Harnessing Microbiome-Mediated and Macrophage-Driven Mechanisms for Oral Wound Healing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review addresses an important and clinically relevant topic by synthesizing current evidence on microbiome–immune interactions, macrophage polarization, and wound healing. The manuscript is comprehensive and well-referenced, with strong mechanistic depth. The focus on microbiome-driven immunomodulation is timely and aligns with emerging translational strategies in regenerative medicine.

Decision: Minor Revision

 

Abstract

1. The abstract should be fully narrative and free of citations.
2. Consider sharpening the final sentence to emphasize therapeutic implications, particularly microbiome-based interventions.
3. The abstract would benefit from explicitly stating knowledge gaps, such as limitations in clinical translation or variability in microbiome responses.
4. Several typographics are present such as (be-tween, Macro-phages, in-terplay)
5. The key words shouldn't be numbered

 

Introduction

1. The introduction is lengthy and partially repetitive. Consider streamlining background content and focusing earlier on the unique contribution of this review.
2. In paragraph that discussing chronic wounds (line 46-48), I suggest supporting this paragraph with an updated reference like  https://doi.org/10.1002/hsr2.2036
3. All bacterial names (Genus and species) should be in italics.

 

Main Body

1. For section 2. Oral Microenvironment, viral components of the microbiome are briefly mentioned but underdeveloped, despite growing evidence of their immunological relevance. So, I suggest to  more discusses viral components of the microbiome (virome)  https://doi.org/10.5662/wjm.v15.i2.92592 . This reference introduces the virome as an active immunomodulatory component, supporting the argument that bacteria alone do not fully explain dysbiosis-driven inflammation. It broadens the mechanistic framework beyond bacteriocentric models.
2. For section 4. Eubiosis, dysbiosis vs probiotics, sentence in line 304, should be re-written in a clearer language.
3. For section 6. Microbiome-Based Therapeutics, when discussing microbiome modulation strategies and immune targeting, the authors should reinforce with https://doi.org/10.3390/immuno4040026. This reference provides a robust framework for immune-targeted microbiome therapies, supporting the feasibility of similar approaches in wound healing contexts.

 

Conclusion

1. The conclusion should clearly summarize key mechanistic insights, and explicitly outline future research directions (e.g., virome inclusion, personalized probiotics, clinical trial gaps).
2. Avoid introducing new mechanistic details not discussed earlier

 

Minor Comments

1. Ensure consistent terminology (e.g., use oral microbiome rather than alternating with oral flora).
2. Correct typographical errors and spacing issues (e.g., missing full stops, inconsistent spacing).
3. Standardize abbreviations at first mention.
4. Avoid redundancy in cytokine and marker descriptions across sections.

 

Author Response

We sincerely thank you for reviewing our manuscript. Our detailed responses are presented below, and the corresponding changes are highlighted in the revised file.

 

Abstract:

Comment 1: The abstract should be fully narrative and free of citations.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have made it fully narrative and free of citation. Comment 2: Consider sharpening the final sentence to emphasize therapeutic implications, particularly microbiome-based interventions. Response 2: Thank you for the suggestion and we did our best in refining the sentence. Comments 3: The abstract would benefit from explicitly stating knowledge gaps, such as limitations in clinical translation or variability in microbiome responses. Response 3: We thank the reviewer for this valuable comment. The abstract has been revised to explicitly state key knowledge gaps, including current limitations in clinical translation, inter-individual variability in microbiome composition, and incomplete mechanistic understanding of host–microbe immune interactions. These additions clarify the unmet needs in the field and strengthen the translational relevance of the review.

 

Comment 4: Several typographics are present such as (be-tween, Macro-phages, in-terplay)

Response 4: We thank the reviewer for pointing this out. We have carefully reviewed the manuscript and corrected all typographical errors, including “be-tween,” “Macro-phages,” and “in-terplay,” to ensure consistent and accurate formatting throughout the text.

Comment 5: The key words shouldn't be numbered

Response: We thank the reviewer for this observation. The numbering of the keywords has been removed, and they are now presented in the standard unnumbered format.

