The Combination of Decellularized Cartilage and Amniotic Membrane Matrix Enhances the Production of Extracellular Matrix Elements in Human Chondrocytes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work presented investigation of the individual and combined effects of decellularized extracellular matrix dECM derived from articular cartilage and amniotic membrane on maintaining the chondrogenic phenotype of human chondrocytes. This study aims to understand whether combining these biomaterials can be enhanced their therapeutic potential for cartilage repair and their regeneration. Here, they produced two decellularized biological matrices: one from decellularized articular cartilage (dACM) and the other from decellularized amniotic membrane (dAMM), where it is seen that Both matrices exhibited biological activity and a high content of biomolecules associated with cartilage generation.

Here, Human articular cartilage slices (2 mm) were obtained from cadaveric donors in an aseptic environment, where the cartilage was collected from the areas of the condyles, patella, and femoral trochlea. Human placentas were taken from the toco-surgery operating room. Both dACM and dAMM were fixed in 4% PFA for 24 hours, then embedded in paraffin blocks using conventional histological methods. Then, the lyophilized dACM and dAMM were sputter-coated with gold and transferred to a scanning electron microscope (SEM). The particle size was then measured through image analysis. Chondrocytes were isolated from the knee joints of healthy human donors and cadaveric donors. The soluble extract of decellularized ECM were prepared by incubating dACM or dAMM with serum-free OptiMEM at 37°C for 24 hours. Then, Chondrogenic gene expression was done.

This study suggests that decellularized matrices derived from articular cartilage and amniotic membrane provide encouraging elements for tissue cartilage engineering by introducing chondrocyte proliferation and maintaining the chondrogenic lineage. Here, decellularized matrices showed biological activity, stimulating chondrocyte proliferation and preserving the chondrogenic phenotype. In short this paper highlighted the importance of effective decellularization, ECM component preservation, and the influence of particle size on tissue development, where combined use of these matrices presented enhanced expression of key markers essential for cartilage repair.

As far as my opinion is concern; This work possess very rich experiments and manuscript presents nice visual materials, however this study needs some quantitative results, which can make the manuscript scientifically richer, such as; Scanning electron micrographs in Figure 1 and 2 need some modelling, which then produce meaningful parameters, so that readers can understand the production of the extracellular matrix elements. In other words, Fractal analysis with box counting method can be used to produce Fractal dimensions, which then can be interpreted to characterize the produced materials.                   

Comments for author File: Comments.pdf

Author Response

Comment 1: As far as my opinion is concern; This work possess very rich experiments and manuscript presents nice visual materials, however this study needs some quantitative results, which can make the manuscript scientifically richer, such as; Scanning electron micrographs in Figure 1 and 2 need some modelling, which then produce meaningful parameters, so that readers can understand the production of the extracellular matrix elements. In other words, Fractal analysis with box counting method can be used to produce Fractal dimensions, which then can be interpreted to characterize the produced materials.

Response 1: Thank you for the positive feedback on our study. We appreciate your suggestions which we have carefully considered. We have performed the image analysis by the box-counting method using the Image J software and the FracLac plug-in to calculate the fractal dimension of the dACM and the dAMM. We set up the program with the basic parameters to perform the calculations and analyze 6 different images of dACM or dAMM. However, we did not include the results from the box-counting method in this revised version of the manuscript. This method is not within our primary field of expertise, and we found it challenging to interpret the results accurately. We understand the relevance of showing quantitative results from SEM images but at this point, we are not able to do so in a reliable fashion. Nonetheless, we are eager to learn more about this technique to enhance our material analysis tools in the future. We will certainly consider incorporating it in future studies, where we plan to characterize the matrices in greater detail. Thank you again for your valuable input. If you have any more suggestions regarding this issue and how we could incorporate fractal analysis results into those currently shown in the manuscript, please let us know.

The results obtained are as follows:

 

dACM
# Image	Fractal Dimension [D]	standard deviation
1	1.5419	0.0257
2	1.5419	0.0257
3	1.5395	0.0257
4	1.5633	0.0233
5	1.5633	0.0233
6	1.5612	0.0233

 

dAMM
# Image	Fractal Dimension [D]	standard deviation
1	1.5939	0.0225
2	1.5881	0.0225
3	1.5477	0.0235
4	1.5364	0.0235
5	1.5342	0.0225
6	1.5342	0.0225

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you to the authors for their work in preparing this publication. It seems interesting, but I have a few comments that I believe will help enhance the quality of the work. Below are my comments:

• The abstract contains too much theoretical content. The abstract should include a maximum of two sentences regarding the theory to guide the reader, while the rest should focus on the methodology used and the results obtained. It is crucial to present numerical results, as this increases the citation potential of the presented work.
• In the introduction, the authors should include information at the very end about the research conducted in this study and the research techniques used.
• In the chapter "2. Materials and Methods," there is a lack of material descriptions. The subsection "Materials" should focus on describing all the reagents used in the study, along with all necessary information about them, such as purity, company, country of origin, and other defining parameters.
• Please add a schematic diagram of the material preparation process to help the reader understand the procedure.
• Please also include a table with the exact compositions of the individual products, their abbreviated names used in the charts, and the synthesis conditions.
• Figure 1 – please include images that are at the same scale. Placing images at different scales and analyzing them is incorrect.
• Figures 4 and 5 – unclear, please sharpen and improve the quality.
• In the IR spectra, please assign the characteristic bands to the groups present in the analyzed material.
• The conclusions should be expanded. They lack numerical values and obtained results. The most important information should be bullet-pointed.

Author Response

Thank you for your enrichment observations, we have carried out several modifications that helped improve the original version.

Comment 1: The abstract contains too much theoretical content. The abstract should include a maximum of two sentences regarding the theory to guide the reader, while the rest should focus on the methodology used and the results obtained. It is crucial to present numerical results, as this increases the citation potential of the presented work.

Response 1: We have modified the abstract following your suggestions (Lines 23-38).

Comment 2: In the introduction, the authors should include information at the very end about the research conducted in this study and the research techniques used.

Response 2: The introduction section was modified as suggested. A new paragraph was introduced (Lines 81-91).

Comment 3: In the chapter "2. Materials and Methods," there is a lack of material descriptions. The subsection "Materials" should focus on describing all the reagents used in the study, along with all necessary information about them, such as purity, company, country of origin, and other defining parameters.

Response 3: The methods section was revised and modified accordingly. Special attention was paid to the description of the company's reagents, their purity, and the volume used. 

Comment 4: Please add a schematic diagram of the material preparation process to help the reader understand the procedure.

Response 4: A new figure describing the workflow and the main stages of the study is now included (Figure 1).

Comment 5: Please also include a table with the exact compositions of the individual products, their abbreviated names used in the charts, and the synthesis conditions.

Response 5: We have added a description of the preparation of the soluble extracts of the individual products and corrected the methods section (Lines 190-193; Lines 394-402). The abbreviated names of each matrix used were revised again in the entire manuscript.

Comment 6: Figure 1 – please include images that are at the same scale. Placing images at different scales and analyzing them is incorrect.

Response 6: Images were revised and changed as suggested (Figure 2).

Comment 7: Figures 4 and 5 – unclear, please sharpen and improve the quality.

Response 7: We have improved the quality of Figures 4 and 5, now Figures 5 and 6.

Comment 8: In the IR spectra, please assign the characteristic bands to the groups present in the analyzed material.

Response 8: The characteristic peaks of each group represented in the analyzed material are now indicated (Figure 4).

Comment 9: The conclusions should be expanded. They lack numerical values and obtained results. The most important information should be bullet-pointed.

Response 9: The conclusions section was rewritten to describe the main results of the study. Bullet points were not added as such as we believe is not allowed by the format of the journal. If considered appropriate by the editors we will be happy to correct it.

Reviewer 3 Report

Comments and Suggestions for Authors The paper The combination of decellularized cartilage and amniotic membrane matrix enhances the production of extracellular matrix elements in human chondrocytes prepared by Antonio Rojas-Murillo et al., present novel and interesting results that deserve to be published after some minor improvements:

1. The paper lacks specific details about some experimental procedures, such as the conditions for centrifugation during the preparation of soluble extracts and the exact parameters used in FT-IR spectroscopy.

2. The statistical analysis section is brief and does not specify the exact tests used for each type of data or the criteria for choosing these tests.

3. The discussion lacks a thorough comparison with existing literature and does not fully explore the implications of the findings or future research directions.

4. The paper does not include data on the growth factors and biochemical composition of the decellularized matrices, which are crucial for understanding their bioactivity.

Author Response

Comment 1: The paper lacks specific details about some experimental procedures, such as the conditions for centrifugation during the preparation of soluble extracts and the exact parameters used in FT-IR spectroscopy.

Response 1: We have corrected the methods section trying to be more specific regarding the procedures carried out. Find changes highlighted in yellow.

Comment 2: The statistical analysis section is brief and does not specify the exact tests used for each type of data or the criteria for choosing these tests.

Response 2: The variables analyzed for the test employed are now described (Lines 225-228). Additionally, we have added in each figure legend the exact statistical analysis test used for each type of data analyzed.

Comment 3: The discussion lacks a thorough comparison with existing literature and does not fully explore the implications of the findings or future research directions.

Response 3: The discussion section was carefully revised and relevant information was discussed and cited as suggested (Lines 404-438).

Comment 4: The paper does not include data on the growth factors and biochemical composition of the decellularized matrices, which are crucial for understanding their bioactivity.

Response 4: We have included such information as part of the discussion section, based on previous reports using these decellularized matrices (Lines 395-403). However, we acknowledge the lack of a deeper characterization of our compounds as one limitation. We understand that this is fundamental to understanding their bioactivity and we are planning such characterization as part of the future work of this project.