Preliminary Effects of Hyperbaric Oxygen Therapy on Hair Follicle Characteristics in Healthy Subjects

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript titled 'Preliminary Effects of Hyperbaric Oxygen Therapy on Hair Follicle Characteristics in Healthy Subjects' presents a preliminary clinical study exploring the effects of hyperbaric oxygen therapy (HBOT) on hair follicle characteristics and scalp condition in healthy adults. The study is carefully conducted from a procedural standpoint, and the combination of objective phototrichogram measures with subjective self-reports represents a reasonable exploratory approach. However, given the very small sample size, lack of a control group, and the largely non-significant objective findings, the manuscript would benefit from more cautious interpretation and clearer positioning as a pilot or hypothesis-generating study.

Major comments:

1. The lack of a control or sham group is a major limitation that substantially constrains causal interpretation. While this is acknowledged, its implications are not fully reflected in the tone of the Results, Discussion, and Conclusions. I would recommend to soften statements suggesting that HBOT “enhances” or “promotes” hair regeneration, to emphasize association and preliminary trends rather than efficacy.

2. With only nine participants completing the protocol, the study is underpowered to detect anything but very large effects. This limitation should be more explicitly integrated into the interpretation of findings, rather than framing consistent trends as indicative of biological effects.

3. The significant decrease in hair shaft thickness is interpreted as evidence of early anagen-phase regrowth. While this is a plausible explanation, authors should consider providing alternative interpretations (e.g., measurement variability, changes in hair cycle synchronization) to avoid overinterpretation of a single significant outcome.

4. Subjective assessments show robust and statistically significant improvements across all domains, whereas objective measures largely do not. This divergence deserves more critical discussion, including the possibility of expectancy effects, placebo responses, or heightened self-monitoring during an intensive intervention.

5. The conclusion suggests that HBOT may represent a promising non-pharmacological approach for hair regeneration. Given the healthy sample and preliminary nature of the findings, maybe authors should better frame these implications and clearly separate them from evidence relevant to alopecia or clinical populations.

6. In discussing the mechanisms by which HBOT might influence hair follicle dynamics, it may be useful to briefly acknowledge that such effects likely emerge from coordinated interactions across vascular, metabolic, and regenerative systems rather than from a single isolated pathway (see for example https://doi.org/10.1038/s41598-019-43860-w; https://doi.org/10.1016/j.neubiorev.2024.105542). In my opininon, adding a short reflection could help contextualize the findings while maintaining appropriate caution.

 

Minor comments

1. While visual images are helpful, factors such as lighting, hair styling, and dyeing are not standardized and could influence perception. Authors should acknowledge this more clearly as a source of variability.

2. The subjective questionnaire is straightforward but appears to be study-specific. I recommend a brief justification for its use, or acknowledgment of its non-validated nature, would strengthen transparency.

3. Authors should report effect sizes alongside p-values to help in a better evaluation of the magnitude and relevance of observed changes.

4. The manuscript is generally understandable, but several sentences, particularly in the Discussion, are long and repetitive. A light language edit would improve readability.

Comments on the Quality of English Language

The manuscript is generally understandable; however, the English requires substantial revision to improve clarity, grammar, and flow.

Author Response

Major comments:

1. The lack of a control or sham group is a major limitation that substantially constrains causal interpretation. While this is acknowledged, its implications are not fully reflected in the tone of the Results, Discussion, and Conclusions. I would recommend to soften statements suggesting that HBOT “enhances” or “promotes” hair regeneration, to emphasize association and preliminary trends rather than efficacy.

☞ [Response] We sincerely thank the reviewer for this insightful and constructive comment. We fully agree that the absence of a control or sham group limits causal inference and warrants a more cautious interpretation of the findings. In response to this valuable suggestion, we have carefully revised the tone throughout the Results, Discussion, and Conclusion sections to avoid causal or efficacy-implying language. Specifically, terms such as “enhances,” “promotes,” and “therapeutic potential” have been replaced with more conservative expressions such as “was associated with,” “showed a trend toward,” or “may be related to.” We have also strengthened the discussion of this limitation to explicitly acknowledge that the observed changes cannot be definitively attributed to HBOT due to the non-controlled design. These revisions aim to align the manuscript more appropriately with the exploratory and preliminary nature of this study. We appreciate the reviewer’s guidance in improving the scientific rigor and interpretative balance of the manuscript.

☞ [Before]

<Results>

(Line 226-228) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters may indicate a meaningful biological response to HBOT.”

(Line 245-247) “These changes align with the quantitative trends observed in hair volume, follicle density, and subjective perception, thus reinforcing the therapeutic potential of HBOT for hair regeneration”

<Discussion>

(Line 288-290) “Enhanced vascularity and oxygenation may thus create a favorable microenvironment for the activation of hair follicle stem cells and dermal papilla cells. [18]”

(Line 301-303) “These visual changes were consistent with both subjective feedback and objective data trends, further supporting the hypothesis that HBOT may contribute to follicular health and scalp rejuvenation. [21]”

<Conclusions>

(Line 348-350) “These preliminary findings suggest that HBOT may contribute to scalp health and follicular activation, offering a promising non-pharmacological approach to hair care.”

☞ [Revised]

<Results>

(Line 263-268) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters should be interpreted with caution. Given the absence of a control group and the limited sample size, the study is underpowered to support causal inference, and these findings should be considered exploratory rather than indicative of definitive biological effects.”

(Line 286-289) “These changes align with the quantitative trends observed in hair volume, follicle density, and subjective perception, suggesting a possible association between HBOT exposure and changes in hair-related parameters.”

<Discussion>

(Line 340-343) “Enhanced vascularity and oxygenation may create a microenvironment theoretically favorable for hair follicle stem cell and dermal papilla activity; however, the present study design does not allow confirmation of this mechanistic relationship. [20]”

(Line 366-368) “These visual changes were consistent with both subjective feedback and objective data trends, further supporting a potential association between HBOT exposure and observed changes in scalp and hair-related parameters. [25]”

<Conclusions>

(Line 429-435) “These preliminary findings suggest that HBOT exposure was associated with changes in scalp and hair-related parameters in healthy adults. However, given the absence of a control group, the exploratory design, and the inclusion of participants without diagnosed alopecia, these results should be interpreted cautiously and should not be considered evidence of therapeutic efficacy in clinical hair loss conditions. Rather, the present findings are hypothesis-generating and may inform future controlled studies in patients with alopecia.”

2. With only nine participants completing the protocol, the study is underpowered to detect anything but very large effects. This limitation should be more explicitly integrated into the interpretation of findings, rather than framing consistent trends as indicative of biological effects.

☞ [Response] We sincerely thank the reviewer for highlighting the important issue of statistical power. We fully agree that the small sample size (n=9) substantially limits the ability of this study to detect anything other than large effect sizes and increases the risk of both type II error and overinterpretation of directional trends. In response to this comment, we have revised the Results, Discussion, and Conclusion sections to more explicitly acknowledge the limited statistical power of the study. Statements previously suggesting that consistent trends may reflect biological effects have been modified to emphasize the exploratory and hypothesis-generating nature of the findings. We now clarify that the observed directional changes should be interpreted cautiously and cannot be considered confirmatory evidence of biological efficacy. Additionally, we have strengthened the limitations section by explicitly discussing statistical power constraints and the need for adequately powered randomized controlled trials to validate these preliminary observations. We appreciate the reviewer’s constructive guidance, which has helped us present a more balanced and methodologically rigorous interpretation of our findings.

☞ [Before]

<Results>

(Line 226-228) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters may indicate a meaningful biological response to HBOT.”

