Osteopontin and Vascular Endothelial Growth Factor-Immunoreactivity in Critical Bone Defects Matrix Production: A Nano-Hydroxyapatite/Beta-Tricalcium Phosphate and Xenogeneic Hydroxyapatite Comparison

Round 1

Reviewer 1 Report

Review Report

This is an interesting study on the "Participation of osteoprogenitor cells and extracellular matrix in the regeneration of critical bone defects with application of  composite grafts of synthetic Nano-hydroxyapatite/Beta tricalcium phosphate and xenogeneic hydroxyapatite. It was a pleasure to evaluate your publication. The subject of  your publication has been considered by numerous researchers for years, but the continuous development of materials engineering makes it still relevant today. Reading your work, I noticed some important issues that should be changed before publication and a few smaller inaccuracies.

1. The authors quote that the porosity increases the affinity of cell attachment. What was the % porosity of the used in the graft materials? Was it manufacture specified? If so kindly mention in the text.
2. What was the size of the individual pores for the 2 tested materials?
3. There need to be a figure showing the 2 test materials.
4. A Figure showing the surgical site in the rat possibly with the defect site with the test materials placed.
5. Hu J et al, analyzed the morphology response - Reference [16] is missing in the text.
6. The authors have kept their focus on the histological aspect in the entire study. I’m sure they would have analysed micro CT showing the amount of bone regeneration and BV/TV. If its published elsewhere, add the reference.

Author Response

Reviewer 1 answers:

1. The authors quote that the porosity increases the affinity of cell attachment. What was the % porosity of the used in the graft materials? Was it manufacture specified? If so kindly mention in the text.

Authors answer: As reviewer requested, was included the information’s in lines 106 and 108.

1. What was the size of the individual pores for the 2 tested materials?

Authors answer: As reviewer requested, was included the information’s in lines 106 and 108.

1. There need to be a figure showing the 2 test materials.

Authors answer: As reviewer requested, was included the figures (figure 1) in line 99

1. A Figure showing the surgical site in the rat possibly with the defect site with the test materials placed.

Authors answer: As reviewer requested, was included the figures (figure 1) in line 99

1. Hu J et al, analyzed the morphology response - Reference [16] is missing in the text.

Authors answer: As reviewer requested, was included the reference in line 263

1. The authors have kept their focus on the histological aspect in the entire study. I’m sure they would have analysed micro CT showing the amount of bone regeneration and BV/TV. If its published elsewhere, add the reference.

Authors answer: thank you for the indication of the test.Certainly, in the evolution of the work we will consider the possibility of micro tomography.

however, this study is really focused on histological analysis.

I will accept the recommendation of the reviewer’s edition to perform the English correction with mdpi.

Reviewer 2 Report

This manuscript needs a significant revision for grammatical correction and spelling mistakes. Several statements on discussion section lack proper grammatical comprehension.

1. I would suggest authors to revise their title and make it more concise.
2. Section 2.6 Please correct heading.
3. The authors should discuss all the figures on results section not just a sentence referring statistical significance.
4. Sentence 235: What does ‘ultrastructural research’ refer to?
5. The defect was 8 mm in size, whereas the histology images are presented at a scale of 100 µ I understand you have mentioned them as representative images but trichrome stained images covering whole 8 mm defect should be presented. This will help understand the results better.
6. Please label the significance among groups on all the relevant bar graphs.
7. It is difficult to understand what information is being conveyed on figure 1, 2, and 4. Please make them clearer with proper discussion on results section.
8. The upregulated VEGF expression after 60 days of implantation would make very little contribution in accelerated bone regeneration. In general, higher VEGF expression at initial stages of bone healing would contribute more pertinent to natural bone healing process. The authors can refer these articles and discuss more about their results comparatively. https://doi.org/10.1016/j.msec.2021.112075, https://doi.org/10.1093/rb/rby013
9. Proper discussion is lacking on why synthetic nHA would upregulate the angiogenic markers such as VEGF compared to xenogenic HA. What is the biological mechanism involved? Please discuss.

Author Response

Reviewer 2 answers:

1. I would suggest authors to revise their title and make it more concise.

Authors answer: As reviewer requested the title was revised and reduced.

1. Section 2.6 Please correct heading.

Authors answer: As reviewer requested heading was amended.

1. The authors should discuss all the figures on results section not just a sentence referring statistical significance.

Authors answer: As reviewer requested the results  were discussed. Section 4.1  1st and 2nd paragraphs; Section 4.2 first paragraph; Section 4.3 first paragraph.

1. Sentence 235: What does ‘ultrastructural research’ refer to?

Authors answer: As reviewer requested the term ‘ultrastructural research’ was replaced to a more precise descriptive term.

1. The defect was 8 mm in size, whereas the histology images are presented at a scale of 100 µ I understand you have mentioned them as representative images but trichrome stained images covering whole 8 mm defect should be presented. This will help understand the results better.

Authors answer: We agree with the reviewer, however, 8mm is huge dimension in microscopic field. Even using the smaller objective lens available we could not capture the entire bone defect. 

1. Please label the significance among groups on all the relevant bar graphs.

Authors answer: As reviewer requested, the significance were registered in the graphs.

1. It is difficult to understand what information is being conveyed on figure 1, 2, and 4. Please make them clearer with proper discussion on results section.

Authors answer: We rearranged figures captions to attend reviewer request and we include texts in results section.

1. The upregulated VEGF expression after 60 days of implantation would make very little contribution in accelerated bone regeneration. In general, higher VEGF expression at initial stages of bone healing would contribute more pertinent to natural bone healing process. The authors can refer these articles and discuss more about their results comparatively. https://doi.org/10.1016/j.msec.2021.112075, https://doi.org/10.1093/rb/rby013

Authors answer: We agree with reviewer argument, however, we considered two additional points: (1) we are dealing with biomaterial influence in the natural process and (2) we consider that this late period (60 days) may be more related to bone remodeling fase of the regenerative process. We include the suggested references in Discussion section (4th paragraph).

1. Proper discussion is lacking on why synthetic nHA would upregulate the angiogenic markers such as VEGF compared to xenogenic HA. What is the biological mechanism involved? Please discuss.

Authors answer: As reviewer requested, paragraphs were added to discuss the mentioned subject. Discussion section, 2nd paragraph.

I will accept the recommendation of the reviewer’s edition to perform the English correction with mdpi.

Round 2

Reviewer 2 Report

I have no further comments. Manuscript has been well-revised by the authors.