  can be seen from histologies.

Please widen the topic of histomorphometric results in your discussion.

Line 278 added : This article compares this new technique with a traditional technique using a patient’s tooth as biomaterial. It is also possible to compare the results obtained by Grassi (15), who used the same new  technique but with a market xenograft biomaterial.

IT is interesting to observe that: the mean value among eight patients showed by Grassi is very different to that found in this research. The residual graft was 31,8+-8,3 and the vital bone was 25,4+- 8,7. In our study the obtained results, as showed before, are better. The residual graft is  5,24 (GA) and 5,29 (GB) and the  Vital bone is  57,53 (GA) and 42.41 (GB) . Probably the difference is due  to the autologous graft material wich is very similar to the bone.

There are at least dozen of the articles about demineralized dentin as scaffold which are present in PubMed and weren’t mentioned in Your discussion.

This is an interesting question. We spent some years in our university  trying to find the best way to treat the dentin . The topic point is that the bone regeneration results are influenced by the demineralization process. The surface of the dentin after the treatments can be dirty or clean and that influences the cells adhesion and that depends to the different treatment. If the surface is clean the adhesion is better. But if the treatment is too aggressive (even with only 0,1M difference of the same acid)  the dentin loses some proteins. So, the demineralized dentin is not always the  same graft material and determines the same results. We are about to  publish an article were we analyze this aspect. I hereby inclose an image where is possible to note the different surfaces using different molarity of the same acid. These images are not printables neither by reviewers nor by editor because are in press. 

Please discuss the limitations of your pilot study

Line 298 added: This study has some limitations: the first being the small number of samples and therefore the poor statistics; other limitations could be the lack of comparison with different biomaterials as well as the use of only one type of absorbable membrane. Therefore, new randomized clinical trials with a greater sample size and different defect shapes are required to evaluate the potentially advantageous properties of the present technique.

in conclusions You mentioned the difference in morbidity in two groups. However, You did not mention how possible morbidity was measured in results section. Please re-write the conclusions and present them in form of bullet points.

Line 255 :we accepted your indication and delete the part

Please adjust the reference style to cope with journal instructions for authors. (MDPI style)

References are adjusted follow the MDPI style

There are a lot of syntax error within the text. Many looks like artificially translated. Please revise the syntax carefully. Also in the discussion, I feel the lack of preservation of the train of thought. 

The article is proofed from a professional mother tongue

The manuscript looks like as if it has been transferred by the authors without due care and respect to the journal.

the authors have great respect for the prestigious magazine to which they are entrusting the publishing of important clinical and scientific results derived from their research.

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

Thank You for improvements made.

Again - in materials and methods section. First inclusion and exclusion criteria, then the number of patient enrolled. Remember that is should describe You enrollment process and looks much more professional. Please revise.

The images that You made in the response to my comments are really interesting. However, You did not put any additional information within manuscript in regard what was asked from You. You don't have put there any image. The publication of an article in the future must not affect the quality of this manuscript. Just describe briefly what I asked You for. Please revise discussion in this point.

The limitations of the study should not be included in Conclusions. Please put them at the end of the discussion.

Again - change the references for MDPI style.

This is AMA style: Lekholm U, Gro¨ndahl K, Jemt T. Outcome of oral implant treatment in partially edentulous jaws 311 followed 20 years in clinical function. Clin Implant Dent Relat Res 2006;8:178–86.

This is MDPI style Lekholm, U.; Gro¨ndahl, K.; Jemt, T. Outcome of oral implant treatment in partially edentulous jaws 311 followed 20 years in clinical function. Clin Implant Dent Relat Res 2006, 8, 178–86.

Author Response

Thank you for your kind suggestions

1- in materials and methods section. First inclusion and exclusion criteria, then the number of patient enrolled. Remember that is should describe You enrollment process and looks much more professional. Please revise.

The inclusion and exclusion criteria are moved to first.

Line 85 : General inclusion criteria (asa-1 and asa-2) were adopted. The exclusion criteria were: patients with a history of allergies, diabetes, HIV, cancer, bone or metabolic diseases, pregnancy, use of immunosuppressive agents, systemic corticosteroids, intramuscular/intravenous bisphosphonates, tobacco, radio or chemotherapy patients (within the last six months).

2-The images that You made in the response to my comments are really interesting. However, You did not put any additional information within manuscript in regard what was asked from You. You don't have put there any image. The publication of an article in the future must not affect the quality of this manuscript. Just describe briefly what I asked You for. Please revise discussion in this point.

Line 238-246  added: No inflammation, infection or graft rejection  were present after 14 implants withouth a bone buccal wall filled using autologous tooth extracted‐derived material. After 6 months the bone resorption was measured : mesial 0.39±1.19mm and distal 0.42±0.90mm.[24]

Murata and Kim, using different demineralization procedure, have achieved similar results in terms of quality and amount of newly formed bone tissue.[25-26]

In a recent literature review the conclusions are: autogenous teeth have superior clinical performance when compared with other grafts.[27]

24. Taschieri, S.; Morandi, B.;Alberti, A.; Tarasenko, S.;Diachkova, E.; Francetti, L.;Corbella, S. Immediate implant positioning using tooth‐derived bone substitute material for alveolar ridge preservation: Preliminary results at 6 months . Clin Exp Dent Res. 2022,1–8.

25-Murata, M.;Kabir, A.M.D.;Hirose, Y.;Ochi, M.;Okubo, N.;Akazawa, T.; Kashiwazaki, H. Histological Evidences of Autograft of Dentin/Cementum Granules into Unhealed Socket at 5 Months after Tooth Extraction for Implant Placement . J. Funct. Biomater. 2022,13, 66.

26- Kim, Y.; Ku, J-K.;Um, I.W.;Seok, H.;Leem, D.H. Impact of Autogenous Demineralized Dentin Matrix on Mandibular Second Molar after Third Molar Extraction: Retrospective Study. J. Funct. Biomater. 2023, 14, 4.

27-Cenicante, J.; Botelho, J.; Machado, F.; Mendes, J.J.; Mascarenhas, P.; Alcoforado, G.; Santos, A. The Use of Autogenous Teeth for Alveolar Ridge Preservation: A Literature Review. Appl. Sci. 2021, 11, 1853.

3-The limitations of the study should not be included in Conclusions. Please put them at the end of the discussion.

Line 277 the limitations are moved to line 277

This study has some limitations: the first being the small number of samples and therefore the poor statistics; other limitations could be the lack of comparison with different biomaterials as well as the use of only one type of absorbable membrane. Therefore, new randomized clinical trials with a greater sample size and different defect shapes are required to evaluate the potential properties of the present bone preservation technique.

4-Again - change the references for MDPI style.

This is AMA style: Lekholm U, Gro¨ndahl K, Jemt T. Outcome of oral implant treatment in partially edentulous jaws 311 followed 20 years in clinical function. Clin Implant Dent Relat Res 2006;8:178–86.

This is MDPI style Lekholm, U.; Gro¨ndahl, K.; Jemt, T. Outcome of oral implant treatment in partially edentulous jaws 311 followed 20 years in clinical function. Clin Implant Dent Relat Res 2006, 8, 178–86.

I’m very sorry was my mistake. Thank you for your suggestion

The style now was MDPI

Round 3

Reviewer 4 Report

The authors sufficiently addressed my suggestions. Hereby, I recommend the acceptance of the manuscript.