Efficacy of a New Non-Invasive System Delivering Microwave Energy for the Treatment of Abdominal Adipose Tissue: Results of an Immunohistochemical Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is relevant and contains new scientific information about evaluation the feasibility of a localized, non-invasive microwave (MW) device for preferential heating of subcutaneous adipose tissue using a controlled electromagnetic field. It should be noted the originality of used methods. Paper can be published in the Cosmetics (mdpi) after minor changes:

·         The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used; 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations.

·         Perhaps the authors should move the information about CD68 higher in the introduction, rather than at the end.

·         It is suggested to confront the advantages and disadvantages of the proposed technique with other methods for disease diagnostics in detail. Please, assess the risks of the approach.

·         No physical/mathematical model has been suggested. What temperature were the fat cells when exposed?

·         “The technology used in this study is non-invasive, with clinically proven rapid results in a highly controlled and safe manner”. Why? If authors write about inflammation in tissue.

·         The text contains typos (for example, p.3 line 143, p 5 line 210, p 6 line 270, etc.)

·         Please provide the Ethics Commission approval number.

·         “For each enrolled patient, at the first visit demographic data and body mass index (BMI) were collected.” But the text of the article does not contain this information and analysis. There is no information about what BMI the volunteers had.

·         Why was the microwave application mode used chosen? In medicine, three frequencies and, accordingly, three microwave lengths are used: 2450 MHz (12.245 cm), 915 MHz (32.79 cm) and 433.9 MHz (69.14 cm). 2.45 GHz is the operating frequency of microwave ovens. How deeply can radiation of a given frequency penetrate into biological tissue? Microwave energy absorption occurs primarily in tissues rich in water. The skin contains up to 72% of all water, while fatty tissue contains less than 10%. That is, absorption should mainly occur in the skin layers, not in the fatty tissue. Depending on the thickness of the subcutaneous fat (on the buttocks and abdomen), the skin layer can be more than 10 cm. If only the superficial layer is damaged, and the deep layers are not affected by microwaves, then how effective is the proposed device.

·         Authors should add an image of the control sample with CD68 antigen (“Control samples are negative for CD68”). Authors should indicate the changes described in the figures.

·         Similar changes in adipose tissue are described in the works: M. Wanner, M. Avram, D. Gagnon, M. C. Mihm Jr., D. Zurakowski, K. Watanabe, Z. Tannous, R. R. Anderson, D. Manstein, Lasers Surg. Med. 2009, 41, 401; M. A. Trelles, S. R. Mordon, Aesthet. Plast. Surg. 2009, 33, 125; Yanina, IY, Navolokin, NA, Bucharskaya, AB, Мaslyakova, GN, Tuchin, VV. Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo. J. Biophotonics. 2019, 12 (12), e201900117. The authors are asked to analyze and compare

·         Reference format does not correspond to the requirements of this journal.

• Authors should take into account recent scientific and review papers by other research groups.

Authors should add more references to articles on this topic published in the Cosmetics recently

Comments on the Quality of English Language

No

Author Response

Comments 1: The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used; 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations.

• Response 1: Thank you for your suggestion. We have revised the abstract to conform to the journal's structured abstract format without headings. The updated version now includes a clear sequence of background, methods, results, and conclusions, providing a concise yet comprehensive summary of the study's objectives and findings. 

Comments 2: Perhaps the authors should move the information about CD68 higher in the introduction, rather than at the end.

• Response 2: We agree that introducing the information about CD68 earlier in the Introduction can enhance the logical flow of the manuscript and provide better context for the study. We have accordingly repositioned the discussion of CD68 to an earlier part of the Introduction, ensuring a more coherent narrative that aligns with the study objectives. (p.2, lines 100-105, highlighted in the manuscript)

Comments 3: It is suggested to confront the advantages and disadvantages of the proposed technique with other methods for disease diagnostics in detail. Please, assess the risks of the approach.

• Response 3: We recognize the importance of comparing the advantages and disadvantages of our proposed technique with other established methods for fat reduction and diagnostics. In response to your comment, we have expanded the Discussion and Conclusions section to include a more detailed comparison of our approach with other techniques. Additionally, we have addressed potential risks associated with the microwave-based approach, including thermal effects, patient variability, and the need for long-term studies to assess sustained efficacy and safety. (p.7, lines 311-318, and p.9, lines 445-450, highlighted in the manuscript)

Comments 4: No physical/mathematical model has been suggested. What temperature were the fat cells when exposed?

• Response 4: While the study primarily focused on the biological and histological effects of the proposed technique, we acknowledge the importance of including a physical/mathematical model to better contextualize the results. We have now included a discussion on the temperature ranges likely achieved during the procedure and the potential thermal thresholds required to induce adipocyte damage. This addition helps to bridge the gap between the physical parameters and the observed biological outcomes. (p.3 lines 147-150, and p.9 lines 409-415, highlighted in the manuscript)

Comments 5: “The technology used in this study is non-invasive, with clinically proven rapid results in a highly controlled and safe manner”. Why? If authors write about inflammation in tissue

• Response 5: We acknowledge the apparent contradiction and appreciate the opportunity to clarify this aspect. While the study reports the presence of an inflammatory response in the treated adipose tissue, it is important to distinguish between the expected, transient inflammatory reaction following targeted adipocyte injury and adverse inflammatory events that may compromise patient safety. The observed inflammation is a physiological response to adipocyte damage and is part of the natural remodeling process rather than an indicator of an unsafe treatment. 

