Alveolar Bone Preservation Using a Combination of Nanocrystalline Hydroxyapatite and Injectable Platelet-Rich Fibrin: A Study in Rats

Round 1

Reviewer 1 Report

In this manuscript the authors aimed to examining the effects of combined nanocrystalline HA and IPRF implantation on the expressions of osteoclastogenic and osteoblastogenic markers during bone healing in wistar rats. However, there are major comments, which need to be met before the manuscript can be considered for publication.

Introduction:

Line 66: Please correct the word “inxlusing” to “including”

Line 73: If you once used the abbreviation e.g. HA for hydroxyapatite, please continue with the abbreviation until the end of the manuscript

Line 78: is this really called “hidroxyapatite”

Material and Methods:

General comment: The M&M part is just sparsely written and is not well-structured. There are not details on especially immunohistochemical techniques, neither citations to support the methods used. Therefore, I kindly ask the authors to rewrite the M&M section (also the statistical analysis part).

IHC: Why did the authors just perform an observational calculation of the stainings? Why didn’t the authors quantify e.g. via Image J?

Line 140: latin names like “ad libitum” are usually written in italics

Figure 4: the images of Eppendorf (a-c) are far too small, especially the numbers and letters are hardly readable.

Line 152: There is something wrong in the description of this M&M part. Here you start with “Decapitation was carried out by administering a lethal dose of anesthetics.” However, I don’t think that you mean decapitation.

Results:

General comments: Proper quantification needs to be performed for the IHC samples. The method is inappropriately used in this paper. The graphs are not nicely presented and are not comparable to each other (e.g. letters are different in size, capitals, etc.).

Line 210: As already mentioned, if the authors use the abbreviation TRAP already throughout the text, they should avoid to use the full name in between.

 

Some parts of manuscript are written like a story, which lowers the scientific soundness of the manuscript. 

Author Response

 Dear Reviewer 1

I do respect and appreciate the reviewer for very valuable and constructive comments to my article.

1. Line 66: "incluxing" has been corrected to "including"
2. Line 73: the abbreviation of hydroxyapatite has been adjusted
3. Line 78: hydroxyapatite has been corrected into  hydroxyapatite
4. The immunohistochemistry section in the M&M has been revised by adding supporting literature and revising statistical analysis
5. IHC: in this study the authors did calculations using the combinative semiquantitative scoring method (Fedchenko et al., 2014). The calculations were assisted with the image raster 3 program. Sorry that we did not write this in the article, but it has been corrected.
6. Line 140: correction have been made to  "ad libitum" to be "ad libitum"
7. Figure 4 has been corrected by enlarging the letter a-c
8. Line 156: "decapitation" has been corrected into "euthanasia"
9. Result:  Adding information on the method has been done that the calculation method used combinative semiquantitative scoring (Fedchenko et al., 2014), while measurement used the image raster 3 program.
10. The graph has been corrected by equalizing the size and markings
11. Line 210: the abbreviation of TRAP has been corrected

Reviewer 2 Report

Dear authors,

What is new in your work? Nanohydroxyapatite and platelet rich fibrin are commonly used in regenerative medicine. The experiments are of little interest and the graphical presentation of the results is of low quality. Generally, the work is read without much interest. Please improve the quality of work. Indicate the novelty of research.

Moderate changes should be taken into account.

Author Response

Dear Reviewer 2

I do respect and thank the reviewer for very critical and constructive comments to my article.

1. The new point in my article is testing the effects of nanocrystalline hydroxyapatite made from pensi shells (a shellfish originating from West Sumatra, Indonesia) which was combined with IPRF (PRF in liquid form obtained through the concept of low speed centrifugation). It is hoped that the local ingredients in this material can provide added value and benefits

Reviewer 3 Report

Dear Authors, this paper about alveolar bone preservation using a combination of nano crystalline hydroxyapatite and injectable platelet rich fibrin: a study in rats, is really well performed and well done. I am pretty sure that both scientists and dentists will find it helpful for their job.

Some issues need to be solved before its final publication in the journal.

Abstract: please divide it into introduction, materials and methods, results, conclusions.

Introduction: this part is well written but some more attention on the use of alloplastic materials that can be used in alveolar preservation should be added. This paper maybe helpful: Ludovichetti FS, De Biagi M, Bacci C, Bressan E, Sivolella S. Healing of human critical-size alveolar bone defects secondary to cyst enucleation: a randomized pilot study with 12 months follow-up. Minerva Stomatol. 2018 Aug;67(4):148-155

Materials and methods and results are well described.

Discussion: Lack of contextualization: The discussion lacks context and background information about the topic of the study. It would be beneficial to provide an introduction that explains the importance of tissue regeneration, the role of hydroxyapatite and platelet-rich fibrin, and any existing research or gaps in the field.

Insufficient data analysis: The discussion primarily focuses on describing the results of the study without providing a comprehensive analysis or interpretation of the findings. It would be helpful to include statistical analyses, discuss the significance of the results, and compare them with previous studies to provide a more robust interpretation.

Incomplete discussion of limitations: The author briefly mentions limitations at the end of the passage but fails to address them adequately. It would be valuable to expand on the limitations of the study, such as the small sample size, the limited observation period, and any potential sources of bias or confounding factors. Additionally, suggestions for future research directions or improvements to overcome these limitations would enhance the discussion.

