Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article investigates how extreme temperatures affect the viability and oxidative stress response in human bronchial epithelial (HBE) cells using a novel approach involving 3D-printed cell-laden bioinks. The study exposes these cells to various temperatures and measures their responses through Hoechst, Sytox, and Mitosox staining. The findings provide valuable insights into the cellular mechanisms of thermal injury, particularly nuclear damage, cell death, and mitochondrial oxidative stress in HBE cells. This research contributes to the field by expanding our understanding of the effects of thermal stress on respiratory cells, with implications for occupational health and respiratory disease management. In general, this manuscript is well-organized and clearly written. However, there are several shortcomings that need to be addressed before it can be accepted.

1.  Could the authors provide a more thorough explanation of the experimental setup for temperature control? Specifically, how was the temperature in each well plate consistently maintained during exposure, and was there any potential variability between wells?

2.      The decrease in Mitosox intensity at higher temperatures is discussed as an indication of reduced oxidative stress due to cell death. However, further analysis on why some residual Mitosox signal was observed at 55°C could strengthen this interpretation. Could the authors expand on the cellular processes involved or consider additional assays to verify this interpretation?

3.   This study has potential applications in the field of 3D bioprinting, particularly for occupational health monitoring and tissue engineering related to thermal stress. It is recommended that the authors discuss specific application contexts, such as how this technique could be applied in occupational health monitoring or explore its potential development in artificial airway tissue models or drug testing platforms.

4.       Reference: There are some typographical errors in the references. Please review and correct them.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The methodology described in the paper is very clear; however, the conclusion is relatively simple. The idea that cells are more prone to damage at high temperatures is quite common knowledge. While the study does achieve certain results, the conclusions reached are not significantly different from what one might expect even before conducting the experiments.

 Whether clearer and more impactful conclusions can be drawn in the discussion and conclusion sections could enhance the overall contribution of the paper.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Abstract (Lines 10-22)

The abstract provides a concise overview of the study. However, consider enhancing the clarity of the specific aim. The current abstract suggests that the study focuses on the effects of extreme temperature on HBE cells but doesn’t mention if it investigates specific cellular mechanisms, such as oxidative stress and cell viability, until the latter part.

• Line 16: "Increasing the temperature from 37 °C to 55 °C led to a significant reduction in Hoechst 33342 intensity." It might be helpful to state the implications of reduced Hoechst 33342 intensity (indicating nuclear damage) in simpler terms for broader accessibility.
• Suggestion: Rephrase for clarity, e.g., "A temperature increase from 37 °C to 55 °C significantly reduced nuclear integrity as observed through Hoechst 33342 intensity."

Introduction (Lines 26-55)

The introduction provides a solid background on the physiological implications of hyperthermia and previous studies in similar areas.

• Line 29: "Hyperthermia poses severe challenges to the human body’s ability to maintain homeostasis…" This sentence could be made more concise for readability. Consider defining homeostasis in a brief, integrated manner if it’s necessary for readers less familiar with the term.
• Line 40-44: "Most of the studies (included in Table 1) relied on animal models…" Some references lack direct association with HBE cells or human-related temperature impacts, which may not support the argument strongly. Ensure references directly link to the study's aim.

Materials and Methods (Lines 56-134)

This section is well-organized, but further detail on certain procedures would improve reproducibility and transparency.

• Line 64: “They were cultured in α-MEM medium…” Specify whether this medium was supplemented with any additional components (e.g., fetal bovine serum or antibiotics).
• Lines 69-79: The preparation of the bioink is clear, but more information on maintaining the bioink's temperature during preparation could improve accuracy, as temperature can affect bioink properties.
• Line 119: “Extrusion height of 0.20 mm” – if this height impacts the overall cellular embedding or viability, explain why this specific distance was selected.
• Suggestion: Overall, consider clarifying any potential temperature effects on the bioink's properties during preparation.

Assessment of HBE Cells’ Responses (Lines 137-175)

The process for cell staining and data collection is described adequately. However, adding rationale for specific choices, like the selected fluorescence intensities and staining times, could enhance the reader’s understanding.

• Lines 153-154: Mention of “Texas red filter” might be confusing; elaborate briefly on the detection criteria to clarify.
• Line 171: Mentioning the format in “plain text (.txt)” is detailed but could be less technical for broader readership if not essential.
• Line 173: The calculation formula is correctly included, but further explaining the choice of parameters for image selection could improve clarity on how randomness was ensured.

Results and Discussion (Lines 184-258)

This section presents data adequately; however, combining some figures into one graph might increase readability.

• Line 193: It’s helpful that p-values for effects are presented, but discussing effect sizes would also improve the interpretation of these statistical results.
• Line 204: The explanation regarding Hoechst intensity and cellular membrane damage is effective but would benefit from explicit mention of how this data correlates with previous studies or anticipated outcomes.
• Lines 225-230: The explanation of Mitosox intensity is comprehensive but could be more concise. Consider summarizing key points without losing depth to make the passage more reader-friendly.

Figures and Tables (Lines 195-261)

The figures are generally well-labeled, although they could be consolidated where possible to streamline data presentation.

• Figures 5-8: Presenting all effects of temperature in one or two combined figures, perhaps with subplots for each measurement, might make it easier to compare trends.
• Table 2: The detailed RFU values are helpful, but for readers unfamiliar with RFU, provide a brief contextualization.

Conclusion (Lines 262-276)

The conclusion restates the findings but could emphasize future research implications more robustly.

• Line 272-275: "Future studies include investigating cells’ responses to high temperature at different levels of exposure duration…" This part is insightful but would be stronger if it included more specific potential applications (e.g., implications in biomedical engineering or occupational health fields).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf