Optimization of a Tricalcium Phosphate-Based Bone Model Using Cell-Sheet Technology to Simulate Bone Disorders

Round 1

Reviewer 1 Report

In the paper submitted to Processes entitled “Optimization of a tricalciumphosphate-based bone model using cell-sheet technology to simulate bone disorders” the authors presented cell-sheet-based process for optimized mesenchymal stromal cell (MSC) seeding of a β-tricalciumphosphate (TCP) scaffold to generate 3D-models of cancellous bone tissue.

The article is well written and structured, and the methodology is well implemented, but there are significant shortcomings in the design of the experiment, and it is necessary to better elaborate why such models were used. Also, I believe that in vitro 3D models can hardly replace animal models and their specifics. In addition, the authors mentioned various bone pathologies from nonunion fractures to osteoporosis, which differ in pathogenesis and clinical features and the authors should better elaborate how such 3D model could be universal.

Regarding the experimental design, I believe it is crucial to justify why the granules of β-tricalcium phosphate were used in the specific size range from 1000 to 2000 µm. In bone, calcium phosphate is present in the form of hydroxyapatite (HA), so the question arises why HA-based granules were not used. It should be elaborated in the article why specific chemical composition and size of particles were used; I would suggest to cite article by Stokovic et al. „Autologous bone graft substitute containing rhBMP6 within autologous blood coagulum and synthetic ceramics of different particle size determines the quantity and structural pattern of bone formed in a rat subcutaneous assay“ (Bone. 2020 Dec;141:115654. doi: 10.1016/j.bone.2020.115654.) in which the ability of different particles (TCP vs HA; small vs large particles) to promote osteogenesis was evaluated and obtained findings might justify your choice of TCP 1000-2000 µm particles.

The authors should also elaborate why deferoxamine was used? It would be interesting to see results if such constructs were treated with some of the osteoinductive BMP molecules, like for example rhBMP2. I believe that this might be discussed in the paper.

In addition, apart from the potential of this construct as an in vitro 3D model, this formulation may be an option for bone regeneration with the possible addition of some osteoinductive molecules, so I believe it should be disscused in the introduction/discussion section.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The article traces an interesting experimental study about optimization of a tricalcium phosphate-based bone model using cell-sheet technology to simulate bone disorders. The methods are properly conducted. The availability of data adheres to the expected standards of your research community. The claims are moderatey discussed in the context of previous literature. The manuscript is clearly written and represents a thought-provoking article. There is adequacy to bioethical parameters with human beings. Plagiarism detector program showed exceptionally low content similarity (<1.0%), guaranteeing the originality of this article. Article with merit for publication and compatible proposal within the scope of the journal Processes. Point-to-point instructions for improving the text follow in the comments to the author. After minor revisions required to the content of this version, the manuscript will be ready for publication.

 

Title:

Replace “tricalciumphosphate-based” with “tricalcium phosphate-based”.

 

Section “Introduction” and/or “Discussion”:

•          The article agrees with the design of a calcium-deficient apatite, closer to natural apatite to simulate a microenvironment with characteristics closer to native human bone. However, it does not address the potential of biphasic ceramics (HA:β-TCP) to improve mechanical and biological performance with the association, which is well explored in the literature in the search for osteosubstitutes. In this context, the following article is suggested, which demonstrates the favoring of adhesion and spreading of mesenchymal cells and stimulation of osteogenesis in vitro and in vivo using HA:β-TCP carrier. de Oliveira Lomelino, R., Castro-Silva, I.I., Linhares, A.B.R. et al. The association of human primary bone cells with biphasic calcium phosphate (βTCP/HA 70:30) granules increases bone repair. J Mater Sci: Mater Med 23, 781–788 (2012). https://doi.org/10.1007/s10856-011-4530-1  
• The article does not discuss in depth other 3D in vitro biological simulation models of bone tissue that have been highlighted in the thematic literature in recent years, as is the case of osteogenic cell culture in a spheroid models with or without mineral carrier. I suggest for reading the following articles: Brochado, A.C.B.; de Souza, V.H.; Correa, J.; dos Anjos, S.A.; de Almeida Barros Mourão, C.F.; Cardarelli, A.; Montemezzi, P.; Gameiro, V.S.; Pereira, M.R.; Mavropoulos, E.; Alves, G.G. Osteosphere Model to Evaluate Cell–Surface Interactions of Implantable Biomaterials. Materials 2021, 14, 5858. https://doi.org/10.3390/ma14195858. VPA Lemos, AM Rossi, JM Granjeiro, MD Calasans-Maia, SC Sartoretto, P Sesterheim, DG Boeckel, I Grivicich, M Camassola, OSTEOGENIC POTENTIAL OF NANOSTRUCTURED CARBONATED HYDROXIAPATITE MICROSPHERES ASSOCIATED WITH MESENCHYMAL STEM CELL IN VITRO AND IN VIVO, Cytotherapy, Volume 23, Issue 4, Supplement, 2021,Page 29, ISSN 1465-3249, https://doi.org/10.1016/j.jcyt.2021.02.085. 
• The mesenchymal stromal cell reparative pattern is suitable for understanding of the osteodifferentiation/angiogenesis process and bone tissue construction. However, the article does not explore the in vitro immunomodulatory routes contextualized to bone tissue or osteosubstitutes, including the participation of hematopoietic and inflammatory cells presents in bone marrow and in immune response processes and remodeling against an artificial bone matrix. Considering the purpose of the title of the study “to simulate bone disorders”, I believe that the introduction and the discussion about in vitro co-culture need to be better developed, to also contemplate this topic that was rarely addressed.

 

Sections “Materials and Methods” and “Results”:

The sections are robust and describe protocols based on relevant science. The biomarkers used are feasible to interpret osseodifferentiation. The protocol with deferoxamine is a suitable strategy to assess responsiveness of the cellular model.

 

Section: “Conclusion”

- The authors must describe the inherent limitation of translational research in bone bioenginneering, which involves promising results in cells and/or animals but may not faithfully represent the response of the human organism.

 

Section: “References”

- It is strongly recommended to add more references/citations in the text, to make the article discussion more robust and evidence-based.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have implemented all the possible suggestions and answered very thoroughly to the all questions. After the changes are made, the article is suitable for publication.