Author Response

Dear Reviewer 3

 I really thank and appreciate the reviewer for highly critical and constructive comments to my article.

1. I broke down the abstract into the introduction, materials and methods, results, and conclusions and adjusted the style of the abstract as requested in the format:  the heading should not be included.
2. Line: 54-57I: reasons for using alloplastic material have been added
3. Line 344-347, 365-369: thediscussion section has been revised and added.
4. Addition has been given to thelimitation of the study.

Reviewer 4 Report

In their study, the authors investigated the effects of nanocrystalline hydroxyapatite applied alone or in combination with injectable platelet rich fibrin on alveolar bone resorption and reformation. They analyzed differences in TRAP-positive osteoclasts, the intensity of ALP and OCN, and bone reformation in male rats. Basically, I unfortunately find little to no new value in this study in its current state. This is due to the already extensive studies on both materials in similar contexts.

The authors also write this: "However, HA has limited osteoinductive properties, because of which researchers try to combine HA with various materials to improve its osteoinductive ability such as injectable platelet rich fibrin (IPRF)." This is very unfavorably stated, as it strongly questions the necessity of this study. Due to the extensive studies dealing with the combination of nHA and IPRF, the authors need to not only introduce accurately known knowledge here but also state the gap in knowledge. Based on this, the question should be presented in more detail, because in its current form, unfortunately, the study does not seem to me to offer sufficient new value.

Beyond that, there are a several general issues that I find very questionable, which I will outline below.

2. Alveolar bone loss is not only problematic in the context of extractions, but in a variety of different oral treatments, as well as concomitant with various local and systemic diseases. Even though the authors have a relatively clear clinical application in mind here, they should keep the beginning of their introduction a bit more general in order to maintain interest in other disciplines.

3. Nanocrystalline hydroxyapatite should not be abbreviated as HA, as this is generally only used for hydroxyapatite. An appropriate abbreviation with nHA, NHA, nano-HA or something like that is recommended and should be changed throughout the manuscript.

4. The analysis of TRAP, ALP and OCN staining seems to me not very valid and quite arbitrary in terms of interpretation of expression levels. This makes me doubt the results. Are there any reference studies with similar evaluation for this? It seems to me that this is highly dependent on the individual performing the study.

5. How exactly was a distinction made between "new" bone material and existing bone material, this should also be stated at the appropriate place in the results.

6. Statistical information should be included throughout the diagrams. This seems to be missing and incomplete in some places. For example, in Figure 9.

7. Why exactly were the two study time points chosen? This should be included in the results, so it currently seems very arbitrary.

8. The method presentation is very flawed and should be corrected.

9. Important information on the test animals is missing. For example, at least the average age of each experimental group should be given. Was this the same? We know about bone loss with age. In addition, it should at least be shown to what extent the ARRIVE guidelines were fulfilled.

1. The writing style seems to me partly not very scientific, this should be extensively improved. For example: "...changes in alveolar bone have been known and explained by...", "...because of which researchers try to combine HA with various materials...", "...all of which can overlap...". Overall, some sentences in the introduction are difficult to understand. For example: "Alternatively, bone grafts can also be obtained from other individuals or allografts and taken from different species or xenografts".  Furthermore, I strongly recommend to have a native speaker with a scientific context proofread the text.

Author Response

Dear Reviewer 4

I respect and appreciate the reviewer highly for critical and constructive comments to my article.

1. The new point in my article is testing the effects of nanocrystalline hydroxyapatite made from pensi shells (a shellfish originating from West Sumatra, Indonesia) which was combined with IPRF (PRF in liquid form obtained through the concept of low speed centrifugation). It is hoped that the local ingredients in this material can provide added value and benefits. Revision has  been done  by giving more detailed information about the problem to be solved, “Despite a great deal of relative satisfaction with the use of nHA, several studies showed the limitations of the osteoinductive properties of nHA and so far no bone graft material has been found to have biological properties as good as autograft”
2. Addition has been given to problems in the wider alveolar bone to line 34-36
3. The abbreviation HA has been corrected into nHA
4. Assessment of TRAP, ALP, OCN was carried out using the method of immunohistochemical analysis with combinative semiquantitative scoring. (Fedchenko, 2014). This information has been added to line 180-181. The assessment was carried out by an experienced anatomical pathologist who has no vested interest in the results of the study
5. New bone material and existing bone material are different. New bone material is obtained from woven bone which is formed in the socket as immature bone tissue. Calculation of the area was measured using the image raster program 3 as stated in  Line 189-191
6. Statistical information has been added to each chart
7. Day 14 and 28 were selected as the time points in this study. This selection was based on the fact that on day 14 the bone healing process entered the proliferative phase where woven bone formation had started to form while on day 28 the bone healing process entered a remodeling period where the growth of woven bone was able to fill the tooth socket. It was added to line 365-369
8. Thank you, we have revised the presentation method
9. Thank you, we have corrected some information about the animals,
10. Thank you, we have made the sentences  more scientific

Round 2

Reviewer 2 Report

Dear Authors,

Thank you for corrections. The paper is suitable for publicaction now.

Language can be improved. Minor corrections are required.