(Line 231-233) “Taken together, these objective measurements suggest a trend toward improved hair characteristics, reinforcing the positive outcomes observed in the subjective assessments.”

<Discussion>

(Line 338-340) “However, the convergence of subjective improvements, positive trends in objective parameters, and visual enhancements supports the hypothesis that HBOT may play a supportive role in hair follicle activation and scalp health.”

<Conclusions>

(Line 350-352) “While the current study had limitations, including a small sample size, absence of long-term follow-up, and use of a modified HBOT protocol, the observed trends warrant further investigation.”

☞ [Revised]

<Results>

(Line 263-268) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters should be interpreted with caution. Given the absence of a control group and the limited sample size, the study is underpowered to support causal inference, and these findings should be considered exploratory rather than indicative of definitive biological effects.”

(Line 271-274) “Taken together, these objective measurements suggest directional changes in hair-related parameters; however, given the limited sample size and reduced statistical power, these findings should be considered exploratory and hypothesis-generating rather than confirmatory.”

<Discussion>

(Line 414-421) “Although subjective improvements and directional trends in objective parameters were observed, the small sample size substantially limits inferential strength. Therefore, these findings should be interpreted as preliminary observations that warrant validation in adequately powered controlled studies. Moreover, with only nine participants completing the protocol, the study was likely underpowered to detect small-to-moderate effect sizes. As such, the absence of statistical significance in several parameters may reflect limited power rather than true absence of effect, while observed directional trends should not be overinterpreted as evidence of biological efficacy.”

<Conclusions>

(Line 435-439) “While the current study had limitations, including a small sample size, absence of long-term follow-up, and use of a modified HBOT protocol, the observed trends, although derived from a small and underpowered sample, warrant further investigation in larger, adequately powered randomized controlled trials.”

3. The significant decrease in hair shaft thickness is interpreted as evidence of early anagen-phase regrowth. While this is a plausible explanation, authors should consider providing alternative interpretations (e.g., measurement variability, changes in hair cycle synchronization) to avoid overinterpretation of a single significant outcome.

☞ [Response] We sincerely thank the reviewer for this thoughtful and important comment. We agree that interpreting the significant reduction in hair shaft thickness solely as evidence of early anagen-phase regrowth may risk overinterpretation, particularly given that this was the only objectively significant outcome in a small sample. In response, we have revised the Discussion section to present alternative explanations for this finding. Specifically, we now acknowledge the possibility of measurement variability, changes in hair cycle synchronization, and technical factors related to phototrichogram-based analysis. We also clarify that the observed reduction in thickness should be interpreted cautiously and cannot be definitively attributed to early regenerative processes. These revisions aim to provide a more balanced and scientifically rigorous interpretation of the data.

☞ [Before] (Line 279-282) “Notably, a significant decrease in hair shaft thickness was observed, which may be indicative of early anagen-phase regrowth, where newly formed vellus or intermediate hairs are typically finer in diameter. This observation aligns with early signs of follicular reactivation.”

☞ [Revised] (Line 324-333) “Notably, a statistically significant decrease in hair shaft thickness was observed. One possible interpretation is that this finding reflects early anagen-phase regrowth, during which newly emerging vellus or intermediate hairs are typically finer in diameter. However, alternative explanations should also be considered. The observed reduction may be influenced by measurement variability inherent to phototrichogram-based analysis, changes in hair cycle synchronization, or shifts in the proportion of growing versus resting hairs within the sampled area. Given that this was the only objectively significant parameter and the study was conducted in a small cohort without a control group, this finding should be interpreted cautiously and cannot be conclusively attributed to follicular regeneration.”

4. Subjective assessments show robust and statistically significant improvements across all domains, whereas objective measures largely do not. This divergence deserves more critical discussion, including the possibility of expectancy effects, placebo responses, or heightened self-monitoring during an intensive intervention.

☞ [Response] We sincerely thank the reviewer for this insightful observation. We agree that the divergence between robust subjective improvements and largely non-significant objective findings warrants more critical examination. In response, we have substantially revised the Discussion section to address this discrepancy. We now explicitly consider alternative explanations, including expectancy effects, placebo responses, increased self-monitoring, and the potential psychological impact of participating in an intensive intervention (50 sessions over three months). We also clarify that, in the absence of a control or sham group, the subjective improvements cannot be definitively attributed to physiological effects of HBOT. These revisions aim to provide a more balanced interpretation of the findings and to transparently acknowledge the possibility of perceptual and contextual influences on self-reported outcomes.

☞ [Before] (Line 291-298) “Subjective assessments in this study revealed statistically significant improvements across all evaluated domains, including perceived hair density, thickness, reduced shedding, and improved anterior hairline. These self-reported outcomes are consistent, noting improvements in hair thickness and perifollicular microcirculation following HBOT in patients with alopecia areata.[19] Furthermore, the observed enhancement in the frontal hairline, often refractory to conventional treatments such as topical minoxidil, suggests that HBOT may have a unique capacity to improve perfusion in typically unresponsive regions of the scalp.”

☞ [Revised] (Line 349-360) “Subjective assessments in this study revealed statistically significant improvements across all evaluated domains, including perceived hair density, thickness, reduced shedding, and improved anterior hairline. However, these robust subjective improvements contrasted with the largely non-significant objective findings, highlighting a divergence that warrants careful interpretation. These self-reported outcomes are consistent with prior reports suggesting that enhanced oxygenation may influence scalp microcirculation and perceived hair characteristics [21]; however, the present findings were observed in healthy individuals and should not be directly extrapolated to clinical alopecia populations. Although participants reported improvement in the frontal hairline, this observation should be interpreted cautiously. In the absence of controlled comparison and objective confirmation, it remains unclear whether this reflects physiological change or perceptual bias.”

5. The conclusion suggests that HBOT may represent a promising non-pharmacological approach for hair regeneration. Given the healthy sample and preliminary nature of the findings, maybe authors should better frame these implications and clearly separate them from evidence relevant to alopecia or clinical populations.

☞ [Response] We sincerely thank the reviewer for this important comment regarding external validity and clinical interpretation. We fully agree that the present study was conducted exclusively in healthy adults and was designed as a preliminary exploratory investigation. Therefore, any implications for alopecia or clinical populations should be interpreted with caution. In response, we have revised the Conclusion section to clearly distinguish between findings observed in healthy individuals and potential applications in clinical populations. Statements suggesting therapeutic promise for hair regeneration have been softened and reframed as exploratory observations. We now explicitly state that the results cannot be generalized to patients with alopecia and that dedicated studies in clinical populations are required before drawing any treatment-related conclusions. We appreciate the reviewer’s guidance in improving the precision and appropriateness of the manuscript’s implications.

☞ [Before] (Line 348-350) “These preliminary findings suggest that HBOT may contribute to scalp health and follicular activation, offering a promising non-pharmacological approach to hair care.”

☞ [Revised] (Line 429-435) “These preliminary findings suggest that HBOT exposure was associated with changes in scalp and hair-related parameters in healthy adults. However, given the absence of a control group, the exploratory design, and the inclusion of participants without diagnosed alopecia, these results should be interpreted cautiously and should not be considered evidence of therapeutic efficacy in clinical hair loss conditions. Rather, the present findings are hypothesis-generating and may inform future controlled studies in patients with alopecia.”

6. In discussing the mechanisms by which HBOT might influence hair follicle dynamics, it may be useful to briefly acknowledge that such effects likely emerge from coordinated interactions across vascular, metabolic, and regenerative systems rather than from a single isolated pathway (see for example https://doi.org/10.1038/s41598-019-43860-w; https://doi.org/10.1016/j.neubiorev.2024.105542). In my opininon, adding a short reflection could help contextualize the findings while maintaining appropriate caution.

☞ [Response] We sincerely thank the reviewer for this thoughtful and constructive suggestion. We agree that the biological effects of hyperbaric oxygen therapy are unlikely to operate through a single isolated pathway and are more plausibly mediated through coordinated interactions among vascular, metabolic, inflammatory, and regenerative systems. In response, we have revised the Discussion section to incorporate a more integrative mechanistic perspective. We now acknowledge that any potential influence of HBOT on hair follicle dynamics likely reflects complex, multi-system interactions rather than direct stimulation of a singular molecular pathway. We have also incorporated the suggested references to contextualize this systems-level framework. We believe that this addition strengthens the biological plausibility of the manuscript while maintaining appropriate interpretative caution.

☞ [Before] (Line 285-290) “HBOT has been shown to upregulate angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), both of which are critical for maintaining perifollicular vascularization and follicle health. [16,17] Enhanced vascularity and oxygenation may thus create a favorable microenvironment for the activation of hair follicle stem cells and dermal papilla cells. [18]”

☞ [Revised] (Line 336-348) “HBOT has been shown to upregulate angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), both of which contribute to perifollicular vascularization and follicle homeostasis. [16,17] However, it is unlikely that any observed changes in hair follicle dynamics arise from a single isolated molecular pathway. Enhanced vascularity and oxygenation may create a microenvironment theoretically favorable for hair follicle stem cell and dermal papilla activity; however, the present study design does not allow confirmation of this mechanistic relationship. [18] Recent literature emphasizes that hyperoxic exposure triggers system-level adaptations involving neurovascular coupling, metabolic reprogramming, and regenerative signaling cascades rather than linear cause–effect mechanisms. [19,20] In this context, any potential effects of HBOT on hair physiology should be understood as emerging from integrated biological networks rather than from direct follicular stimulation alone.”

 

Minor comments

1. While visual images are helpful, factors such as lighting, hair styling, and dyeing are not standardized and could influence perception. Authors should acknowledge this more clearly as a source of variability.

☞ [Response] We thank the reviewer for this valuable comment. We have revised the manuscript to more explicitly acknowledge that photographic assessments were not fully standardized with respect to lighting, hair styling, and cosmetic treatments, and that these factors may have influenced visual interpretation.

☞ [Before] (Line 303-306) “However, the use of photographic assessment also presents a potential source of variability. Factors such as lighting, camera angle, and hair dye could influence perceived changes, particularly in a study without a control group. [22]”

☞ [Revised] (Line 368-373) “However, the use of photographic assessment represents an important source of variability. Despite attempts to maintain consistent imaging conditions, factors such as lighting, camera angle, hair styling, and cosmetic treatments (including hair dyeing) were not fully standardized and may have influenced visual perception of hair density and coverage, particularly in the absence of a control group. [26]”

2. The subjective questionnaire is straightforward but appears to be study-specific. I recommend a brief justification for its use, or acknowledgment of its non-validated nature, would strengthen transparency.

☞ [Response] We thank the reviewer for this important comment. We acknowledge that the questionnaire used in this study was developed specifically for this exploratory investigation and has not undergone formal validation. We have revised the Methods section to clarify its study-specific nature and to emphasize that subjective findings should be interpreted with appropriate caution.

☞ [Before] (Line 155-158) “Table 1 shows the hair condition (scalp appearance, crown, amount of hair, hair thickness, hair growth rate, and frontal hairline) before and after hyperbaric oxygen therapy was evaluated using a 7-point Likert scale from “very worse (1 point)” to “very improved (7 points).””

☞ [Revised] (Line 187-193) “Table 1 shows that hair condition (scalp appearance, crown, amount of hair, hair thickness, hair growth rate, and frontal hairline) before and after hyperbaric oxygen therapy was evaluated using a 7-point Likert scale ranging from “very worse (1 point)” to “very improved (7 points).” This questionnaire was developed specifically for the present exploratory study and has not undergone formal psychometric validation. It was used as a pragmatic tool to capture participant-perceived changes; therefore, the subjective findings should be interpreted with appropriate caution.”

3. Authors should report effect sizes alongside p-values to help in a better evaluation of the magnitude and relevance of observed changes.

☞ [Response] We thank the reviewer for this valuable suggestion. In response, we have calculated and reported effect sizes alongside p-values for the primary outcome measures to provide a clearer understanding of the magnitude and potential clinical relevance of the observed changes. The corresponding effect size metrics have been added to the Results section, and the statistical analysis subsection has been updated accordingly.

☞ [Before] (Line 201-202) “All statistical tests were two-tailed, and a p-value of less than 0.05 was considered statistically significant.”

☞ [Revised] (Line 236-240) “All statistical tests were two-tailed, and a p-value of less than 0.05 was considered statistically significant. In addition to p-values, effect sizes were calculated to estimate the magnitude of observed changes. Cohen’s d was used for paired comparisons and interpreted as small (0.2), medium (0.5), or large (0.8) effects.”

4. The manuscript is generally understandable, but several sentences, particularly in the Discussion, are long and repetitive. A light language edit would improve readability.

☞ [Response] We thank the reviewer for this helpful comment. We have carefully reviewed the manuscript and performed a light language edit to improve clarity and readability, particularly in the Discussion section. Several long and repetitive sentences were shortened or restructured to enhance flow and reduce redundancy while preserving the original meaning.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The communication “Preliminary Effects of Hyperbaric Oxygen Therapy on Hair Follicle Characteristics in Healthy Subjects“ depicts a study showing  a preliminary study on the HBOT treatment applied on hair follicle.

 

The merit of this study is that it investigates a general population problem with a promising approach using HBOT, but its experimental robustness is too limited.

The study is well conducted, but the study group is too small, and many of the observations made by authors in the discussion section, significantly reduce the weight of the study.

The abstract aims to introduce a clear scientific route and materials and methods are clear, and data collection is well explained. The application of the Becon system has several weaknesses.

Furthermore, the study is too biased toward qualitative rather than quantitative aspects. Even though the statistics are applied correctly, the observations are not shown to be sufficiently significant and meaningful because the numbers are too limited.

In the discussion section, the authors showed the study limits, the results are encouraging, but at the limit of publishability in this journal.

Furthermore, the appropriateness of the study with respect to bioengineering is not understood.

The literature cited is appropriate.

For the reasons mentioned above, the manuscript cannot be accepted for publication in this journal but it is suggested that it be transferred to other publications of the same publisher.

Author Response

The merit of this study is that it investigates a general population problem with a promising approach using HBOT, but its experimental robustness is too limited.

☞ [Response] We thank the reviewer for recognizing the potential relevance of our study and for the thoughtful comment regarding its experimental robustness. We acknowledge that the exploratory design, small sample size, and absence of a control group limit the strength of causal inference and generalizability. As suggested, we have revised the manuscript to more clearly emphasize these limitations throughout the Discussion and Conclusions, and to frame our findings as preliminary and hypothesis-generating. We agree that further randomized controlled studies with larger sample sizes are necessary to establish the robustness and clinical relevance of HBOT in this context.

☞ [Before] (Line 348-350) “These preliminary findings suggest that HBOT may contribute to scalp health and follicular activation, offering a promising non-pharmacological approach to hair care.”

☞ [Revised] (Line 429-435) “These preliminary findings suggest that HBOT exposure was associated with changes in scalp and hair-related parameters in healthy adults. However, given the absence of a control group, the exploratory design, and the inclusion of participants without diagnosed alopecia, these results should be interpreted cautiously and should not be considered evidence of therapeutic efficacy in clinical hair loss conditions. Rather, the present findings are hypothesis-generating and may inform future controlled studies in patients with alopecia.”

The study is well conducted, but the study group is too small, and many of the observations made by authors in the discussion section, significantly reduce the weight of the study.

☞ [Response] We thank the reviewer for acknowledging the overall conduct of the study and for highlighting these important concerns. We fully agree that the small sample size limits statistical power and reduces the strength of inference. In response, we have further emphasized this limitation in the Discussion and Conclusion sections. Additionally, we have carefully revised the Discussion to reduce speculative interpretations and to avoid overstating the implications of our findings. Statements suggesting efficacy or therapeutic potential have been softened, and the results are now framed as preliminary and hypothesis-generating. We believe these revisions more appropriately reflect the exploratory nature of the study and its limited sample size.

☞ [Before] (Line 348-350) “These preliminary findings suggest that HBOT may contribute to scalp health and follicular activation, offering a promising non-pharmacological approach to hair care.”

☞ [Revised] (Line 429-435) “These preliminary findings suggest that HBOT exposure was associated with changes in scalp and hair-related parameters in healthy adults. However, given the absence of a control group, the exploratory design, and the inclusion of participants without diagnosed alopecia, these results should be interpreted cautiously and should not be considered evidence of therapeutic efficacy in clinical hair loss conditions. Rather, the present findings are hypothesis-generating and may inform future controlled studies in patients with alopecia.”

The abstract aims to introduce a clear scientific route and materials and methods are clear, and data collection is well explained. The application of the Becon system has several weaknesses.

☞ [Response] We thank the reviewer for this important comment. We acknowledge that the Becon imaging system has methodological limitations, including potential variability in image acquisition and reliance on visual interpretation. We have revised the manuscript to avoid overstating the imaging findings and to clarify that these images provide descriptive, rather than confirmatory, evidence.

☞ [Before] (Line 299-301) “Standardized scalp imaging using the Becon system provided visual corroboration of therapeutic effect, revealing apparent increases in hair density and coverage, particularly in the vertex and crown regions. [20]”

☞ [Revised] (Line 361-366) “Standardized scalp imaging using the Becon system provided visual corroboration of therapeutic effect, revealing apparent increases in hair density and coverage, particularly in the vertex and crown regions. [24] However, these observations were based on photographic comparison and should be interpreted descriptively, as the system is subject to variability in image acquisition and does not provide definitive confirmation of therapeutic efficacy.”

Furthermore, the study is too biased toward qualitative rather than quantitative aspects. Even though the statistics are applied correctly, the observations are not shown to be sufficiently significant and meaningful because the numbers are too limited.

☞ [Response] We thank the reviewer for this important comment. We agree that the relatively small sample size limits statistical power and constrains the strength and interpretability of the quantitative findings. We also acknowledge that subjective outcomes were more consistently significant than objective measures, which may create an impression of qualitative bias. In response, we have revised the manuscript to clearly emphasize the exploratory and hypothesis-generating nature of the study, explicitly acknowledge the limited statistical power, report effect sizes alongside p-values, and provide a more cautious interpretation of directional trends. We now clearly distinguish between subjective perceptions and objective measurements and discuss the divergence between these domains, including potential expectancy or contextual effects. These revisions aim to ensure that the findings are not overstated and that the manuscript accurately reflects the preliminary nature of the data.

☞ [Before]

<Results>

(Line 226-228) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters may indicate a meaningful biological response to HBOT.”

(Line 231-233) “Taken together, these objective measurements suggest a trend toward improved hair characteristics, reinforcing the positive outcomes observed in the subjective assessments.”

<Discussion>

(Line 291-298) “Subjective assessments in this study revealed statistically significant improvements across all evaluated domains, including perceived hair density, thickness, reduced shedding, and improved anterior hairline. These self-reported outcomes are consistent, noting improvements in hair thickness and perifollicular microcirculation following HBOT in patients with alopecia areata.[19] Furthermore, the observed enhancement in the frontal hairline, often refractory to conventional treatments such as topical minoxidil, suggests that HBOT may have a unique capacity to improve perfusion in typically unresponsive regions of the scalp.”

(Line 338-340) “However, the convergence of subjective improvements, positive trends in objective parameters, and visual enhancements supports the hypothesis that HBOT may play a supportive role in hair follicle activation and scalp health.”

<Conclusions>

(Line 350-352) “While the current study had limitations, including a small sample size, absence of long-term follow-up, and use of a modified HBOT protocol, the observed trends warrant further investigation.”

☞ [Revised]

<Results>

(Line 263-268) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters should be interpreted with caution. Given the absence of a control group and the limited sample size, the study is underpowered to support causal inference, and these findings should be considered exploratory rather than indicative of definitive biological effects.”

(Line 271-274) “Taken together, these objective measurements suggest directional changes in hair-related parameters; however, given the limited sample size and reduced statistical power, these findings should be considered exploratory and hypothesis-generating rather than confirmatory.”

<Discussion>

 (Line 349-360) “Subjective assessments in this study revealed statistically significant improvements across all evaluated domains, including perceived hair density, thickness, reduced shedding, and improved anterior hairline. However, these robust subjective improvements contrasted with the largely non-significant objective findings, highlighting a divergence that warrants careful interpretation. These self-reported outcomes are consistent with prior reports suggesting that enhanced oxygenation may influence scalp microcirculation and perceived hair characteristics [23]; however, the present findings were observed in healthy individuals and should not be directly extrapolated to clinical alopecia populations. Although participants reported improvement in the frontal hairline, this observation should be interpreted cautiously. In the absence of controlled comparison and objective confirmation, it remains unclear whether this reflects physiological change or perceptual bias.”

(Line 414-421) “Although subjective improvements and directional trends in objective parameters were observed, the small sample size substantially limits inferential strength. Therefore, these findings should be interpreted as preliminary observations that warrant validation in adequately powered controlled studies. Moreover, with only nine participants completing the protocol, the study was likely underpowered to detect small-to-moderate effect sizes. As such, the absence of statistical significance in several parameters may reflect limited power rather than true absence of effect, while observed directional trends should not be overinterpreted as evidence of biological efficacy.”

<Conclusions>

(Line 435-439) “While the current study had limitations, including a small sample size, absence of long-term follow-up, and use of a modified HBOT protocol, the observed trends, although derived from a small and underpowered sample, warrant further investigation in larger, adequately powered randomized controlled trials.”

In the discussion section, the authors showed the study limits, the results are encouraging, but at the limit of publishability in this journal.

Furthermore, the appropriateness of the study with respect to bioengineering is not understood.

☞ [Response] We sincerely thank the reviewer for this thoughtful assessment. We appreciate the recognition that the findings are encouraging, while also acknowledging the concern regarding the study’s scope and strength in relation to the journal’s standards. In response, we have further refined the Discussion and Conclusion sections to ensure that the manuscript is appropriately positioned as a preliminary, exploratory investigation rather than a definitive or high-impact efficacy study. We have carefully moderated interpretative statements, emphasized methodological limitations—including small sample size and lack of a control group—and clearly framed the findings as hypothesis-generating observations intended to inform future adequately powered controlled trials. We hope that these revisions align the manuscript more appropriately with the journal’s expectations and improve its interpretative balance and scientific rigor.

The literature cited is appropriate.

For the reasons mentioned above, the manuscript cannot be accepted for publication in this journal but it is suggested that it be transferred to other publications of the same publisher.

☞ [Response] We sincerely thank the reviewer for the careful evaluation of our manuscript and for the suggestion regarding transfer. We fully acknowledge the methodological limitations of the study, including the small sample size, absence of a control group, and exploratory design.

However, we respectfully wish to clarify the intended positioning and contribution of this work. To our knowledge, systematic clinical investigations examining the effects of hyperbaric oxygen exposure on hair follicle characteristics in healthy adults are extremely limited. Rather than presenting definitive therapeutic evidence, this study was designed as an initial translational exploration integrating objective phototrichogram parameters with subjective assessments under controlled HBOT exposure.

In response to both reviewers’ comments, we have substantially revised the manuscript to:

• Explicitly frame the study as exploratory and hypothesis-generating
• Remove causal or efficacy-implying language
• Emphasize statistical power limitations
• Distinguish clearly between subjective and objective findings
• Moderate mechanistic interpretation
• Clarify the limited external validity to alopecia populations

We believe that, in its revised form, the manuscript no longer overstates its implications and instead offers incremental but meaningful preliminary data in an underexplored area. Given the growing clinical interest in non-pharmacological and regenerative approaches affecting tissue oxygenation and vascular dynamics, we respectfully suggest that these findings may still fall within the scope of the journal as an exploratory contribution that can inform future controlled studies.

We therefore kindly ask the reviewer and the editorial team to reconsider the suitability of the revised manuscript for publication in this journal.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The current work focuses on the Preliminary Effects of Hyperbaric Oxygen Therapy on Hair Follicle Characteristics in Healthy Subjects. The authors put some effort into the manuscript, but major issues should be addressed. 

Introduction
-    The introduction doesn't provide sufficient background, and all relevant references are not included. 

-    The novelty of this work is not highlighted, and the author's contribution was unclear compared to other previous works.

- Compare and highlight the novelty of the current work to recent research papers

•    Giardiello F, De Medeiros Quirino L, Brigante R, Chumak M. Hyperbaric Oxygen Therapy for Enhanced Postoperative Recovery in Hair Transplantation. Cureus. 2025 Dec 19;17(12):e99635. doi: 10.7759/cureus.99635. PMID: 41556017; PMCID: PMC12812318.

•    Fan Z-X, Gan Y, Qu Q, et al. The effect of hyperbaric oxygen therapy combined with hair transplantation surgery for the treatment of alopecia. J Cosmet Dermatol. 2021; 20: 917–921. https://doi.org/10.1111/jocd.13665

-    Line 51, The first appearance of the abbreviation in the introduction section should have a full definition, e.g., FDA.

Discussion section
-    Line 271-274, Authors responsible for taking care of the manuscript, this template sentence should be removed: "Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted."

-    One of the main problems in the manuscript is that the authors show only results without interpreting the details or confirming them by citation. More details are required to explain the results.

-    - To strengthen the manuscript, the authors should identify gaps or areas less thoroughly addressed in these articles and incorporate additional insights to provide novel contributions. Comparison with recent citation: They used only 2 references from 2022 until now!!

-    The main limitation is the amount of results and findings obtained, which is clear from the conclusion section.
-    Some study limitations should be addressed: larger-scale studies are needed to confirm long-term effects on hair characteristics. Potential side effects of overstimulation, in some cases, could include increased scalp sebum production, and sessions may cause mild side effects like headache or dizziness. 

References
The number of references is low, and most of them are old. References need to be more complete with recent citations.

Author Response

Introduction
-    The introduction doesn't provide sufficient background, and all relevant references are not included. 

☞ [Response] We thank the reviewer for this important suggestion. In response, we have expanded the Introduction to provide a more comprehensive background on the relationship between tissue oxygenation and hair follicle biology. Specifically, we incorporated additional discussion on the metabolic demands of the anagen phase, the role of oxygen tension in follicular stem cell activity and angiogenic signaling, and the potential relevance of microvascular support in hair cycling.

We also reviewed and supplemented the reference list to include additional relevant and recent studies, thereby strengthening the scientific context and completeness of the Introduction section.

☞ [Before] (Line 70-75) “Emerging evidence suggests that the secondary mechanisms of HBOT, such as anti-hypoxia effects, capillary regeneration, and mitigation of ischemia-reperfusion injury, may extend benefits to other tissues, including the scalp. [9] These mechanisms have demonstrated efficacy in treating spinal cord injuries and compromised skin grafts or flaps, suggesting a potential role in supporting hair follicle health and promoting hair regrowth. [10]”

☞ [Revised] (Line 74-92) “Emerging evidence suggests that the secondary mechanisms of HBOT, such as anti-hypoxia effects, capillary regeneration, and mitigation of ischemia-reperfusion injury, may extend benefits to other tissues, including the scalp. [11] Experimental evidence further supports the biological plausibility of oxygen modulation in hair follicle regulation. Kato et al. reported that ischemic conditions adversely affected hair growth and cycling, whereas hyperoxygenation was associated with modulation of hair cycle dynamics and enhanced follicular activity. [12] Moreover, oxygen-enriched environments have been shown to influence the expression of growth factors closely associated with hair follicle maintenance and regeneration, including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF). These mediators are known to contribute to angiogenesis, dermal papilla signaling, and regulation of the anagen phase. Recent regenerative medicine literature has further emphasized the importance of microvascular support and growth factor–mediated signaling pathways in sustaining hair follicle vitality. [13] Collectively, these findings suggest that oxygen-based interventions, including hyperbaric or normobaric oxygen exposure, may influence hair follicle physiology through modulation of the perifollicular microenvironment. These mechanisms have demonstrated efficacy in treating spinal cord injuries and compromised skin grafts or flaps, suggesting a potential role in supporting hair follicle health and promoting hair regrowth. [14]”

-    The novelty of this work is not highlighted, and the author's contribution was unclear compared to other previous works.

☞ [Response] We thank the reviewer for this important comment. We agree that the novelty and specific contribution of the present study should be more clearly articulated in the Introduction. In response, we have revised the final paragraph of the Introduction to more explicitly distinguish our work from prior studies, particularly those investigating HBOT as an adjunct to hair transplantation surgery. We clarified that, unlike previous research focused on postoperative graft survival or surgical outcomes in patients with alopecia, the present study explores the isolated effects of controlled HBOT exposure on hair-related parameters in non-surgical, healthy individuals. We have also more clearly defined the translational gap addressed by this study and emphasized its exploratory contribution to understanding the potential relationship between HBOT and scalp physiology.

☞ [Before] (Line 68-79) “Despite the proven benefits of HBOT in tissue regeneration and inflammation control, few studies have examined its potential effects on hair follicle physiology. Emerging evidence suggests that the secondary mechanisms of HBOT, such as anti-hypoxia effects, capillary regeneration, and mitigation of ischemia-reperfusion injury, may extend benefits to other tissues, including the scalp. [9] These mechanisms have demonstrated efficacy in treating spinal cord injuries and compromised skin grafts or flaps, suggesting a potential role in supporting hair follicle health and promoting hair regrowth. [10] Given this background, the present study aims to evaluate the effects of hyperbaric oxygen therapy on hair follicles and hair characteristics in healthy adult subjects. By analyzing pre- and post-therapy morphological changes, this research seeks to explore the regenerative potential of HBOT in a novel domain and assess its implications for both medical and aesthetic applications.”

☞ [Revised] (Line 70-111) “Despite the proven benefits of HBOT in tissue regeneration and inflammation control, few studies have examined its potential effects on hair follicle physiology. Recent investigations have explored HBOT primarily as an adjunctive intervention in the context of hair transplantation surgery, focusing on postoperative recovery and graft survival in patients with alopecia. [9,10] Emerging evidence suggests that the secondary mechanisms of HBOT, such as anti-hypoxia effects, capillary regeneration, and mitigation of ischemia-reperfusion injury, may extend benefits to other tissues, including the scalp. [11] Experimental evidence further supports the biological plausibility of oxygen modulation in hair follicle regulation. Kato et al. reported that ischemic conditions adversely affected hair growth and cycling, whereas hyperoxygenation was associated with modulation of hair cycle dynamics and enhanced follicular activity. [12] Moreover, oxygen-enriched environments have been shown to influence the expression of growth factors closely associated with hair follicle maintenance and regeneration, including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF). These mediators are known to contribute to angiogenesis, dermal papilla signaling, and regulation of the anagen phase. Recent regenerative medicine literature has further emphasized the importance of microvascular support and growth factor–mediated signaling pathways in sustaining hair follicle vitality. [13] Collectively, these findings suggest that oxygen-based interventions, including hyperbaric or normobaric oxygen exposure, may influence hair follicle physiology through modulation of the perifollicular microenvironment. These mechanisms have demonstrated efficacy in treating spinal cord injuries and compromised skin grafts or flaps, suggesting a potential role in supporting hair follicle health and promoting hair regrowth. [14] However, these prior studies evaluated HBOT in conjunction with surgical intervention rather than examining the isolated effects of HBOT exposure itself. Moreover, limited research has investigated the impact of HBOT on hair-related parameters in non-surgical settings, particularly among otherwise healthy individuals without clinically diagnosed alopecia. As a result, the independent contribution of controlled hyperbaric oxygen exposure to intrinsic hair follicle dynamics remains insufficiently characterized. In contrast, the present study was not designed to assess surgical outcomes or graft viability, but instead to explore whether controlled HBOT exposure alone, in the absence of surgical manipulation, is associated with measurable changes in intrinsic hair follicle–related parameters in healthy individuals.

Given this background, the present study aims to evaluate the effects of hyperbaric oxygen therapy on hair follicles and hair characteristics in healthy adult subjects. By analyzing pre- and post-therapy morphological changes, this research seeks to explore the regenerative potential of HBOT in a novel domain and assess its implications for both medical and aesthetic applications. By integrating objective phototrichogram-based measurements with structured subjective assessments, this study seeks to address this translational gap and provide preliminary data on HBOT exposure independent of procedural confounders. Rather than establishing therapeutic efficacy, this exploratory investigation seeks to generate preliminary observational data that may inform future controlled studies in clinical hair loss populations.”

- Compare and highlight the novelty of the current work to recent research papers

•    Giardiello F, De Medeiros Quirino L, Brigante R, Chumak M. Hyperbaric Oxygen Therapy for Enhanced Postoperative Recovery in Hair Transplantation. Cureus. 2025 Dec 19;17(12):e99635. doi: 10.7759/cureus.99635. PMID: 41556017; PMCID: PMC12812318.
•    Fan Z-X, Gan Y, Qu Q, et al. The effect of hyperbaric oxygen therapy combined with hair transplantation surgery for the treatment of alopecia. J Cosmet Dermatol. 2021; 20: 917–921. https://doi.org/10.1111/jocd.13665

☞ [Response] We sincerely thank the reviewer for highlighting these important recent studies. We have carefully reviewed the cited articles and agree that they contribute valuable insights into the role of hyperbaric oxygen therapy (HBOT) in the context of hair transplantation procedures. However, the focus and design of the present study differ substantially from these previous reports. The cited studies primarily investigated HBOT as an adjunctive therapy to enhance postoperative recovery or graft survival following hair transplantation surgery in patients with alopecia. In contrast, our study was designed to explore the effects of HBOT exposure alone, independent of surgical intervention, on hair follicle–related parameters in healthy adults. Rather than evaluating wound healing or transplant outcomes, we assessed intrinsic hair characteristics using phototrichogram-based measures and subjective scalp assessments. To clarify this distinction, we have revised the Introduction to explicitly compare our study with these prior investigations and to highlight that our work represents an exploratory attempt to examine isolated HBOT exposure and hair-related parameters in a non-surgical, non-alopecia population. We believe this conceptual difference underscores the novelty and complementary nature of the present investigation.

☞ [Before] (Line 68-79) “Despite the proven benefits of HBOT in tissue regeneration and inflammation control, few studies have examined its potential effects on hair follicle physiology. Emerging evidence suggests that the secondary mechanisms of HBOT, such as anti-hypoxia effects, capillary regeneration, and mitigation of ischemia-reperfusion injury, may extend benefits to other tissues, including the scalp. [9] These mechanisms have demonstrated efficacy in treating spinal cord injuries and compromised skin grafts or flaps, suggesting a potential role in supporting hair follicle health and promoting hair regrowth. [10] Given this background, the present study aims to evaluate the effects of hyperbaric oxygen therapy on hair follicles and hair characteristics in healthy adult subjects. By analyzing pre- and post-therapy morphological changes, this research seeks to explore the regenerative potential of HBOT in a novel domain and assess its implications for both medical and aesthetic applications.”

☞ [Revised] (Line 70-111) “Despite the proven benefits of HBOT in tissue regeneration and inflammation control, few studies have examined its potential effects on hair follicle physiology. Recent investigations have explored HBOT primarily as an adjunctive intervention in the context of hair transplantation surgery, focusing on postoperative recovery and graft survival in patients with alopecia. [9,10] Emerging evidence suggests that the secondary mechanisms of HBOT, such as anti-hypoxia effects, capillary regeneration, and mitigation of ischemia-reperfusion injury, may extend benefits to other tissues, including the scalp. [11] Experimental evidence further supports the biological plausibility of oxygen modulation in hair follicle regulation. Kato et al. reported that ischemic conditions adversely affected hair growth and cycling, whereas hyperoxygenation was associated with modulation of hair cycle dynamics and enhanced follicular activity. [12] Moreover, oxygen-enriched environments have been shown to influence the expression of growth factors closely associated with hair follicle maintenance and regeneration, including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF). These mediators are known to contribute to angiogenesis, dermal papilla signaling, and regulation of the anagen phase. Recent regenerative medicine literature has further emphasized the importance of microvascular support and growth factor–mediated signaling pathways in sustaining hair follicle vitality. [13] Collectively, these findings suggest that oxygen-based interventions, including hyperbaric or normobaric oxygen exposure, may influence hair follicle physiology through modulation of the perifollicular microenvironment. These mechanisms have demonstrated efficacy in treating spinal cord injuries and compromised skin grafts or flaps, suggesting a potential role in supporting hair follicle health and promoting hair regrowth. [14] However, these prior studies evaluated HBOT in conjunction with surgical intervention rather than examining the isolated effects of HBOT exposure itself. Moreover, limited research has investigated the impact of HBOT on hair-related parameters in non-surgical settings, particularly among otherwise healthy individuals without clinically diagnosed alopecia. As a result, the independent contribution of controlled hyperbaric oxygen exposure to intrinsic hair follicle dynamics remains insufficiently characterized. In contrast, the present study was not designed to assess surgical outcomes or graft viability, but instead to explore whether controlled HBOT exposure alone, in the absence of surgical manipulation, is associated with measurable changes in intrinsic hair follicle–related parameters in healthy individuals.

