The Effect of Mineralized Plasmatic Matrix and Chitosan on the Healing of Critical-Sized Mandibular Bone Defects in a Rabbit Model

Round 1

Reviewer 1 Report

Dear Authors,

Thank you for your submission.

Please see my comments in the attached document.

Comments for author File: Comments.pdf

Author Response

Response to reviewers

Reviewer 1:

We would like to introduce our deep appreciation of your fruitful revision of our manuscript. We changed all required comments according the respectful reviewer’s view.  

Introduction

Line 28: small variable areas of still radio-lucence. Do you mean “persistent small radiolucent areas”?

Response: Thanks for your comment, it was replaced.

 

 

Line 45: Is this statement about the mandible unique to the rabbit or to the man, or to all mammals?

Response: Thanks for your comment, it is suitable for both, we added it in the text.

 

Lines 50 to 52: repetition in these 2 sentences. Maybe say “In reconstructive orthopedic and maxillofacial surgery, bone grafts play a significant role and is required to reconstruct large bone defects”.

Response: Thanks for your comment, it was removed.

 

Line 53: are types of bone grafts. Maybe say “have all been reported”?

Response: Thanks for your comment, it was replaced.

 

Line 56: “However” can be deleted.

Response: Thanks for your comment, it was deleted.

 

Line 60: Please move reference 12

Response: Thanks for your comment, it was moved.

 

Line 61 and 62: “can be used to 61 overcome these limitations”

 Response: Thanks for your comment, it was replaced.

 

It would be beneficial for your study if you could explain what a critical-sized bone defect is.

Response: Thanks for your comment, it was explained.

 

Materials and Methods

 

Lines 86-87: Maybe not necessary to repeat the approval number twice.

Response: Thanks for your comment, repetition was deleted.

 

Line 89: Please rephrase “This study used 45 skeletally mature white adults New Zealand rabbits …” and remove “were used in this study”

Response: Thanks for your comment, it was rephrased.

 

Line 91: remove “their cages”

 Response: Thanks for your comment, it was removed.

 

Line 100: a skin incision is always extra-oral I believe, please adjust

Response: Thanks for your comment, it was adjusted.

 

Line 101: “incision was made 2-3mm away from the inferior mandibular border”. Please change “away from” to “2-3 mm dorsal to the ventral border of the mandible”

 Response: Thanks for your comment, it was changed.

 

Line 107: “According to the group of the animal”

Response: Thanks for your comment, it was corrected.

 

Lines 109-110: Make it simpler “simple continuous pattern using 4-0 polyglycolic acid sutures (SURGICRYL…”

Response: Thanks for your comment, it was adjusted.

 

Lines 111-112: Make it “simple interrupted suture pattern using 2-0 polyglycolic acid sutures.

Response: Thanks for your comment, it was adjusted.

 

You should call Figure 1 in the text. Additionally in Figure 1H you say the subcutis was sutured whereas in the text you only mention the muscle and the skin. Please adjust.

 Response: Thanks for your comment, it was adjusted, and Figure 1 was called in the text

 

 

Furthermore, I wonder why you mention a depth of 3mm. In your Figure 1 it looks like you removed all the outer cortex of the mandible, up to the molar roots.

 Response: Thanks for your interesting comment, we removed the depth.  

 

Lines 126-127: Please adjust brackets.

Response: Thanks for your comment, it was adjusted

 

Lines 128-129: Please make it clearer. The Chitosan was prepared beforehand and prepared during the surgery?

Response: Thanks for your comment, The Chitosan was prepared beforehand and it was cleared

 

Line 132: Spinning time is 2 to 4 minutes? Why so much difference?

Response: Thanks for your comment, it was adjusted.

 

Lines 134-135: Add a verb to your sentence. How many minutes is “a few”?

Response: Thanks for your comment, a verb was added, and mixing time was adjusted

 

Line 146: Is that your generator? A DR system? Please elaborate

Response: Thanks for your comment, it was added.

