The Incorporation of Marine Coral Microparticles into Collagen-Based Scaffolds Promotes Osteogenesis of Human Mesenchymal Stromal Cells via Calcium Ion Signalling

Round 1

Reviewer 1 Report

Interesting work, but some things could be improved.

How were scaffolds sterilized? What influence did this have on mechanical properties?

Make the XRD figures clearer. Increase the size of the numbers. 

In the results section, the rationale for looking for calcium ethanoate is not clear. Make clear that the collagen solution has ethanoic acid as a solvent at the start of the results section.

The conclusions section is missing!

Author Response

Reviewer 1: Interesting work, but some things could be improved.

Thank you to reviewer 1 for their time in reviewing our manuscript

How were scaffolds sterilized? What influence did this have on mechanical properties?

Scaffolds were sterilised using a dehydrothermal treatment process which involves heating the scaffolds to a temperature of 105°C for 24 h at 0.05 bar. As well as sterilising the scaffolds, the elevated temperature causes condensation reactions between the carboxyl groups of aspirate or glutamate residues and the amino acids of lysine or hydroxylysine, resulting in the formation of intermolecular cross-links. The use of this process in addition to treatment with 6 mM N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride and 5 mM N-Hydroxysuccinimide, which forms zero-length cross-links in collagen between the carboxyl and amino groups of varying residues, increases the mechanical properties of the scaffolds. This has been clarified in section 4.5. of the revised version of the manuscript.

Make the XRD figures clearer. Increase the size of the numbers. 

In order to make Figure 2A clearer, we have replaced the original image with an enlarged portion of that image illustrating the relevant peaks in the material and control spectra. The original image is now included as Supplementary Figure S1.

In the results section, the rationale for looking for calcium ethanoate is not clear. Make clear that the collagen solution has ethanoic acid as a solvent at the start of the results section.

Thank you to the reviewer for this recommendation. In the revised manuscript we have included additional text in section 2.1. to clarify that the use of ethanoic/acetic acid as a solvent in the scaffold fabrication process motivated the investigation into the crystalline structure of the resultant biomaterial.

The conclusions section is missing!

A conclusions section has been included in the revised manuscript.

Reviewer 2 Report

Dear authors, the manuscript entitled “The incorporation of marine coral microparticles into collagen-based scaffolds promotes osteogenesis of human mesenchymal stromal cells via calcium ion signaling.” Is a well written study. In this study the marine coral microparticles can be used for scaffold production, that efficiently could promote the osteocyte differentiation of MSCs and additionally the release of calcium. These scaffolds can be used in several tissue engineering application such as the regeneration of bone and cartilage defects.

Only a number of minor corrections should be performed to this study, which I think the authors are capable to perform, in order to further processed.

Macroscopic images of the different categories of produced scaffolds must be added to the results section, for being easier for the readers to understand how these scaffolds look like.

 

In Materials and Methods section, 4.2. Fabrication of collagen and collagen/coral scaffolds, lines 366 – 377, the exact dimensions of the scaffolds should be added (e.g. length, width and thickness).

 

Additionally, in materials and Methods section, 4.6. Mechanical testing of scaffolds, lines 409- 417, only compressive tests were performed? I think also, extension tests, involving engineering strain (ε), engineering stress (σ, in MPa). Elastin (El-E) and collagen (Col-E) phase slope, transition stress (σTrans) and strain (εTrans), ultimate tensile strength (σUTS) and failure strain (εUTS) would be very informative for the readers. (Compressions tests have been well performed, and this is only an optional minor revision. If the authors think that no additional information will be extracted from the extension tests, you can overpass this correction)

 

The rehydration rate of the scaffolds should be an important parameter that could be added in the materials and methods and results section.