Comments 6: The text contains typos (for example, p.3 line 143, p 5 line 210, p 6 line 270, etc.)

• Response 6: We have carefully reviewed the entire text and corrected all identified typos, including those mentioned on pages 3, 5, and 6. Additionally, we have conducted a thorough proofreading to ensure clarity and accuracy throughout the document. 

Comments 7: Please provide the Ethics Commission approval number.

• Response 7: The study was conducted in accordance with the Declaration of Helsinki and received approval from the Institutional Review Board of Calabria Centro. The Ethics Commission approval number is 373/19, granted on 4 December 2019. We have updated the manuscript to include this information in the appropriate section.
  

Comments 8: “For each enrolled patient, at the first visit demographic data and body mass index (BMI) were collected.” But the text of the article does not contain this information and analysis. There is no information about what BMI the volunteers had.

• Response 8: Thank you for your observation. We acknowledge the lack of specific details regarding the BMI of the enrolled volunteers in the initial version of the manuscript. To address this, we have now included a description of the BMI values of the study participants and their analysis in the revised manuscript. (p.3 lines 128-131, highlighted in the manuscript)

Comments 9: Why was the microwave application mode used chosen? In medicine, three frequencies and, accordingly, three microwave lengths are used: 2450 MHz (12.245 cm), 915 MHz (32.79 cm) and 433.9 MHz (69.14 cm). 2.45 GHz is the operating frequency of microwave ovens. How deeply can radiation of a given frequency penetrate into biological tissue? Microwave energy absorption occurs primarily in tissues rich in water. The skin contains up to 72% of all water, while fatty tissue contains less than 10%. That is, absorption should mainly occur in the skin layers, not in the fatty tissue. Depending on the thickness of the subcutaneous fat (on the buttocks and abdomen), the skin layer can be more than 10 cm. If only the superficial layer is damaged, and the deep layers are not affected by microwaves, then how effective is the proposed device.

• Response 9: The selection of the 2.45 GHz frequency was based on several factors, including its established safety profile, targeted absorption characteristics, and clinical efficacy in previous studies. While it is true that water-rich tissues such as the skin exhibit higher microwave absorption, recent literature and experimental findings have demonstrated that the dielectric properties of adipose tissue allow for selective heating at this frequency.
  Specifically, the penetration depth of 2.45 GHz microwaves into biological tissue has been shown to reach approximately 1.2 cm to 1.5 cm in adipose tissue under controlled conditions, which is sufficient to induce thermal effects leading to adipocyte disruption. The device used in this study incorporates an advanced cooling system that minimizes excessive heating of the skin, allowing the microwaves to preferentially target the underlying fat layer without causing significant epidermal damage.
  Moreover, the efficacy of the proposed device is supported by histological findings demonstrating selective adipocyte damage and the absence of substantial epidermal changes, confirming the depth of penetration is adequate to achieve the intended therapeutic effect. This was further validated through immunohistochemical analysis showing perilipin-1 depletion and macrophage infiltration in treated adipose regions, which suggests that deep layers are effectively impacted.
  Regarding alternative frequencies, while 915 MHz and 433.9 MHz offer deeper penetration, they also require different power densities and applicator designs, which could increase treatment duration and reduce the precision of adipose targeting. The 2.45 GHz frequency, in contrast, provides an optimal balance between penetration depth and focused energy delivery, making it a suitable choice for localized fat reduction.

Comments 10: Authors should add an image of the control sample with CD68 antigen (“Control samples are negative for CD68”). Authors should indicate the changes described in the figures.

• Response 10: Thank you for your suggestion. Unfortunately, an image of the control sample with CD68-negative staining was not initially acquired during the study. However, based on our histological analysis and observations, no CD68-positive cells were detected in the control samples.

Comments 11: Similar changes in adipose tissue are described in the works: M. Wanner, M. Avram, D. Gagnon, M. C. Mihm Jr., D. Zurakowski, K. Watanabe, Z. Tannous, R. R. Anderson, D. Manstein, Lasers Surg. Med. 2009, 41, 401; M. A. Trelles, S. R. Mordon, Aesthet. Plast. Surg. 2009, 33, 125; Yanina, IY, Navolokin, NA, Bucharskaya, AB, Мaslyakova, GN, Tuchin, VV. Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo. J. Biophotonics. 2019, 12 (12), e201900117. The authors are asked to analyze and compare

• Response 11: We have carefully reviewed the suggested references and, among them, we found the study by Trelles et al. (Aesthet. Plast. Surg. 2009, 33, 125) to be the most relevant to our work. This study explores the effects of radiofrequency energy on adipose tissue, describing changes such as adipocyte membrane lysis and structural modifications. We have now incorporated a comparison of our findings with those reported in Trelles et al., emphasizing the similarities and differences in the mechanisms of action and histological outcomes. We believe this addition strengthens our manuscript by providing a broader perspective on energy-based fat reduction techniques. (p.7 lines 327-335, highlighted in the manuscript)

Comments 12: Reference format does not correspond to the requirements of this journal.

• Response 12: Thank you for pointing out the discrepancies in the reference formatting. We have carefully reviewed the journal's guidelines and have reformatted all references accordingly to ensure compliance with the required style. We appreciate your attention to detail and believe that the updated reference section now aligns with the journal’s standards.

Reviewer 2 Report

Comments and Suggestions for Authors

The study under review presents a novel approach to addressing a common aesthetic concern: unwanted abdominal fat. By leveraging a localized, non-invasive microwave (MW) device designed for the preferential heating of subcutaneous adipose tissue, the researchers introduce a potential alternative to conventional surgical and energy-based fat reduction techniques.