 

Lines 150-151: The grading system described by (ref 27) was used as a basis to assess the quality and quantity of radiographic bone development

Response: Thanks for your comment, it was adjusted

 

Line 153: immediate postoperative appearance

Response: Thanks for your comment, it was added

 

Line 163: Which molecule did you use?

Response: Thanks for your comment, thiopental sodium was mentioned in line 165

 

Results

 

Lines 194-196: why didn’t you give non-steroidal anti-inflammatory drugs for perioperative pain?

Response: Thanks for your comment, it was not used to avoid its effect on bone healing. We have previously published data about the effect of non-steroidal anti-inflammatory drugs on bone healing.

 

Line 199: Figures

Response: Thanks for your comment, it was corrected

 

Line 207: “so did” instead of “as do”

Response: Thanks for your comment, it was corrected

 

Why do you always put the scores between brackets?

Response: Thanks for your comment, brackets were used to highlight the score to be more obvious

 

Lines 217-218: “differed” and “so did” to stay in the past tense

Response: Thanks for your comment, it was corrected

 

Line 224: “still radio-lucence” Please improve this wording

Response: Thanks for your comment, it was improved

 

Figure 2: This figure is difficult to look at. You should maybe zoom in and focus on the region of interest. Another advice would be to use images of the same individual if possible (so, radiographs of 1 rabbit for control group, images of 1 rabbit of CH group and images of 1 rabbit for MPM group).

Response: Thanks for your comment, the X-ray figure was changed.

 

Figure 4: Maybe more legends could be inserted on the histologic images than just osseous tissue and granulation tissue

Response: Thanks for your comment, more legends were added.

 

Figure 5: Please make sure you check your letters and their meaning, and also please add what happened at 8 weeks.

 

Line 296: please check your sentence

 Response: Thanks for your comment, it was corrected

 

Line 303: please check your sentence

Response: Thanks for your comment, it was corrected

 

Figure 6: Probably it would be better to make 2 figures from this one, as you did before: one for microphotographs and 1 for graphs. Indeed, the graphs are very small here. Previously you wrote photomicrographs. Please stay consistent.

Response: Thanks for your comment, it was split.

 

Figure 7: Maybe a better idea to split it in 2 figures.

Figure 8: Same remark.

Response: Thanks for your comment, it was split.

 

Discussion

Line 363: Please remove the “u

Response: Thanks for your comment, it was removed

 

Line 363: remove “include the repair of large bone defects”

Response: Thanks for your comment, it was removed

 

Line 372: please change “are infected” to “got” or “became infected”

Response: Thanks for your comment, it was changed

 

Lines 375-377: if you saw periapical lysis, how can you be sure it was not an infection starting? I would then modify this chapter accordingly.

 Response: Thanks for your interesting comment, the periapical lesions noticed in some animals were mostly related to inflammation rather than infection. A routine oral examination of the defect area didn’t show any signs or features of infection as an abscess or purulent exudate, and a sequential histopathological examination did not reveal any neutrophilic cell infiltration.

Line 381: Maybe say that your findings correlate with Amine’s about positive effects of MPM. The goal is to link your findings with the ones reported previously.

Response: Thanks for your comment, it was adjusted

 

Line 387: I am afraid you did not mention necrotic tissue in the previous part. Please clarify.

Response: Thanks for your comment, it was removed.

 

Line 395: Why “previous results” I believe these are your “current” results

Response: Thanks for your comment, previous was removed.

 

Line 411: “which might explain”

Response: Thanks for your comment, it was corrected

 

 

Line 421: “according to Intini”

Response: Thanks for your comment, to was added

 

Line 423: no subject to the sentence. Please adjust

Response: Thanks for your comment, it was modified.

 

 

Line 433: not clear if this sentence “MMP-9 was expressed in osteoblasts, osteoclasts, osteocytes, and bone marrow cells, indicating that it is a bone resorption enzyme.” Is linked to your results or just a fact from previous research (which I believe it is). Maybe better to say “MMP-9 is expressed…”

Response: Thanks for your comment, references were added.