Given this background, the present study aims to evaluate the effects of hyperbaric oxygen therapy on hair follicles and hair characteristics in healthy adult subjects. By analyzing pre- and post-therapy morphological changes, this research seeks to explore the regenerative potential of HBOT in a novel domain and assess its implications for both medical and aesthetic applications. By integrating objective phototrichogram-based measurements with structured subjective assessments, this study seeks to address this translational gap and provide preliminary data on HBOT exposure independent of procedural confounders. Rather than establishing therapeutic efficacy, this exploratory investigation seeks to generate preliminary observational data that may inform future controlled studies in clinical hair loss populations.”

-    Line 51, The first appearance of the abbreviation in the introduction section should have a full definition, e.g., FDA.

☞ [Response] We thank the reviewer for this helpful comment. The abbreviation has been revised to include its full definition at first mention in the Introduction. The text now reads: “U.S. Food and Drug Administration (FDA)–approved treatments.”

☞ [Before] (Line 51-52) “Currently, FDA-approved treatments include oral finasteride and topical minoxidil. [4,5]”

☞ [Revised] (Line 51-53) “Currently, U.S. Food and Drug Administration (FDA)-approved treatments include oral finasteride and topical minoxidil. [4,5]”

Discussion section

-    Line 271-274, Authors responsible for taking care of the manuscript, this template sentence should be removed: "Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted."

☞ [Response] We sincerely thank the reviewer for identifying this oversight. The template sentence was unintentionally retained during manuscript preparation and has now been removed from the Discussion section. We appreciate the reviewer’s careful reading of the manuscript.

-    One of the main problems in the manuscript is that the authors show only results without interpreting the details or confirming them by citation. More details are required to explain the results.

☞ [Response] We thank the reviewer for this important comment. We agree that clearer interpretation of the findings in relation to previous studies and working hypotheses would strengthen the Discussion section. In response, we have revised the Discussion to provide more detailed interpretation of the observed results, including:

• a clearer explanation of potential biological mechanisms underlying the directional trends in objective parameters
• a more structured discussion of the divergence between subjective and objective findings
• additional references to relevant literature to contextualize our observations

These revisions aim to improve the depth of interpretation while maintaining appropriate caution given the exploratory design and limited sample size.

☞ [Before] (Line 276-278) “Although most objective parameters, including overall hair health score, follicle density, hair volume, and the average number of hairs per follicle, did not reach statistical significance, they exhibited consistent positive trends following HBOT.”

☞ [Revised] (Line 313-323) “Although most objective parameters, including overall hair health score, follicle density, hair volume, and the average number of hairs per follicle, did not reach statistical significance, they exhibited consistent positive trends following HBOT. These directional changes may reflect subtle modulation of the follicular microenvironment, potentially related to improved tissue oxygenation and microvascular support. Hyperoxic exposure has been associated with angiogenic signaling and capillary remodeling in other tissues, suggesting a plausible biological pathway through which HBOT could influence perifollicular conditions. However, given the exploratory design, limited sample size, and absence of a control group, these findings should be interpreted as preliminary and hypothesis-generating rather than confirmatory evidence of efficacy.”

-  To strengthen the manuscript, the authors should identify gaps or areas less thoroughly addressed in these articles and incorporate additional insights to provide novel contributions. Comparison with recent citation: They used only 2 references from 2022 until now!!

☞ [Response] We sincerely thank the reviewer for emphasizing the importance of incorporating recent literature. In revising the manuscript, we made a concerted effort to include more up-to-date references to better reflect the current research landscape. Although the reviewer suggested focusing on studies published within the past three years (from 2022 until now), we broadened our consideration to include relevant research published from 2019 to the present to ensure comprehensive coverage of recent developments in the field. In the revised manuscript, 10 out of the 26 references (approximately one-third of the total citations) were published within this recent 5–6 year period. We respectfully hope that this expanded inclusion of contemporary literature demonstrates our effort to strengthen the manuscript’s relevance and contextual positioning within the evolving scientific framework.

-    The main limitation is the amount of results and findings obtained, which is clear from the conclusion section.

☞ [Response] We thank the reviewer for this important observation. We agree that the limited number of statistically significant findings and the exploratory nature of the results represent a major limitation of the present study. In response, we have revised the Conclusion section to more clearly acknowledge that the findings are preliminary, based on a small sample size, and should be interpreted as hypothesis-generating rather than definitive evidence. The revised conclusion now emphasizes the need for larger, controlled studies to validate and expand upon the observed trends.

☞ [Before] (Line 348-350) “These preliminary findings suggest that HBOT may contribute to scalp health and follicular activation, offering a promising non-pharmacological approach to hair care.”

☞ [Revised] (Line 429-435) “These preliminary findings suggest that HBOT exposure was associated with changes in scalp and hair-related parameters in healthy adults. However, given the absence of a control group, the exploratory design, and the inclusion of participants without diagnosed alopecia, these results should be interpreted cautiously and should not be considered evidence of therapeutic efficacy in clinical hair loss conditions. Rather, the present findings are hypothesis-generating and may inform future controlled studies in patients with alopecia.”

-    Some study limitations should be addressed: larger-scale studies are needed to confirm long-term effects on hair characteristics. Potential side effects of overstimulation, in some cases, could include increased scalp sebum production, and sessions may cause mild side effects like headache or dizziness. 

☞ [Response]

☞ [Before] (Line 313-318) “Third, although the study involved 50 treatment sessions over three months, this duration may have been insufficient to observe the full extent of follicular transformation, particularly the conversion of vellus hairs into terminal hairs. Hair growth is a slow process, and longer follow-up periods are necessary to evaluate the sustainability and progression of treatment effects.”

☞ [Revised] (Line 380-394) “Third, although the study involved 50 treatment sessions over three months, this duration may have been insufficient to observe the full extent of follicular transformation, particularly the conversion of vellus hairs into terminal hairs. Hair growth is a slow process, and longer follow-up periods are necessary to evaluate the sustainability and progression of treatment effects. In addition, the present study did not assess long-term outcomes beyond the intervention period. Therefore, it remains unclear whether the observed directional trends would persist, stabilize, or diminish over time. Larger-scale longitudinal studies are required to confirm the durability of any potential effects on hair characteristics. Although no serious adverse events were reported during the intervention, repeated HBOT exposure may be associated with mild transient symptoms such as headache or dizziness. Furthermore, theoretical concerns regarding potential physiological overstimulation, including possible alterations in scalp sebum production, should be considered. Future investigations should incorporate systematic safety monitoring to better characterize both short- and long-term tolerability.”

References
The number of references is low, and most of them are old. References need to be more complete with recent citations.

☞ [Response] We thank the reviewer for this valuable comment. In response, we have carefully re-examined the reference list and incorporated additional recent studies to better reflect current developments in the field. In the revised manuscript, 10 out of 26 references (approximately one-third) were published between 2019 and the present, including several from the past three years. These more recent citations were added to strengthen the contextualization of HBOT-related regenerative mechanisms and dermatologic applications. At the same time, several earlier references were retained because they represent foundational studies on hair follicle biology, alopecia classification, and established HBOT mechanisms that remain scientifically relevant. We aimed to maintain a balanced reference list that integrates both classical foundational knowledge and contemporary research. We hope that the revised reference section now more comprehensively reflects the current state of the literature.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,
I have read the revised version of your manuscript  and appreciate the effort made to tone down causal language and to more clearly frame the study as exploratory and hypothesis-generating. The discussion of limitations is more balanced, and the discrepancy between subjective and objective findings is handled more cautiously.

I just have few additional minor comments that could further strengthen clarity and methodological transparency. First, in the Methods section (2.1), there is some redundancy in the description of the study design (the sentence describing the prospective preliminary trial appears twice in slightly different wording). I would suggest streamlining this paragraph to improve readability and avoid repetition.