The application of a controlled electromagnetic field to selectively target subcutaneous adipose tissue represents an innovative and promising avenue in non-invasive body contouring. This method could potentially fill a gap in current aesthetic treatments by offering a precise and less invasive solution. The use of Hematoxylin and Eosin staining, alongside immunohistochemical markers such as Perilipin-1 and CD68, provides robust evidence for the biological effects of the MW treatment. These analyses effectively demonstrate adipocyte disruption while confirming the preservation of the epidermal and dermal layers, which underscores the specificity of the MW device. The study contributes to our understanding of the physiological responses to adipocyte injury. The observation of CD68-positive macrophage recruitment to the site of adipocyte disruption is a significant finding, suggesting an active clearance process and potential implications for tissue remodeling post-treatment. The study’s evidence that epidermal and dermal layers remain unaffected by the MW device is particularly encouraging, as it aligns with the safety profile expected of non-invasive interventions.

While the study offers important preliminary insights, the small sample of five female participants significantly limits the generalizability of the findings. Future studies must include larger and more diverse cohorts to validate these results. The evaluation of tissue effects only five days post-treatment provides a narrow temporal window, leaving long-term outcomes and potential delayed adverse effects unexplored. The lack of a control group undermines the ability to attribute observed changes solely to the MW treatment. Comparisons with alternative fat-reduction modalities or untreated controls are necessary to contextualize the efficacy. Although the histological data are compelling, the study lacks reporting on clinically relevant outcomes such as measurable fat volume reduction, patient satisfaction, and aesthetic improvement, which are critical in evaluating practical efficacy. The reliance on a single treatment session limits the exploration of dose-response effects. Understanding whether multiple sessions yield enhanced or cumulative results is essential for optimizing treatment protocols.

Future studies should involve a larger cohort that reflects a more heterogeneous population in terms of age, gender, and BMI to ensure broader applicability. Longer follow-up periods are necessary to evaluate the sustainability of fat reduction and monitor any late-onset adverse effects or complications. Quantitative measurements of fat reduction (e.g., via imaging modalities like ultrasound or MRI) and patient-reported outcomes should complement histological findings to provide a holistic evaluation of treatment efficacy. Comparative trials assessing the MW device against established non-invasive fat reduction technologies, such as cryolipolysis or radiofrequency, would clarify its relative effectiveness and safety.Investigating the effects of repeated MW treatments could reveal whether cumulative benefits can be achieved without compromising safety or tolerability.

This study represents an important step forward in the development of non-invasive fat reduction technologies. The demonstrated ability of the MW device to induce selective adipocyte injury, while preserving adjacent tissue, is a compelling proof of concept. However, the study's limitations—most notably the small sample size, lack of clinical outcomes, and short follow-up duration—necessitate further investigation. Future research should focus on addressing these gaps to fully elucidate the potential of this innovative approach in clinical practice.

Author Response

Comments 1: The study under review presents a novel approach to addressing a common aesthetic concern: unwanted abdominal fat. By leveraging a localized, non-invasive microwave (MW) device designed for the preferential heating of subcutaneous adipose tissue, the researchers introduce a potential alternative to conventional surgical and energy-based fat reduction techniques.
The application of a controlled electromagnetic field to selectively target subcutaneous adipose tissue represents an innovative and promising avenue in non-invasive body contouring. This method could potentially fill a gap in current aesthetic treatments by offering a precise and less invasive solution. The use of Hematoxylin and Eosin staining, alongside immunohistochemical markers such as Perilipin-1 and CD68, provides robust evidence for the biological effects of the MW treatment. These analyses effectively demonstrate adipocyte disruption while confirming the preservation of the epidermal and dermal layers, which underscores the specificity of the MW device. The study contributes to our understanding of the physiological responses to adipocyte injury. The observation of CD68-positive macrophage recruitment to the site of adipocyte disruption is a significant finding, suggesting an active clearance process and potential implications for tissue remodeling post-treatment. The study’s evidence that epidermal and dermal layers remain unaffected by the MW device is particularly encouraging, as it aligns with the safety profile expected of non-invasive interventions.
While the study offers important preliminary insights, the small sample of five female participants significantly limits the generalizability of the findings. Future studies must include larger and more diverse cohorts to validate these results. The evaluation of tissue effects only five days post-treatment provides a narrow temporal window, leaving long-term outcomes and potential delayed adverse effects unexplored. The lack of a control group undermines the ability to attribute observed changes solely to the MW treatment. Comparisons with alternative fat-reduction modalities or untreated controls are necessary to contextualize the efficacy. Although the histological data are compelling, the study lacks reporting on clinically relevant outcomes such as measurable fat volume reduction, patient satisfaction, and aesthetic improvement, which are critical in evaluating practical efficacy. The reliance on a single treatment session limits the exploration of dose-response effects. Understanding whether multiple sessions yield enhanced or cumulative results is essential for optimizing treatment protocols.
Future studies should involve a larger cohort that reflects a more heterogeneous population in terms of age, gender, and BMI to ensure broader applicability. Longer follow-up periods are necessary to evaluate the sustainability of fat reduction and monitor any late-onset adverse effects or complications. Quantitative measurements of fat reduction (e.g., via imaging modalities like ultrasound or MRI) and patient-reported outcomes should complement histological findings to provide a holistic evaluation of treatment efficacy. Comparative trials assessing the MW device against established non-invasive fat reduction technologies, such as cryolipolysis or radiofrequency, would clarify its relative effectiveness and safety.Investigating the effects of repeated MW treatments could reveal whether cumulative benefits can be achieved without compromising safety or tolerability.
This study represents an important step forward in the development of non-invasive fat reduction technologies. The demonstrated ability of the MW device to induce selective adipocyte injury, while preserving adjacent tissue, is a compelling proof of concept. However, the study's limitations—most notably the small sample size, lack of clinical outcomes, and short follow-up duration—necessitate further investigation. Future research should focus on addressing these gaps to fully elucidate the potential of this innovative approach in clinical practice.

• Response 1: Thank you for your constructive feedback. We appreciate your recognition of the study's contributions and its potential implications in non-invasive fat reduction. We acknowledge the limitations you have pointed out, including the small sample size, short follow-up duration, and the lack of a control group. These aspects have been highlighted in the revised manuscript to ensure transparency and clarity. We have also expanded the discussion addressing the advantages and limitations of our approach. Furthermore, we recognize the importance of incorporating clinically relevant outcomes, such as volumetric fat reduction and patient satisfaction, in future studies. Your insights will help us shape subsequent research efforts aimed at optimizing the clinical application of this technology. (p.10 lines 451-456, highlighted in the manuscript)

Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting report about the effect of microwave application to the adipose tissue  before abdominoplasty. Immunohistochemical study through Perilipin-1 and CD68 showed the effectiveness of subcutaneous fat reduction by microwave application. The readers may want to know why the timing of microwave application is 5 days before abdominoplasty? neither 1 week or 2 weeks? In the session of "Discussion" , it is suggested to analyse the advantages and possible complications clinically by routine use of the microwave before abdominoplasty. 

Author Response

Comments 1: This is an interesting report about the effect of microwave application to the adipose tissue  before abdominoplasty. Immunohistochemical study through Perilipin-1 and CD68 showed the effectiveness of subcutaneous fat reduction by microwave application. The readers may want to know why the timing of microwave application is 5 days before abdominoplasty?

• Response 1: Thank you for your question regarding the timing of microwave application before abdominoplasty. The decision to apply the treatment five days prior to surgery was based on preliminary observations and clinical experience, which suggested that this timeframe allows sufficient time for initial adipocyte disruption and macrophage infiltration while minimizing potential confounding effects of prolonged inflammatory responses. We have now clarified this rationale in the manuscript to enhance the understanding of the experimental timeline. (p.3 lines 152-157, highlighted in the manuscript)

Comments 2: In the session of "Discussion" , it is suggested to analyse the advantages and possible complications clinically by routine use of the microwave before abdominoplasty. 

• Response 2: Thank you for your suggestion. In response to your feedback, we have expanded the Discussion section to include an analysis of the clinical benefits, such as improved fat tissue remodeling and potential skin tightening effects, as well as possible complications, including localized erythema, transient edema, and variability in patient response. (p.7 lines 297-310, highlighted in the manuscript)

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Not all of the reviewer's comments were taken into account in the text of the paper.

Not all responses to comments have been added to the paper, and accordingly there are no references confirming the authors' assertions.

 

• Comments 1: The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used; 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations.

 Response 1: Thank you for your suggestion. We have revised the abstract to conform to the journal's structured abstract format without headings. The updated version now includes a clear sequence of background, methods, results, and conclusions, providing a concise yet comprehensive summary of the study's objectives and findings.

 

Additional comments 1: The abstract is unchanged from the original paper.

 

• Comments 2: Perhaps the authors should move the information about CD68 higher in the introduction, rather than at the end.

 Response 2: We agree that introducing the information about CD68 earlier in the Introduction can enhance the logical flow of the manuscript and provide better context for the study. We have accordingly repositioned the discussion of CD68 to an earlier part of the Introduction, ensuring a more coherent narrative that aligns with the study objectives. (p.2, lines 100-105, highlighted in the manuscript)

 

Additional comments 2: +

 

• Comments 3: It is suggested to confront the advantages and disadvantages of the proposed technique with other methods for disease diagnostics in detail. Please, assess the risks of the approach.

 Response 3: We recognize the importance of comparing the advantages and disadvantages of our proposed technique with other established methods for fat reduction and diagnostics. In response to your comment, we have expanded the Discussion and Conclusions section to include a more detailed comparison of our approach with other techniques. Additionally, we have addressed potential risks associated with the microwave-based approach, including thermal effects, patient variability, and the need for long-term studies to assess sustained efficacy and safety. (p.7, lines 311-318, and p.9, lines 445-450, highlighted in the manuscript)

 

Additional comments 3:+

 

• Comments 4: No physical/mathematical model has been suggested. What temperature were the fat cells when exposed?

 Response 4: While the study primarily focused on the biological and histological effects of the proposed technique, we acknowledge the importance of including a physical/mathematical model to better contextualize the results. We have now included a discussion on the temperature ranges likely achieved during the procedure and the potential thermal thresholds required to induce adipocyte damage. This addition helps to bridge the gap between the physical parameters and the observed biological outcomes. (p.3 lines 147-150, and p.9 lines 409-415, highlighted in the manuscript)

 

Additional comments 4: Unfortunately, the authors do not provide any insight into the physical processes that lead to biological effects. Include additional outcome measures. It is not clear how the temperature of the fat on the patient was measured. Is there any confirmation that this is the temperature of fat and not skin? (p.3 lines 147-150)

 

• Comments 5: “The technology used in this study is non-invasive, with clinically proven rapid results in a highly controlled and safe manner”. Why? If authors write about inflammation in tissue

 Response 5: We acknowledge the apparent contradiction and appreciate the opportunity to clarify this aspect. While the study reports the presence of an inflammatory response in the treated

adipose tissue, it is important to distinguish between the expected, transient inflammatory reaction following targeted adipocyte injury and adverse inflammatory events that may compromise patient safety. The observed inflammation is a physiological response to adipocyte damage and is part of the natural remodeling process rather than an indicator of an unsafe treatment.

Additional comments 5: Taking a biopsy from the area of inflammation raises questions. This may lead to sepsis in the patient. Extend the observation period to assess the durability of fat reduction and monitor any long-term adverse effects.

 

• Comments 6: The text contains typos (for example, p.3 line 143, p 5 line 210, p 6 line 270, etc.)

 Response 6: We have carefully reviewed the entire text and corrected all identified typos, including those mentioned on pages 3, 5, and 6. Additionally, we have conducted a thorough proofreading to ensure clarity and accuracy throughout the document.

 

Additional comments 6:+

 

• Comments 7: Please provide the Ethics Commission approval number.

 Response 7: The study was conducted in accordance with the Declaration of Helsinki and received approval from the Institutional Review Board of Calabria Centro. The Ethics Commission approval number is 373/19, granted on 4 December 2019. We have updated the manuscript to include this information in the appropriate section.

 

Additional comments 7: Not all information has been added to the text of the paper.

 

• Comments 8: “For each enrolled patient, at the first visit demographic data and body mass index (BMI) were collected.” But the text of the article does not contain this information and analysis. There is no information about what BMI the volunteers had.

 

 Response 8: Thank you for your observation. We acknowledge the lack of specific details regarding the BMI of the enrolled volunteers in the initial version of the manuscript. To address this, we have now included a description of the BMI values of the study participants and their analysis in the revised manuscript. (p.3 lines 128-131, highlighted in the manuscript)

 

Additional comments 8:+

 

• Comments 9: Why was the microwave application mode used chosen? In medicine, three frequencies and, accordingly, three microwave lengths are used: 2450 MHz (12.245 cm), 915 MHz (32.79 cm) and 433.9 MHz (69.14 cm). 2.45 GHz is the operating frequency of microwave ovens. How deeply can radiation of a given frequency penetrate into biological tissue? Microwave energy absorption occurs primarily in tissues rich in water. The skin contains up to 72% of all water, while fatty tissue contains less than 10%. That is, absorption should mainly occur in the skin layers, not in the fatty tissue. Depending on the thickness of the subcutaneous fat (on the buttocks and abdomen), the skin layer can be more than 10 cm. If only the superficial layer is damaged, and the deep layers are not affected by microwaves, then how effective is the proposed device.

 Response 9: The selection of the 2.45 GHz frequency was based on several factors, including its established safety profile, targeted absorption characteristics, and clinical efficacy in previous studies. While it is true that water-rich tissues such as the skin exhibit higher microwave absorption, recent literature and experimental findings have demonstrated that the dielectric properties of adipose tissue allow for selective heating at this frequency. Specifically, the penetration depth of 2.45 GHz microwaves into biological tissue has been shown to reach approximately 1.2 cm to 1.5 cm in adipose tissue under controlled conditions, which is sufficient to induce thermal effects leading to adipocyte disruption. The device used in this study incorporates an advanced cooling system that minimizes excessive heating of the skin, allowing the microwaves to preferentially target the underlying fat layer without causing significant epidermal damage. Moreover, the efficacy of the proposed device is supported by histological findings demonstrating selective adipocyte damage and the absence of substantial epidermal changes, confirming the depth of penetration is adequate to achieve the intended therapeutic effect. This was further validated through immunohistochemical analysis showing perilipin-1 depletion and macrophage infiltration in treated adipose regions, which suggests that deep layers are effectively impacted. Regarding alternative frequencies, while 915 MHz and 433.9 MHz offer deeper penetration, they also require different power densities and applicator designs, which could increase treatment duration and reduce the precision of adipose targeting. The 2.45 GHz frequency, in contrast, provides an optimal balance between penetration depth and focused energy delivery, making it a suitable choice for localized fat reduction.

 

Additional comments 9: The authors do not provide references to the works they mention. How can the veracity of the statements be verified? Readers would be interested in this information.

Comments 10: Authors should add an image of the control sample with CD68 antigen (“Control samples are negative for CD68”). Authors should indicate the changes described in the figures.

• Response 10: Thank you for your suggestion. Unfortunately, an image of the control sample with CD68-negative staining was not initially acquired during the study. However, based on our histological analysis and observations, no CD68-positive cells were detected in the control samples.

 

Additional comments 10:+

 

• Comments 11: Similar changes in adipose tissue are described in the works: M. Wanner, M. Avram, D. Gagnon, M. C. Mihm Jr., D. Zurakowski, K. Watanabe, Z. Tannous, R. R. Anderson, D. Manstein, Lasers Surg. Med. 2009, 41, 401; M. A. Trelles, S. R. Mordon, Aesthet. Plast. Surg. 2009, 33, 125; Yanina, IY, Navolokin, NA, Bucharskaya, AB, Мaslyakova, GN, Tuchin, VV. Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo. Biophotonics. 2019, 12 (12), e201900117. The authors are asked to analyze and compare

 Response 11: We have carefully reviewed the suggested references and, among them, we found the study by Trelles et al. (Aesthet. Plast. Surg. 2009, 33, 125) to be the most relevant to our work. This study explores the effects of radiofrequency energy on adipose tissue, describing changes such as adipocyte membrane lysis and structural modifications. We have now incorporated a comparison of our findings with those reported in Trelles et al., emphasizing the similarities and differences in the mechanisms of action and histological outcomes. We believe this addition strengthens our manuscript by providing a broader perspective on energy-based fat reduction techniques. (p.7 lines 327-335, highlighted in the manuscript)

 

Additional comments 11: Not all proposed articles are reviewed in the text.

 

• Comments 12: Reference format does not correspond to the requirements of this journal.

 Response 12: Thank you for pointing out the discrepancies in the reference formatting. We have carefully reviewed the journal's guidelines and have reformatted all references accordingly to ensure compliance with the required style. We appreciate your attention to detail and believe that the updated reference section now aligns with the journal’s standards.

 

Additional comments 12:+

Comments on the Quality of English Language

-

Author Response

• Comments 1: The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used; 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations.

 Response 1: Thank you for your suggestion. We have revised the abstract to conform to the journal's structured abstract format without headings. The updated version now includes a clear sequence of background, methods, results, and conclusions, providing a concise yet comprehensive summary of the study's objectives and findings.

 

Additional comments 1: The abstract is unchanged from the original paper.

 

 Additional response 1: Dear reviewer, we really appreciate your careful review of our manuscript and your comments. The abstract is already formatted as a single paragraph without headings, following the structure you outlined: it presents the background, describes the methods, summarizes the results, and concludes with the main findings and interpretations.

Nevertheless, if there are specific aspects that you believe require further refinement, we would be grateful if you could provide additional guidance on the exact modifications you have in mind. We are happy to make any necessary adjustments to enhance clarity.

 

 

• Comments 2: Perhaps the authors should move the information about CD68 higher in the introduction, rather than at the end.

 Response 2: We agree that introducing the information about CD68 earlier in the Introduction can enhance the logical flow of the manuscript and provide better context for the study. We have accordingly repositioned the discussion of CD68 to an earlier part of the Introduction, ensuring a more coherent narrative that aligns with the study objectives. (p.2, lines 100-105, highlighted in the manuscript)

 

Additional comments 2: +

 

• Comments 3: It is suggested to confront the advantages and disadvantages of the proposed technique with other methods for disease diagnostics in detail. Please, assess the risks of the approach.

 Response 3: We recognize the importance of comparing the advantages and disadvantages of our proposed technique with other established methods for fat reduction and diagnostics. In response to your comment, we have expanded the Discussion and Conclusions section to include a more detailed comparison of our approach with other techniques. Additionally, we have addressed potential risks associated with the microwave-based approach, including thermal effects, patient variability, and the need for long-term studies to assess sustained efficacy and safety. (p.7, lines 311-318, and p.9, lines 445-450, highlighted in the manuscript)

 

Additional comments 3:+

 

• Comments 4: No physical/mathematical model has been suggested. What temperature were the fat cells when exposed?

 Response 4: While the study primarily focused on the biological and histological effects of the proposed technique, we acknowledge the importance of including a physical/mathematical model to better contextualize the results. We have now included a discussion on the temperature ranges likely achieved during the procedure and the potential thermal thresholds required to induce adipocyte damage. This addition helps to bridge the gap between the physical parameters and the observed biological outcomes. (p.3 lines 147-150, and p.9 lines 409-415, highlighted in the manuscript)

 

Additional comments 4: Unfortunately, the authors do not provide any insight into the physical processes that lead to biological effects. Include additional outcome measures. It is not clear how the temperature of the fat on the patient was measured. Is there any confirmation that this is the temperature of fat and not skin? (p.3 lines 147-150)

 

 Additional response 4: Dear reviewer, the treatment parameters (energy output, frequency, and duration) were selected based on previous studies demonstrating that microwaves at 2.45 GHz can achieve selective heating of subcutaneous fat while preserving the epidermis, as also cited in our manuscript, including Hoffmann, K.; Zappia, E.; Bonan, P.; Coli, F.; Bennardo, L.; Clementoni, M.T.; Pedrelli, V.; Piccolo, D.; Poleva, I.; Salsi, B.; et al. Microwave-Energy-Based Device for the Treatment of Cellulite and Localized Adiposity: Recommendations of the “Onda Coolwaves” International Advisory Board. Bioengineering 2024, 11, 1249. https://doi.org/10.3390/bioengineering11121249. The histological analysis conducted post-treatment provides indirect confirmation of effective adipocyte disruption, as evidenced by the loss of Perilipin-1 expression and the presence of CD68-positive macrophages in treated areas.

Due to the dielectric properties of biological tissues, microwaves at 2.45 GHz are preferentially absorbed by adipose tissue, leading to localized heating at depths of approximately 1.2–1.5 cm. The epidermis and dermis, in contrast, exhibit lower absorption at this frequency, and the integrated cooling system of the device prevents excessive heating of the skin. This thermal selectivity is further supported by histological evidence, as no structural alterations were observed in the epidermis or dermis of treated samples.

While direct temperature measurements of subcutaneous fat were not performed, the observed loss of Perilipin-1 expression in treated adipocytes and the presence of CD68-positive macrophages strongly suggest that the treatment effectively induced controlled thermal adipocyte disruption. These findings are consistent with the expected thermal effects of microwaves in adipose tissue, as documented in previous literature.

