Bone Regeneration by Dedifferentiated Fat Cells Using Composite Sponge of Alfa-Tricalcium Phosphate and Gelatin in a Rat Calvarial Defect Model

Round 1

Reviewer 1 Report

Some comments are made in a separate document "Response Applied Sciences"

Comments for author File: Comments.docx

Author Response

Thank you for your suggestions.

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Reviewer 2 Report

The authors have described the application of a gelatin-αTCP composite scaffold loaded with dedifferentiated fat cells for bone regeneration in critical sized bone defects. While, as the authors agree, the effectiveness of biopolymer and bio ceramic composites including that of αTCP for bone regeneration is not a novel concept, they aver that superior osteogenic differentiation of dedifferentiated fat cells over adipose derived stem cells would enable enhanced bone regeneration. The authors have performed a very thorough analysis of the scaffold ultrastructure, composition and mechanical properties and further show enhanced bone regeneration as well as vascularization in composite samples containing dedifferentiated fat cells over any of the controls consisting of individual scaffold components or in the absence of cells. However, the authors do not address the main hypothesis/premise of the study that dedifferentiated fat cells would support better bone regeneration than adipose derived stem cells through a direct comparison in this study. Moreover, the following aspects would require further clarification to ensure adequate presentation of the methods and interpretation of results.

1. The authors do not discuss the rationale beyond the choice of gelatin biopolymer over other natural polymers including collagen.
2. Page 2, Line 52. " Previous studies have demonstrated the role of αTCP particles in bone rebuilding by the gradual biodegradation and formation of adjacent bone" requires a reference.
3. In the methods, the authors mention 3 different concentrations of αTCP, but it is unclear which amount of αTCP was used in the entire study.

4. It makes sense that cell adhesion efficiency would hit a plateau through increasing cell suspension medium volume due to pore space availability. have the authors attempted to increase the concentration of cell suspension instead. also, it is unclear what concentration of cell suspension was actually used and what the cell adhesion density was.

Author Response

Thank you for your suggestions.

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Reviewer 3 Report

The article entitled "Bone regeneration by dedifferentiated fat cells using composite sponge

of alfa-tricalcium phosphate and gelatin in a rat calvarial defect model" written by Tsumano et al. report the fabrication, characterization and in vivo testing of a porous ceramic-biopolymeric composite scaffold made of alpha tricalcium phosphate and gelatin. The authors tested various combinations of the materials and pre-seeded the scaffold with dedifferentiated fat cells. The authors tested the osteoinductive properties of the scaffold in a rat calvaria defect model. The authors claim that the pre-seeded scaffold possess superior regenerative properties as compared to non-seeded scaffolds.

The article lacks in originality and novelty. The literature on the use of alpha-TCP/GE is very rich and different groups developed divers scaffold types and reported various levels of success. In this paper, Tsumano et al. do not cite relevant works on similar topics and the discussion of their findings in comparison to the current (and past) literature is very limited.

Specific comments:

P4 L141 The flasks were... the flask ceiling: it is not clear how the authors performed the culture. Please specify.

P5 L173 source current (uA) = 85 image. What does image stand for?

P7 figure 6: very unusual shapes of the stress strain curves. Usually, porous material display an initially linear elastic part followed by a zero slope plateau and then a rapid increase in stress due to material densification. Very stiff materials may not display a horizontal plateau. In figure 6 a downward concavity is clearly present. This is usually observed in fibre reinforced composites in tension. Do the authors have an explanation for this behaviour?

P9 L256: The bone density is divided in a coarse manner. The grouping is qualitative in nature. What is the meaning of high, medium and small? Can the author insert some reference scale of known density? Pseudocolors may enable to better spot differences; however, they can also over-amplify negligible differences. The authors should make available also grayscale images of the ct reconstructions.

P10 fig 10. It is not clear how the analysis was performed. In the way it is described, the figures report the (normalized) values of the fraction of the denser, more x-ray absorbing matter. This would make the difference between control/GS/GS+DFAT and aTCP+GS/aTCP+GS+DFAT rather obvious, as in the latter case, dense ceramic material is inserted in the defect. Also, the pixel size used might not be sufficiently small to detect small variation of signal absorption due to the newly formed bone. A sensitivity analysis, with the scanning setting chosen by the authors, should be performed n the first place.

P11 L295: it is hard to draw out definitive conclusions by observing Immunohistochemical images. Digital image analysis that evaluates relative distances and morphology of the blood vessel by the ceramic particle should be performed on a sufficiently large number of images.

Finally, a thorough language polishing should be performed.

Author Response

ご提案ありがとうございます。

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Tsumano et al., I think the manuscript is much more clear now. Thanks for the modifications. Congratulations! 

Author Response

Thank you very much.

The paper has been revised.

Reviewer 2 Report

The authors have endeavored to address concerns and comments raised by the reviewers. The inclusion of clarification statements regarding the rationale and methodology as well as additional figures has helped bolster the study design.

Author Response

Thank you very much.

The paper has been revised.

Reviewer 3 Report

The authors have only partly addressed my questions and comments. The interpretation of the results obtained from morphometric analyses is unclear (calibration levels and not LUT of the microCT scans; cell localization on the SEM images; samples size (n) of the Immunohistochemical analyses). The experimental campaign, in the way it is presented by the authors, does not allow to discern to what extent the α-TCP/GS + DFAT sample is superior to the other substrates and why.

Author Response

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