 

In Materials and Methods section, 4.7. Osteogenic differentiation of human MSCs cultured on scaffolds and in 2D, lines 418 – 434, the authors indicated that seeding of MSCs was performed in the produced scaffolds. It should be added the number of MSCs/ cm² (or cm³ of scaffold) and the exact dimensions of the scaffolds that were used for the seeding assay. In addition, please add the passage of MSCs used for seeding.

 

In materials and methods section, 4.8 Immunohistochemical staining for CaSR and results section, 2.4. Osteogenesis of human MSCs seeded on scaffolds and in 2D insert culture, the performed assay is indirect immunofluorescence, in this way, please change the immunohistochemistry term with indirect immunofluorescence.

 

When a new biomaterial is fabricated, it is reasonable to check if this scaffold has potentially toxic effect to the cells. For this purpose, cell viability assay (either contact or extract cytotoxicity test) must be performed.

 

Proliferation of MSCs or differentiated osteocytes into the scaffolds should also be estimated by MTT assay.

 

In Materials and Methods section, 4.7. qPCR analysis of osteogenic and angiogenic gene expressing, 4.8 Immunohistochemical staining for CaSR, 4.9. Biochemical analysis of MSC seeded on scaffolds and in 2D, MSCs with regular culture medium and validated osteocytes must be used as negative and positive control, respectively. It is known, that MSCs depending to their microenvironment could be differentiated to other cell types. In regards to this study, the produced scaffold may act beneficially to the differentiation of MSCs, or even to the production of calcium, without the external use of growth factors and differentiation medium. For this purpose, the seeding of MSCs in the produced scaffolds and further cultivation without the use of differentiation medium is worth to be performed.

 

In Results section, 2.2. Architectural properties of collagen/coral scaffolds, lines 133 – 147, Figure 4, the original magnification and scale bars of the SEM images, should be added in the figure legend.

 

In Results section, 2.4. Osteogenesis of human MSCs seeded on scaffolds and in 2D insert culture, lines 158 – 222, figure 8, higher magnification of images representing CaSR immunohistochemistry of MSCs seeded scaffolds, should be added. It would be better, if the figures were placed in different channels (e.g. CaSR channel - green, DAPI channel- blue, and the merge of them, CaSR + DAPI channels -green/blue). The latter could be placed as supplementary data. In addition, original magnification and scale bars of the images must be added in the figure legend.

 

In Discussion section, comparison with previous studies should be performed regarding the beneficial effects of the coral-collagen based scaffolds in MSCs osteogenic differentiation process. In this way, the pros and cons of this study will be highlighted.

Comments for author File: Comments.pdf

Author Response

Reviewer 2: Dear authors, the manuscript entitled “The incorporation of marine coral microparticles into collagen-based scaffolds promotes osteogenesis of human mesenchymal stromal cells via calcium ion signaling.” Is a well written study. In this study the marine coral microparticles can be used for scaffold production, that efficiently could promote the osteocyte differentiation of MSCs and additionally the release of calcium. These scaffolds can be used in several tissue engineering application such as the regeneration of bone and cartilage defects.

Only a number of minor corrections should be performed to this study, which I think the authors are capable to perform, in order to further processed.

Thank you to reviewer 2 for taking the time to review our manuscript

Macroscopic images of the different categories of produced scaffolds must be added to the results section, for being easier for the readers to understand how these scaffolds look like.

Macroscopic images of scaffolds have been included as supplementary figure S2 in the revised manuscript. We have also updated the graphical abstract to include macroscopic images of the scaffolds.

In Materials and Methods section, 4.2. Fabrication of collagen and collagen/coral scaffolds, lines 366 – 377, the exact dimensions of the scaffolds should be added (e.g. length, width and thickness).