Introduction

Comment 1: The introduction is lengthy and partially repetitive. Consider streamlining background content and focusing earlier on the unique contribution of this review.

Response 1:   We thank the reviewer for this constructive feedback. The Introduction has been revised to streamline the background content, reduce repetition, and focus earlier on the unique contribution of this review. These changes improve clarity and better highlight the scope and significance of our work.

Comment 2: In paragraph that discussing chronic wounds (line 46-48), I suggest supporting this paragraph with an updated reference like  https://doi.org/10.1002/hsr2.2036

Response 2: We thank the reviewer for this helpful suggestion. In the paragraph discussing chronic wounds (lines 49-51), we have now supported the content with a recent, relevant reference: Hetta, H.F., et al., Mesenchymal stem cell therapy in diabetic foot ulcer: An updated comprehensive review, Health Sci Rep, 2024, 7(4), e2036. This addition strengthens the background by providing up-to-date evidence on chronic wound pathophysiology and therapeutic approaches.

Comment3: All bacterial names (Genus and species) should be in italics.

Response 3: We thank the reviewer for this important observation. All bacterial names, including both genus and species, have been updated to appear in italics throughout the manuscript to ensure consistency with scientific conventions.

Main Body

Comment 1: For section 2. Oral Microenvironment, viral components of the microbiome are briefly mentioned but underdeveloped, despite growing evidence of their immunological relevance. So, I suggest to  more discusses viral components of the microbiome (virome)  https://doi.org/10.5662/wjm.v15.i2.92592 . This reference introduces the virome as an active immunomodulatory component, supporting the argument that bacteria alone do not fully explain dysbiosis-driven inflammation. It broadens the mechanistic framework beyond bacteriocentric models.

Response 1: We thank the reviewer for this insightful comment. In Section 2, “Oral Microenvironment,” we have expanded the discussion(Line 109-115) to include viral components of the microbiome (virome), highlighting their emerging immunological relevance. We have incorporated the suggested reference: Hetta, H.F., Ahmed, R., Ramadan, Y.N., Fathy, H., Khorshid, M., Mabrouk, M.M., Hashem, M., Gut virome: New key players in the pathogenesis of inflammatory bowel disease, World J Methodol, 2025, 15(2), 92592 [PMID: 40548227; DOI: 10.5662/wjm.v15.i2.92592]

Comment2: For section 4. Eubiosis, dysbiosis vs probiotics, sentence in line 304, should be re-written in a clearer language.

Response 2: We thank the reviewer for this suggestion. The sentence in line 305 of Section 4, “Eubiosis, Dysbiosis vs. Probiotics,” has been revised for clarity and readability to better convey the intended message.

 

Comment 3: For section 6. Microbiome-Based Therapeutics, when discussing microbiome modulation strategies and immune targeting, the authors should reinforce with https://doi.org/10.3390/immuno4040026. This reference provides a robust framework for immune-targeted microbiome therapies, supporting the feasibility of similar approaches in wound healing contexts.

Response 3: We thank the reviewer for this valuable suggestion. In Section 6, “Microbiome-Based Therapeutics,” we have reinforced the discussion of microbiome modulation strategies and immune-targeted approaches by incorporating the suggested reference: Hetta, H.F.; Ramadan, Y.N.; Alharbi, A.A.; Alsharef, S.; Alkindy, T.T.; Alkhamali, A.; Albalawi, A.S.; El Amin, H., Gut Microbiome as a Target of Intervention in Inflammatory Bowel Disease Pathogenesis and Therapy, Immuno, 2024, 4, 400–425.

Conclusion

Comment 1: The conclusion should clearly summarize key mechanistic insights, and explicitly outline future research directions (e.g., virome inclusion, personalized probiotics, clinical trial gaps).

Response1: We thank the reviewer for this constructive feedback.We made our best to summarize key mechanistic insights, and explicitly outline future research directions (e.g., virome inclusion, personalized probiotics, clinical trial gaps).