 

 

Line 451-454: Please rephrase

Response: Thanks for your comment, it was rephrased

 

I am surprised you mention in line 269 “a matrix resembling dentine, along with marked odontoblastic, osteoplastic cell proliferation, and marked osteogenesis” and then you never discuss about this fact. In other words, you talk about dental-looking tissues in the healing phase of your bone. I believe you must discuss these findings.

Response: Thanks for your comment, it was discussed.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The article entitled “The effect of Mineralized Plasmatic Matrix and Chitosan on the healing of critical-sized mandibular bone defects in a rabbit model”. This study aimed to evaluate the impact of MPM and Chitosan on the healing of a critical-sized mandibular bone defect in a rabbit model.

 

Below are some suggestions:

 

In the Abstract:

-Due to the importance of the repair process in critical bone defects, the authors could insert a paragraph emphasizing the importance of new techniques and therapeutic approaches already here in the abstract, thus demonstrating the relevance of the research.

Introduction:

- On line 41, I suggest replacing "Massive bone defects" with critical bone defects, a more appropriate terminology within bone repair.

- In lines 54-63, a suggestion would be to reduce the explanation of the types of bone grafts, since they are already known in the tissue bioengineering scenario, and to focus on the biomaterials used in the research, mainly Chitosan, which was not very detailed.

2. Materials and |Methods:

2.4. Animal Grouping:

- An illustrative image of the randomization of the groups could be made as well as the experimental design.

2.5.1. Radiography and Radiographic Scoring:

- Was the grading system used for the radiographic assessment developed by the research group or is it an existing one? I suggest a more detailed explanation.

2.5.2. Histopathology:

- Justify periods of euthanasia.

- In the 12-week period the defect would not be repaired?

3.3. Histological Findings:

- I suggest separating the plate from figure 4, it is difficult to visualize, as well as improving the quality of figure 5.

* Could the authors say why microtomography was not performed?

4. Discussion

- I suggest including the limitations of the study in the discussion.

5. Conclusion

- Change the conclusion into final considerations, including the purpose of the research, its main results, the conclusions and future clinical perspectives.

Author Response

Response to reviewers

Reviewer 2:

We would like to introduce our deep appreciation of your fruitful revision of our manuscript. We changed all required comments according the respectful reviewer’s view.  

In the Abstract:

-Due to the importance of the repair process in critical bone defects, the authors could insert a paragraph emphasizing the importance of new techniques and therapeutic approaches already here in the abstract, thus demonstrating the relevance of the research.

Response: Thanks for your comment, we added it. Unfortunately, we have limited 200 words in the abstract.

Introduction:

- On line 41, I suggest replacing "Massive bone defects" with critical bone defects, a more appropriate terminology within bone repair.

Response: Thanks for your comment, it was replaced.

- In lines 54-63, a suggestion would be to reduce the explanation of the types of bone grafts, since they are already known in the tissue bioengineering scenario, and to focus on the biomaterials used in the research, mainly Chitosan, which was not very detailed.

Response: we appreciate your comment, we added a more detailed data about chitosan.

2. Materials and |Methods:

2.4. Animal Grouping:

- An illustrative image of the randomization of the groups could be made as well as the experimental design.

Response: we appreciate your comment, we added it.

 

2.5.1. Radiography and Radiographic Scoring:

- Was the grading system used for the radiographic assessment developed by the research group or is it an existing one? I suggest a more detailed explanation.

Response: we appreciate your comment, we added it.

 

2.5.2. Histopathology:

- Justify periods of euthanasia.

 Response: Thanks for your comment, the type of anesthesia used in our study was added.

 

- In the 12-week period the defect would not be repaired?