Given that our study was primarily focused on the biological and histological effects of the treatment, we believe that our findings provide a clear indication of the efficacy of the technology. We hope this clarification addresses your concerns.

 

• Comments 5: “The technology used in this study is non-invasive, with clinically proven rapid results in a highly controlled and safe manner”. Why? If authors write about inflammation in tissue

 Response 5: We acknowledge the apparent contradiction and appreciate the opportunity to clarify this aspect. While the study reports the presence of an inflammatory response in the treated

adipose tissue, it is important to distinguish between the expected, transient inflammatory reaction following targeted adipocyte injury and adverse inflammatory events that may compromise patient safety. The observed inflammation is a physiological response to adipocyte damage and is part of the natural remodeling process rather than an indicator of an unsafe treatment.

 

Additional comments 5: Taking a biopsy from the area of inflammation raises questions. This may lead to sepsis in the patient. Extend the observation period to assess the durability of fat reduction and monitor any long-term adverse effects.

 

 Additional response 5: We would like to clarify that the biopsies were not performed directly on inflamed tissue but rather collected from areas already designated for surgical excision during abdominoplasty. This ensures that the biopsy procedure did not pose an additional risk to the patients beyond the standard surgical protocol. The study was conducted under strict aseptic conditions, following ethical guidelines and approved protocols to ensure patient safety.
Regarding the follow-up period, we acknowledge the importance of long-term monitoring to assess the durability of fat reduction. As already discussed in the manuscript (p.10, lines 452-456), we recognize the short follow-up as a limitation and have highlighted the need for further studies with extended observation periods.

 

• Comments 6: The text contains typos (for example, p.3 line 143, p 5 line 210, p 6 line 270, etc.)

 Response 6: We have carefully reviewed the entire text and corrected all identified typos, including those mentioned on pages 3, 5, and 6. Additionally, we have conducted a thorough proofreading to ensure clarity and accuracy throughout the document.

 

Additional comments 6:+

 

• Comments 7: Please provide the Ethics Commission approval number.

 Response 7: The study was conducted in accordance with the Declaration of Helsinki and received approval from the Institutional Review Board of Calabria Centro. The Ethics Commission approval number is 373/19, granted on 4 December 2019. We have updated the manuscript to include this information in the appropriate section.

 

Additional comments 7: Not all information has been added to the text of the paper.

 

 Additional response 7: As stated in our previous response, this information has been included in the manuscript in the dedicated section in accordance with the journal’s guidelines (p.10, lines 472-474). If there are specific details that you believe are still missing or need further clarification, we would be grateful if you could indicate them so that we can address them accordingly.

 

• Comments 8: “For each enrolled patient, at the first visit demographic data and body mass index (BMI) were collected.” But the text of the article does not contain this information and analysis. There is no information about what BMI the volunteers had.

 Response 8: Thank you for your observation. We acknowledge the lack of specific details regarding the BMI of the enrolled volunteers in the initial version of the manuscript. To address this, we have now included a description of the BMI values of the study participants and their analysis in the revised manuscript. (p.3 lines 128-131, highlighted in the manuscript)

 

Additional comments 8:+

 

• Comments 9: Why was the microwave application mode used chosen? In medicine, three frequencies and, accordingly, three microwave lengths are used: 2450 MHz (12.245 cm), 915 MHz (32.79 cm) and 433.9 MHz (69.14 cm). 2.45 GHz is the operating frequency of microwave ovens. How deeply can radiation of a given frequency penetrate into biological tissue? Microwave energy absorption occurs primarily in tissues rich in water. The skin contains up to 72% of all water, while fatty tissue contains less than 10%. That is, absorption should mainly occur in the skin layers, not in the fatty tissue. Depending on the thickness of the subcutaneous fat (on the buttocks and abdomen), the skin layer can be more than 10 cm. If only the superficial layer is damaged, and the deep layers are not affected by microwaves, then how effective is the proposed device.

 Response 9: The selection of the 2.45 GHz frequency was based on several factors, including its established safety profile, targeted absorption characteristics, and clinical efficacy in previous studies. While it is true that water-rich tissues such as the skin exhibit higher microwave absorption, recent literature and experimental findings have demonstrated that the dielectric properties of adipose tissue allow for selective heating at this frequency. Specifically, the penetration depth of 2.45 GHz microwaves into biological tissue has been shown to reach approximately 1.2 cm to 1.5 cm in adipose tissue under controlled conditions, which is sufficient to induce thermal effects leading to adipocyte disruption. The device used in this study incorporates an advanced cooling system that minimizes excessive heating of the skin, allowing the microwaves to preferentially target the underlying fat layer without causing significant epidermal damage. Moreover, the efficacy of the proposed device is supported by histological findings demonstrating selective adipocyte damage and the absence of substantial epidermal changes, confirming the depth of penetration is adequate to achieve the intended therapeutic effect. This was further validated through immunohistochemical analysis showing perilipin-1 depletion and macrophage infiltration in treated adipose regions, which suggests that deep layers are effectively impacted. Regarding alternative frequencies, while 915 MHz and 433.9 MHz offer deeper penetration, they also require different power densities and applicator designs, which could increase treatment duration and reduce the precision of adipose targeting. The 2.45 GHz frequency, in contrast, provides an optimal balance between penetration depth and focused energy delivery, making it a suitable choice for localized fat reduction.