This additional information has been included in section 4.2 of the revised manuscript

Additionally, in materials and Methods section, 4.6. Mechanical testing of scaffolds, lines 409- 417, only compressive tests were performed? I think also, extension tests, involving engineering strain (ε), engineering stress (σ, in MPa). Elastin (El-E) and collagen (Col-E) phase slope, transition stress (σTrans) and strain (εTrans), ultimate tensile strength (σUTS) and failure strain (εUTS) would be very informative for the readers. (Compressions tests have been well performed, and this is only an optional minor revision. If the authors think that no additional information will be extracted from the extension tests, you can overpass this correction)

Our thanks to the reviewer for this recommendation. As the scaffolds described in this study are intended for use as grafts in orthopaedic applications, we considered compressive properties to be the most relevant mechanical properties to report. Although the tensile tests recommended by the reviewer would be interesting, particularly if these scaffolds were being evaluated for applications such as vascular tissue engineering which would necessitate the development of a compliant scaffold, we feel they are beyond the scope of the research question/hypothesis explored herein, which is primarily focused on the optimisation of these scaffolds for bone tissue engineering applications.

The rehydration rate of the scaffolds should be an important parameter that could be added in the materials and methods and results section.

This is indeed an important point. The rehydration rate (or swelling ratio) is an important characteristic of scaffolds for tissue engineering applications, as a scaffold’s capacity to swell upon hydration is typically associated with enhanced cellular adhesion. At the request of the reviewer, we have performed additional tests to evaluate the swelling ratios of our scaffolds and these results are presented in the revised manuscript as Figure 4d and are described in sections 2.2 and 4.4. Briefly, the incorporation of coral microparticles was found to reduce the swelling ratio of collagen-based scaffolds, which goes some way to explaining the initial reduction in cell attachment described below.

In Materials and Methods section, 4.7. Osteogenic differentiation of human MSCs cultured on scaffolds and in 2D, lines 418 – 434, the authors indicated that seeding of MSCs was performed in the produced scaffolds. It should be added the number of MSCs/ cm² (or cm³ of scaffold) and the exact dimensions of the scaffolds that were used for the seeding assay. In addition, please add the passage of MSCs used for seeding.

This additional information has been included in section 4.7 of the revised manuscript

 

In materials and methods section, 4.8 Immunohistochemical staining for CaSR and results section, 2.4. Osteogenesis of human MSCs seeded on scaffolds and in 2D insert culture, the performed assay is indirect immunofluorescence, in this way, please change the immunohistochemistry term with indirect immunofluorescence.

Thank you to the reviewer for this recommendation. In the revised manuscript, we now use ‘indirect immunofluorescence’ to describe CaSR staining.

When a new biomaterial is fabricated, it is reasonable to check if this scaffold has potentially toxic effect to the cells. For this purpose, cell viability assay (either contact or extract cytotoxicity test) must be performed.

Proliferation of MSCs or differentiated osteocytes into the scaffolds should also be estimated by MTT assay.

Our thanks to the reviewer for both these suggestions. We believe both points can be addressed together.  In the present study, we utilised the Picogreen assay to determine the DNA content of human MSCs seeded on scaffolds. Our experience is that this assay provides a more accurate assessment of cell numbers than metabolic assays such as Alamar Blue or MTT where increases in activity do not necessarily correlate with proliferation. After 24 hours, collagen scaffolds demonstrated a significantly higher DNA content compared to collagen/coral S and collagen/coral L scaffolds, which suggests that the incorporation of coral hinders, to a certain extent, initial cell attachment onto collagen-based scaffolds. However, after 28 days in culture, no differences in DNA content were observed between the different groups which demonstrates that collagen/coral scaffolds present an appropriate milleau for MSCs to migrate through and proliferate. Indeed, after 28 days in culture, MSCs seeded on collagen/coral scaffolds were shown to undergo a greater degree of proliferation when compared to MSCs seeded on collagen only scaffolds which demonstrates that collagen/coral scaffolds promote a more proliferative phenotype in MSCs. In the revised manuscript, we have included additional figures 8a and 8b and discuss their findings in section 2.4.