Thanks for your interesting comment. We totally agree this respectful comment. However, we follow the published protocol in healing of the mandibular defect area as mentioned by Al-fotawei et.al (2014). Also, our X-ray findings of the bone defect area showed radiopaque features of bone healing. In addition, the difference in healing criteria may be disappear with the time and it will be difficult to differentiate between different animal groups. 

3.3. Histological Findings:

- I suggest separating the plate from figure 4, it is difficult to visualize, as well as improving the quality of figure 5.

Response: Thanks for your comment, it was split.

 

* Could the authors say why microtomography was not performed?

Response: Thanks for your comment. Unfortunately, it is not available at our institute, and it was added to the limitations of the study.

 

4. Discussion

- I suggest including the limitations of the study in the discussion.

Response: Thanks for your comment, it was added

 

5. Conclusion

- Change the conclusion into final considerations, including the purpose of the research, its main results, the conclusions and future clinical perspectives.

Response: Thanks for your comment, it was changed

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Authors,

Thank you for your submission.

Please see a few comments in the attached document.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

 

First of all, thank you very much for your comments, also we considered all your comments in detail which made our manuscript better and highlighted by yellow colour.

 

Point 1: Line 23: you could maybe say “Recently, bone tissue engineering has provided many benefits in improving bone healing”.

Response 1: We changed this part “Recently, bone tissue engineering has numerous advantages in improvement of bone healing.” With this one “Recently, bone tissue engineering has provided many benefits in improving bone healing” according to your comment.

 

Point 2: Where did you explain what a critical-sized bone defect is?

Response 2: We mentioned a critical-sized bone defect in introduction in line 44 “Critical bone defects can be caused by congenital defects, diseases, infections, trauma or injuries, tumors, osteotomies, and large fracture. Typically, small bone defects heal spontaneously; however, the tissue’s ability to heal is limited in massive defects; therefore, surgical intervention is required. and we added a paragraph in the discussion in line There is no confined definition of a critical-sized defect. generally, a critical size defect is defined as a defect that would not heal spontaneously without surgical stabilization and requires further surgical intervention.

 

Point 3: Lines 110-111: Please improve this sentence, I don’t understand the medium molecular weight and deacetylation degree. Maybe an incorrect bracket position? 

Response 3:  This sentence is corrected as following “chitosan have medium molecular weight of 300 kDa, with deacetylation degree of 75 ± 10%”.

Point 4: Figure 1: I believe letters are not correct (incision is b for example and so on). I cannot see any filling of the defect on g.

Response 4: You are right and we corrected the letters. (g) is corrected as it showing the filling materials that will be added to the defect area

 

Point 5: Line 156: OK if it is a CR system, please elaborate about its brand and model because I believe what you mention is only the generator.

Response 5: the brand and model of the CR system was mentioned as following; CR system (FUJIFILM-FCR PRIMA II. FUJIFILM UK Ltd.UK).

 

Point 6: Line 165: you mention that in grade 4 the defect is filled on the medial and lateral side. You are taking lateral radiographs so you cannot really evaluate what is medial and what is lateral, only rostal and caudal. Please adjust.

Response 6: We corrected this part as following

“the defect is filled with a substance of uniform radiodensity rostrally and caudally”  

 

Point 7: About the scores between brackets, you say it’s to highlight the results. I believe you should check with the editors with regards to this point.

Response 7: thanks for your comment, brackets were removed. The score was already charted in Figure 2.

 

Point 8: Figure 2: This sentence “At 4 and 8 weeks, MPM group show a clear bone formation at the defect site in the MPM, chitosan, and control groups, respectively.” Is still unclear. Please adjust.

Response 8: This part was corrected according to reviewer 1 comments as following;

At 4 and 8 weeks, MPM group demonstrated the most pronounced bone formation with the greatest increase in defect area radio-opacity as compared to chitosan and control groups. At the end of 12 weeks, MPM group demonstrated the highest degree of bone formation with uniform radio-opacity, while in the chitosan group, most of the defect area was filled with radio-opaque bone with persistent small radiolucent areas. The control group showed less bone formation than the MPM and chitosan group, with more radiolucent areas.