 

Additional comments 9: The authors do not provide references to the works they mention. How can the veracity of the statements be verified? Readers would be interested in this information.

 

 Additional response 9: In response to your request, we have now incorporated three additional references that support the dielectric properties of adipose tissue and the selective heating mechanism of microwaves at 2.45 GHz.

The revised section, which includes the appropriate citations, can be found on page 2, lines 97-105 of the manuscript, and has been highlighted in the text for ease of review. These additions provide further scientific context regarding the penetration depth of microwaves in biological tissues and their preferential absorption by adipose layers.

 

 

• Comments 10: Authors should add an image of the control sample with CD68 antigen (“Control samples are negative for CD68”). Authors should indicate the changes described in the figures.

 Response 10: Thank you for your suggestion. Unfortunately, an image of the control sample with CD68-negative staining was not initially acquired during the study. However, based on our histological analysis and observations, no CD68-positive cells were detected in the control samples.

 

Additional comments 10:+

 

• Comments 11: Similar changes in adipose tissue are described in the works: M. Wanner, M. Avram, D. Gagnon, M. C. Mihm Jr., D. Zurakowski, K. Watanabe, Z. Tannous, R. R. Anderson, D. Manstein, Lasers Surg. Med. 2009, 41, 401; M. A. Trelles, S. R. Mordon, Aesthet. Plast. Surg. 2009, 33, 125; Yanina, IY, Navolokin, NA, Bucharskaya, AB, Мaslyakova, GN, Tuchin, VV. Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo. Biophotonics. 2019, 12 (12), e201900117. The authors are asked to analyze and compare

 Response 11: We have carefully reviewed the suggested references and, among them, we found the study by Trelles et al. (Aesthet. Plast. Surg. 2009, 33, 125) to be the most relevant to our work. This study explores the effects of radiofrequency energy on adipose tissue, describing changes such as adipocyte membrane lysis and structural modifications. We have now incorporated a comparison of our findings with those reported in Trelles et al., emphasizing the similarities and differences in the mechanisms of action and histological outcomes. We believe this addition strengthens our manuscript by providing a broader perspective on energy-based fat reduction techniques. (p.7 lines 327-335, highlighted in the manuscript)

 

Additional comments 11: Not all proposed articles are reviewed in the text.

 

 Additional response 11: We appreciate your suggestions regarding additional references. As per the journal’s guidelines, we critically analyzed the proposed studies to assess their relevance to our manuscript. After thorough evaluation, we found the study by Trelles et al. (Aesthet. Plast. Surg. 2009, 33, 125) to be the most relevant, as it explores adipocyte membrane lysis and structural modifications following radiofrequency treatment, allowing a meaningful comparison with our findings. This comparison has been included in the manuscript (p.7, lines 327-335).

The other suggested references were not directly applicable to our study's focus and, therefore, were not incorporated, in accordance with the journal’s guidelines, which state:
"If the reviewer(s) recommended references, critically analyze them to ensure that their inclusion would enhance your manuscript. If you believe these references are unnecessary, you should not include them."

We remain open to further discussion if needed. Thank you for your time and constructive feedback.

 

 

• Comments 12: Reference format does not correspond to the requirements of this journal.

 Response 12: Thank you for pointing out the discrepancies in the reference formatting. We have carefully reviewed the journal's guidelines and have reformatted all references accordingly to ensure compliance with the required style. We appreciate your attention to detail and believe that the updated reference section now aligns with the journal’s standards.

 

Additional comments 12:+

Reviewer 2 Report

Comments and Suggestions for Authors  The manuscript has been sufficiently improved to warrant publication in Cosmetics.

Author Response

Comments 1: The manuscript has been sufficiently improved to warrant publication in Cosmetics.

• Response 1: Thank you for your comments and for recognizing the improvements made in the manuscript. We appreciate your time and effort in reviewing our work and for recommending it for publication in Cosmetics.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

To additional response 4: If no temperature measurements were taken, then the following phrase should be removed from the text: "The device used in this study, equipped with a real-time temperature monitoring system, ensured that the subcutaneous fat temperature reached approximately 45°C to 50°C during treatment. This range aligns with previously reported thresholds for inducing irreversible adipocyte damage without compromising the overlying skin. " (p.4. lines 155-158)

Author Response

Comments: To additional response 4: If no temperature measurements were taken, then the following phrase should be removed from the text: "The device used in this study, equipped with a real-time temperature monitoring system, ensured that the subcutaneous fat temperature reached approximately 45°C to 50°C during treatment. This range aligns with previously reported thresholds for inducing irreversible adipocyte damage without compromising the overlying skin. " (p.4. lines 155-158)

• Response: Dear Reviewer, the previous statement regarding temperature monitoring was based on expected thermal effects derived from prior literature on microwave-induced adipocyte damage rather than real-time temperature recordings from the device itself.
  To ensure clarity and accuracy, we will revise the text to avoid any implication that direct temperature monitoring was conducted. The revised phrase will read:

  "Based on previous literature, microwave-based treatments have been shown to induce adipocyte disruption at temperatures ranging from approximately 45°C to 50°C, without compromising the overlying skin. While direct temperature measurements of the subcutaneous fat were not performed in this study, the observed histological changes, including the loss of Perilipin-1 expression and the presence of CD68-positive macrophages, strongly suggest that the treatment induced a controlled thermal effect consistent with adipocyte injury." (pag. 4, lines 155-161)

  We appreciate your feedback and hope this revision adequately addresses your concern.