In Materials and Methods section, 4.7. qPCR analysis of osteogenic and angiogenic gene expressing, 4.8 Immunohistochemical staining for CaSR, 4.9. Biochemical analysis of MSC seeded on scaffolds and in 2D, MSCs with regular culture medium and validated osteocytes must be used as negative and positive control, respectively. It is known, that MSCs depending to their microenvironment could be differentiated to other cell types. In regards to this study, the produced scaffold may act beneficially to the differentiation of MSCs, or even to the production of calcium, without the external use of growth factors and differentiation medium. For this purpose, the seeding of MSCs in the produced scaffolds and further cultivation without the use of differentiation medium is worth to be performed.

We agree with the point the reviewer makes regarding the importance of validating the osteogenic phenotype of human MSCs. Prior to their seeding onto scaffolds, tri-potentiality assays were performed on human MSCs to confirm their capacity to undergo osteogenic differentiation according to protocols previously published [1].  With regards to the use of regular medium vs. differentiation medium, in order to facilitate initial cell attachment, MSC-seeded scaffolds were maintained in a regular medium for the first 24 h. Biochemical assays performed at this point (Figure 7) demonstrated a significant increase in the alkaline phosphatase activity of collagen/coral scaffolds when compared to collagen only scaffolds, illustrating the capacity of these scaffolds to enhance osteogenesis in the absence of exogenous growth factors. Thereafter, scaffolds were switched to a differentiation medium for the duration of the experiment with increases in BMP2 expression observed in collagen/coral scaffolds at day 7 (Figure 6) and increases in calcium accumulation observed in collagen/coral scaffolds at day 28 (Figure 8). In the discussion section of the revised manuscript, we have emphasised this capacity of collagen/coral scaffolds to promote osteogenesis of MSCs in the absence of exogenous growth factors.

In Results section, 2.2. Architectural properties of collagen/coral scaffolds, lines 133 – 147, Figure 4, the original magnification and scale bars of the SEM images, should be added in the figure legend.

 In the revised manuscript, this additional information has been added to the legend in Figure 4.

In Results section, 2.4. Osteogenesis of human MSCs seeded on scaffolds and in 2D insert culture, lines 158 – 222, figure 8, higher magnification of images representing CaSR immunohistochemistry of MSCs seeded scaffolds, should be added. It would be better, if the figures were placed in different channels (e.g. CaSR channel - green, DAPI channel- blue, and the merge of them, CaSR + DAPI channels -green/blue). The latter could be placed as supplementary data. In addition, original magnification and scale bars of the images must be added in the figure legend.

At the reviewer’s request, in the revised manuscript we have included supplementary figure S3 which illustrates the different channels used to represent CaSR indirect immunofluorescence. We have also included the magnification used in the legend of fig 8 and have increased the sizes of the images to improve the quality of their visual representation.

In Discussion section, comparison with previous studies should be performed regarding the beneficial effects of the coral-collagen based scaffolds in MSCs osteogenic differentiation process. In this way, the pros and cons of this study will be highlighted.

In the Discussion section of the revised manuscript, we have compared the effects on MSC proliferation observed herein to those observed in previous studies from our group which focussed on the fabrication of natural polymer/bioceramic composite scaffolds. Furthermore, we have elaborated on the role CaSR plays in regulating osteogenesis and angiogenesis and have compared our findings to previous work in the field which have examined the fabrication of composite scaffolds for musculoskeletal tissue engineering.

 

References

Barreto, S.; Gonzalez-Vazquez, A.; Cameron, A. R.; Cavanagh, B.; Murray, D. J.; O'Brien, F. J., Identification of the mechanisms by which age alters the mechanosensitivity of mesenchymal stromal cells on substrates of differing stiffness: Implications for osteogenesis and angiogenesis. Acta Biomater. 2017, 53, 59-69.

Round 2

Reviewer 2 Report

Dear Authors,

The majority of my comments have been well addressed.

Your manuscript is a quite interesting work which is worth to get published.

Yours sincerely