Transient Early Mechanical Loading Induces Hypertrophic Chondrocyte Differentiation of Human Mesenchymal Stromal Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Building on previous work from the same group, this manuscript by Enzmann et al. investigates how timing, duration and frequency of mechanical stimulation influences the hypertrophic chondrocyte differentiation of osteoprogenitor cells during endochondral bone formation, using an in vitro bioreactor model and human primary MSCs encapsulated in GelMA hydrogels

This work addresses an important topic, which concerns the cellular and molecular basis of endochondral ossification during fracture repair.

Experimental design is sound, and the conclusions are overall supported by the data.

While the findings are sufficiently novel and the manuscript is well written, A few improvements will increase the rigor and the discussion of the data:

• Limiting the impact and generalizability of the findings is the choice of assessing a single time point at Day14: intermediate endpoint would have clarified whether early mechanostimulation initiates a cascade or only correlates with later markers. The author may briefly discuss this choice in the discussion section
• The claim that findings Supports early mobilization” is speculative without in vivo validation and should be more accurately put in context of the in vitro model used in this work

Author Response

please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This article investigates how the number of deformation cycles, the length of the inter-cycles break and the duration of the deformation period affect MSCs differentiation into hypertrophic cartilage. The text is well written and the experiments are well described, well presented and easy to follow and understand. However, some aspects must be addressed prior to publication.

Major revisions:

1. For this study, the authors used BM-MSC obtained from females between 18 and 73 years old. In the literature, some differences in the differentiation capacity of MSCs have been described depending on the sex and the age of the donor. Taking this into account, why did the authors use only female cells? What about the differences observed between young/old donors (mainly pre/post menopause)? These aspects must be discussed.
2. As indicated by SO staining, GAG deposition and Col2 expression, chondrogenic differentiation was not achieved in either of the mechanically stimulated conditions nor the negative control. This observation could be explained by an inefficient activation of the chondrogenic differentiation of MSC due to the use of a defective chondrogeneic media. A positive control of differentiation must be included (by supplementing the media with TFGβ) to confirm the chondrogenic potential of the MSC lines.
3. MSC constructs consisted of 40M MSC/mL embedded in an 8% GelMA (w/v) matrix. Due to the high density, cellular viability should be addressed in a more direct and specific manner (Live/Dead staining, ATP,...) to assess possible central necrosis, as the initial viability of the cells could compromise both matrix deposition and the response to mechanical stimuli. DNA quantification is adequate for normalization of GAG content but may be insufficient to assess cell viability and proliferation.
4. The title of the manuscript is: "Short early mechanical stimulation equals prolonged loading in promoting hypertrophic chondrocyte differentiation of naïve Mesenchymal Stromal Cells: Implications for secondary bone healing". However, to talk about implications for bone healing, further experiments must be performed to demonstrate that the hypertrophic cartilage obtained by mechanical stimulation is able to promote a mineralized ECM in an enhanced or faster manner. Despite the authors considered this as a future study (line 557), this evidence should be included in this report to further increase its relevance. Assessment of RUNX2 or ALP expression at day 14 is not enough as evidence of functional hypertrophic cartilage and further in vitro experiments (switching the chondrogenic media to an osteogenic one and performing alizarin red/Von Kossa staining) must also be performed to complete the study.

Minor revisions:

1. Abstract (line 16): define post-OP.
2. Title and description for table 1 is missing.
3. In section “2.5. Statistical analysis” authors declared that “all experiments were performed using cells from 6 different donors (n=6 biological replicates) and in duplicates (n=2 technical replicates)”. However, according to the methods section, 2 technical replicates were included only in gene expression assays. DNA content and ECM characterization were performed only in 1 sample per donor and per condition (n=1 technical replicate). Please, clarify this.
4. Avoid including references in the results section (lines 243, 245, 263, 270, 299, 358, 361, ...)

Author Response

Please see attachment

Author Response File: Author Response.pdf