 

Point 9: Line 286: The histopathology score was illustrated Figure 7, at the early stage, a significant difference between the MPM and control groups was noticed. Please adjust. This sentence is incorrect.

Response 9: This part was corrected as following;

The histopathology score was illustrated in Figure 7, at the 4^th week, a significant difference between the MPM and control group was noticed.

 

Point 10: Lines 287-291: please add a verb in each sentence

Response 10: Done.

 

Point 11: Figure 6 : what is Bv?

Response 11: Bv denotes blood vessel (added)

 

Point 12: Line 319: are you taking about the 4th or the 12th postoperative week? Please adjust

Response 12: This part was corrected as following;

deposition at the 4th postoperative week showed that, the MPM and chitosan groups demonstrated early collagen fiber deposition, either interstitial or within the newly formed osseous tissue……………………………….. At the 12th postoperative, the MPM showed deposition of well-organized and abundant collagen fibers

 

Point 13: Lines 317-328: I don’t really understand your paragraph here. Where is the time frame? Please add the postoperative weeks clearly.

Response 13: This part was corrected as following;

deposition at the 4th postoperative week showed that, the MPM and chitosan groups demonstrated early collagen fiber deposition, either interstitial or within the newly formed osseous tissue……………………………….. At the 12th postoperative, the MPM showed deposition of well-organized and abundant collagen fibers

 

Point 14: Line 328 : (Figure 9). Please adjust

Response 14: Done

 

Point 15: Line 404: I am still concerned about the lysis and what you called “periapical lesions”. You are right in your previous comments: an infection is unlikely because there was not neutrophilic infiltrate on histopathology. But then we say it was only inflammation. Can you be more accurate? Since you did histology, you should probably qualify this inflammation.

Response 15: Periapical lesions may be caused by different aetiological agents of endodontic origin. Many issues can result in apical periodontitis for example: poor aseptic measurements, inadequate access cavity design, missed canals, insufficient instrumentation, debridement and leaking temporary or permanent restorations. Even when the strictest precautions are taken, apical periodontitis may still persist as asymptomatic radiolucencies, because of the complexity of the root canal system formed by the main and accessory canals. Additional, the inflammatory periapical tissue contains extra radicular causes that may prevent post-treatment recovery. This part was added.

 

Point 16: Line 422: as previously requested, please clarify “previous results”

Response 16: radiographic and histopathological results. This part was added.

 

Point 17: Lines 451-456: Do you mean that dentine is appearing first, then replaced by bone??? Please clarify this paragraph.

Response 17: Thanks for your comment, Yes, dentin was first appeared and then replaced with osseous matrix, we mentioned it in the discussion section.

 

Point 18: Line 491: please add a period before “Our results”.

Response 18: at 4, 8, and 12 post-operative weeks. This part was added.

 

Point 19: It would be beneficial for your study if you could explain what a critical-sized bone defect is please.

Response 19: Done

We mentioned a critical-sized bone defect in the introduction in the following paragraphs:

- Critical bone defects can be caused by congenital defects, diseases, infections, trauma or injuries, tumors, osteotomies, and large fractures. Typically, small bone defects heal spontaneously; however, the tissue’s ability to heal is limited in massive defects; therefore, surgical intervention is required.

-There is no confined definition of a critical-sized defect. generally, a critical size defect is defined as a defect that would not heal spontaneously without surgical stabilization and requires further surgical intervention.

 

-Hollinger and Kleinschmidt defined the critical size defect as the smallest intraosseous wound that will not spontaneously heal by bone formation. during the animal’s lifetime.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

No comments

Author Response

First of all, thank you very much for your fruitful revision of our manuscript. We checked our manuscript by enago proofreading service. the attach file is the certificate of the proofreading.   

Author Response File: Author Response.pdf