Second, regarding statistical analysis (Section 2.6), you report Cohen’s d for paired comparisons, but I think that you should clarify whether this was calculated using the pooled SD or the SD of the difference scores. In small samples (n=9), effect size estimation can vary depending on the formula used, so a brief specification would enhance reproducibility.

Third, while you appropriately caution against overinterpretation of non-significant trends (Results, Table 3), the repeated mention of “consistent directional improvements” may still be perceived as suggestive. You might consider adding one explicit sentence noting that, given the small sample and multiple comparisons, these trends may also reflect random variability.

Finally, in the Conclusions, the statement that HBOT is “underutilized” in South Korea may be slightly tangential to the primary outcomes of this study. You might consider either supporting this claim with a reference or slightly softening it to maintain focus on your data.

Overall, the manuscript is clearer and more cautious than the previous version, and the exploratory framing is now much more appropriate. With these minor clarifications, the methodological transparency and interpretative balance would be further strengthened.

Comments on the Quality of English Language

The manuscript is generally understandable; however, the English still some revision to improve clarity, grammar, and flow.

Author Response

First, in the Methods section (2.1), there is some redundancy in the description of the study design (the sentence describing the prospective preliminary trial appears twice in slightly different wording). I would suggest streamlining this paragraph to improve readability and avoid repetition.

☞ [Response] We sincerely thank the reviewer for this insightful comment. We agree that the description of the study design in Section 2.1 contained redundant wording, which may have affected readability. Accordingly, we have streamlined the paragraph by removing repetitive phrasing and consolidating the description of the prospective preliminary clinical trial into a single, concise statement to improve clarity and flow.

☞ [Before] (Line 114-119) “This study is a prospective, preliminary clinical trial involving 50 sessions of hyperbaric oxygen therapy (HBOT) conducted over three months in nine healthy adult participants at two HBOT centers. It was designed to observe and analyze changes in the condition of hair follicles and hair following HBOT. This study is a prospective, preliminary clinical trial conducted over three months at two HBOT centers, involving a total of 50 HBOT sessions in nine healthy adult participants.”

☞ [Revised] (Line 114-117) “This study is a prospective, preliminary clinical trial conducted over three months at two HBOT centers, involving a total of 50 hyperbaric oxygen therapy (HBOT) sessions in nine healthy adult participants. It was designed to observe and analyze changes in hair follicle–related and hair characteristics following HBOT exposure.”

Second, regarding statistical analysis (Section 2.6), you report Cohen’s d for paired comparisons, but I think that you should clarify whether this was calculated using the pooled SD or the SD of the difference scores. In small samples (n=9), effect size estimation can vary depending on the formula used, so a brief specification would enhance reproducibility.

☞ [Response] We sincerely thank the reviewer for this important methodological comment. We agree that the method used to calculate Cohen’s d in paired comparisons should be clearly specified, particularly given the small sample size. In the revised manuscript, we have clarified that Cohen’s d was calculated using the standard deviation of the paired difference scores (mean difference divided by the standard deviation of the differences). This clarification enhances transparency and reproducibility of the statistical analysis.

☞ [Before] (Line 237-241) “In addition to p-values, effect sizes were calculated to estimate the magnitude of observed changes. Cohen’s d was used for paired comparisons and interpreted as small (0.2), medium (0.5), or large (0.8) effects. Statistical methods were reviewed and refined as necessary based on consultation with domain experts.”

☞ [Revised] (Line 235-239) “In addition to p-values, effect sizes were calculated to estimate the magnitude of observed changes. For paired comparisons, Cohen’s d was calculated by dividing the mean of the paired differences by the standard deviation of the difference scores (dz) and interpreted as small (0.2), medium (0.5), or large (0.8) effects.”

Third, while you appropriately caution against overinterpretation of non-significant trends (Results, Table 3), the repeated mention of “consistent directional improvements” may still be perceived as suggestive. You might consider adding one explicit sentence noting that, given the small sample and multiple comparisons, these trends may also reflect random variability.

☞ [Response] We sincerely thank the reviewer for this thoughtful suggestion. We agree that repeated references to “consistent directional improvements” could potentially be interpreted as suggestive. In response, we have added an explicit statement clarifying that, given the small sample size and the presence of multiple comparisons, the observed trends may also reflect random variability rather than true biological effects. This revision further reinforces the exploratory nature of the findings and reduces the risk of overinterpretation.

☞ [Before] (Line 263-268) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters should be interpreted with caution. Given the absence of a control group and the limited sample size, the study is underpowered to support causal inference, and these findings should be considered exploratory rather than indicative of definitive biological effects.”

☞ [Revised] (Line 261-268) “While these changes were not statistically significant, the consistent directional improvements across multiple parameters should be interpreted with caution. Given the absence of a control group and the limited sample size, the study is underpowered to support causal inference, and these findings should be considered exploratory rather than indicative of definitive biological effects. Furthermore, given the small sample size and the multiple statistical comparisons performed, these observed trends may also reflect random variability rather than true underlying biological change.”

Finally, in the Conclusions, the statement that HBOT is “underutilized” in South Korea may be slightly tangential to the primary outcomes of this study. You might consider either supporting this claim with a reference or slightly softening it to maintain focus on your data.

☞ [Response] We sincerely thank the reviewer for this valuable comment. We agree that the statement describing HBOT as “underutilized” in South Korea may be perceived as tangential to the primary outcomes of this exploratory study. To maintain focus on the study findings and avoid overstating contextual implications, we have revised the Conclusion section by softening this statement and re-centering the discussion on the data generated in the present investigation.

☞ [Before] (Line 426-427) “Hyperbaric oxygen therapy (HBOT) remains an underutilized modality in South Korea, largely due to limited awareness of its therapeutic potential.”

☞ [Revised] (Line 428-430) “Hyperbaric oxygen therapy (HBOT) is increasingly being explored across various medical fields, yet its potential applications in trichology remain insufficiently investigated.”

Overall, the manuscript is clearer and more cautious than the previous version, and the exploratory framing is now much more appropriate. With these minor clarifications, the methodological transparency and interpretative balance would be further strengthened.

☞ [Response] We sincerely thank the reviewer for the careful re-evaluation of our manuscript and for acknowledging the improvements made in this revised version. We greatly appreciate the positive assessment that the manuscript is now clearer, more cautious, and appropriately framed as exploratory. In response to the additional minor suggestions, we have further clarified the statistical methodology and strengthened statements to ensure balanced interpretation and enhanced transparency. We believe that these revisions have improved the overall rigor and readability of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Communication has been improved in all areas criticized, and the authors have addressed many of the identified weaknesses. Although the work still has limitations, as the authors noted, the approach has improved significantly.

Author Response

Communication has been improved in all areas criticized, and the authors have addressed many of the identified weaknesses. Although the work still has limitations, as the authors noted, the approach has improved significantly.

☞ [Response] We sincerely thank the reviewer for the thoughtful reassessment of our manuscript. We greatly appreciate the recognition that the clarity and communication have improved and that the previously identified weaknesses have been addressed. We fully acknowledge that limitations remain, as discussed in the revised manuscript, and we have made every effort to present the findings with appropriate caution and methodological transparency. We are grateful for the reviewer’s constructive feedback, which has substantially strengthened the quality and balance of this work.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

OK...The added explantation may be good enough. 

Author Response

OK...The added explanation may be good enough.

☞ [Response] We sincerely thank the reviewer for the positive feedback. We are pleased that the added explanation was considered sufficient. We appreciate the reviewer’s careful evaluation throughout the revision process, which has helped improve the clarity and overall quality of the manuscript.

Author Response File: Author Response.pdf