Regenerative Potential of Blood-Derived Products in 3D Osteoarthritic Chondrocyte Culture System
Round 1
Reviewer 1 Report
An interesting article about the use of platelet-rich plasma in an osteoarthritic chondrocyte pellet model by assessing the metabolic cell activity, cartilage-related gene expression, and extracellular matrix deposition within the pellets. Although the article is interesting I think that the statistics and results section should be clarified before considering the paper eligible for publication:
Why did you consider significant p:0,1? I suggest you change this part in the statistical analysis and maybe consider it a tendency; you should consider significant at least p:0,05. Also, you should add the version and the maker, and the maker's location of the statistical program.
In the result section, when you consider a difference statistically significant, you should also report the p value
Page 1 line 41 you should add: "Different studies have proposed the use of platelet derivatives in order to facilitate the repair system of bones and cartilages. " and cite an article such as doi: 10.1111/dth.13334.
Thank You
Author Response
Dear Reviewer,
Thank you for taking the time to review our work. I greatly appreciate your thoughtful comments that helped to improve the manuscript.
Thank you very much for your effort. In the following text I give a point-by-point reply to your comments:
An interesting article about the use of platelet-rich plasma in an osteoarthritic chondrocyte pellet model by assessing the metabolic cell activity, cartilage-related gene expression, and extracellular matrix deposition within the pellets. Although the article is interesting I think that the statistics and results section should be clarified before considering the paper eligible for publication:
Why did you consider significant p:0,1? I suggest you change this part in the statistical analysis and maybe consider it a tendency; you should consider significant at least p:0,05. Also, you should add the version and the maker, and the maker's location of the statistical program. In the result section, when you consider a difference statistically significant, you should also report the p value
Thank you very much for pointing out the importance of considering the results significant only when p<0,05. Considering also suggestions from Reviewer 2, figure 2 was changed, only results with p<0,05 were considered significant and in case of COL2AB and COL1 expression some additional groups were presented. The requested modifications have been completed. The Results and Discussion sections were updated with the new p values and the description of the significancy was changed. Information about software was added to the Materials and Methods section.
Page 1 line 41 you should add: "Different studies have proposed the use of platelet derivatives in order to facilitate the repair system of bones and cartilages. " and cite an article such as doi: 10.1111/dth.13334.
Thank you for this suggestion and for pointing out a very valuable publication. The requested modifications have been completed.
I trust that all of your comments have been addressed accordingly in a revised manuscript.
Reviewer 2 Report
- A brief summary
The article entitled “Regenerative Potential of Blood-Derived Products in 3D Osteoarthitic Culture System” by Olga Kuten-Pella et al. compares different different types of platelet-rich plasma and hyperacute serum in the in vitro chondrogenic differentiation of OA-derived chondrocytes
- Broad comments
This article shows very clear the effects of the different blood products to enhance differentiation of chondrocytes in vivo for later application in patients with degenerative cartilage disorders, especially OA. This is an important field to investigate. The article shows useful results, which could have consequences on further work in this field.
For me, some controls are missing.
- You don´t use cells from healthy persons.
- You don´t cultivate chondrocytes in 3D without the addition of TGFb, which results in the real chondrogenic differentiation.
Overall, the paper reads well and does fit within the scope of the journal.
- Specific comments
Please find as comments in the text.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
Thank you for taking the time to review our work. I greatly appreciate your thoughtful and detailed comments included in the text. I trust that all of your comments have been addressed accordingly in a revised manuscript. All the requested modifications have been completed together with suggestions from Reviewer 1 and can be seen with the "track changes option" in the main Microsoft Word file.
Thank you very much for your effort. In the following text I included a reply to your comments:
You don´t use cells from healthy persons.
Thank you for highlighting this issue. I agree it would be very helpful to include pellets with chondrocytes obtained from healthy donors. However, human articular cartilage was received from the University Hospital Krems from patients undergoing total knee arthroplasty. Unfortunately, we did not have an access to the cartilage tissue of healthy donors. We work only with the chondrocytes in passage 1 to ensure the high quality of the cells. Cultivation of commercially available chondrocytes from healthy donors generate high costs and in case of pellet culture experiments a high number of cells is required. Therefore there is a serious risk of phenotypic changes and this is the reason we decided to include only OA chondrocytes in our study.
You don´t cultivate chondrocytes in 3D without the addition of TGFb, which results in the real chondrogenic differentiation.
Thank you for raising this point. I am aware of this limitation of our study. The reason we did not include the groups without a chondrogenic medium (including TGF beta) was that the cell number we were able to obtain from the cartilage tissues (which was a surgical waste) was a serious limiting factor. Reduction of cell number to prepare 1 chondrogenic pellet is not possible due to the small size of pellets with less than 250.000 cells per pellet. We tried to obtain the best quality of the pellets to perform successful histological analysis and evaluate ECM deposition.
For that reason, we followed the protocol published by our colleague Ass. Prof. Mag. Andrea De Luna (Luna-Preitschopf A De, Zwickl H, Nehrer S, Hengstschläger M, Mikula M. Rapamycin Maintains the Chondrocytic Phenotype and Interferes with Inflammatory Cytokine Induced Processes. Int J Mol Sci. 2017;18(7).). Mrs De Luna together with her colleagues observed that using the chondrogenic medium for cultivating 3D OA chondrocyte aggregates improves the quality of the structures. Please find the attached picture of pellets we cultivated in normal DMEM with FCS and without the chondrogenic factors. These had very poor quality and we had to modify the medium composition to obtain better structures. Therefore, the composition we described in our manuscript seems to be the most optimal for this 3D culture system for blood products testing.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors responded to all queries. Statistics has been improved since the last version, and requested additions have been made. The paper is in my opinion eligible to be published.