 

Comment 2: Avoid introducing new mechanistic details not discussed earlier

Response 2: We did our best to avoid introducing new mechanistic details .

 

Minor Comments

1. Ensure consistent terminology (e.g., use oral microbiome rather than alternating with oral flora).
2. Correct typographical errors and spacing issues (e.g., missing full stops, inconsistent spacing).
3. Standardize abbreviations at first mention.
4. Avoid redundancy in cytokine and marker descriptions across sections.

Response: We thank the reviewer for these helpful suggestions. We made our best of our knowledge to correct all the above details.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Minor comments:

This review provides a comprehensive overview of the interplay between the oral microbiome and macrophage polarization during the distinct phases of oral wound healing. The manuscript is well-structured and timely, but several mechanistic assertions rely on extrapolation from non-oral models rather than direct oral-specific evidence. Greater clarity is needed in defining eubiosis versus dysbiosis and in distinguishing microbiome-driven effects from host-mediated immune regulation. The translational potential of probiotic and microbiome-based therapies is promising but remains limited by variable clinical outcomes and delivery challenges. Overall, the review would be strengthened by clearer mechanistic boundaries and a more critical discussion of evidence gaps.

Lines 23–31: The abstract emphasizes microbiome–macrophage crosstalk as a central determinant of oral wound healing. Could the authors clarify which conclusions are supported by direct experimental evidence in oral models versus extrapolated from cutaneous or gastrointestinal studies?

Lines 48–52: The concepts of eubiosis and dysbiosis are introduced early in the manuscript. Please specify whether these states are defined primarily by microbial composition, functional output (e.g., metabolite profiles), or host immune responses in the context of oral wounds.

Lines 71–75: The manuscript states that the oral microbiome signals the transition from inflammation to regeneration. Are there specific microbial taxa or molecular mediators that have been causally linked to this transition in oral wound healing?

Lines 77–83: Probiotic-based therapeutic approaches are presented as promising. Could the authors clarify whether the cited evidence derives predominantly from oral wound models or from extrapolation of skin and gut wound studies?

Lines 134–136: Reduced immune cell infiltration is proposed as a mechanism underlying minimal scarring in oral wounds. Are there quantitative or comparative clinical data that directly support this hypothesis?

Lines 146–149: The authors suggest that microbiome composition directly influences macrophage polarization. How do they distinguish microbiome-driven effects from polarization mediated by host-derived cytokines and growth factors?

Lines 152–155: Figure 1 summarizes the phases of oral wound healing. Could the authors more explicitly describe how oral-specific factors, such as saliva, mastication, and biofilm formation, mechanistically modify each phase compared with cutaneous wound healing?

Lines 181–183: Dysbiosis is described as inducing dysfunctional neutrophil responses. Are there oral-specific studies demonstrating impaired neutrophil function, rather than simply enhanced inflammatory burden?

Lines 204–208: Beneficial commensals such as Streptococcus salivarius are reported to promote M2 macrophage polarization. Is this effect supported by in vivo oral wound models, or is it primarily inferred from epithelial or in vitro immune studies?

Lines 226–229: Microbial metabolites, including short-chain fatty acids, are discussed as immunomodulators. Given their relatively low concentrations in the oral cavity, how physiologically relevant are these metabolites to oral wound healing?

Lines 255–264: Dysbiosis is proposed to impair fibroblast activity and extracellular matrix remodeling. Could the authors clarify whether these effects are mediated directly by microbial factors or indirectly via macrophage-derived cytokines?

Lines 269–276: Remodeling is attributed to balanced MMP and TIMP activity. Are there oral wound–specific data linking microbiome composition to regulation of MMP/TIMP expression during the remodeling phase?

Lines 303–312: The manuscript compares microbiome–immune interactions across gut, skin, and oral tissues. How do the authors justify these cross-tissue generalizations given the unique mechanical, salivary, and microbial dynamics of the oral cavity?

Lines 329–359: Macrophage polarization is discussed primarily within the M1/M2 paradigm. Do the authors consider emerging macrophage subpopulations (e.g., M2b, tissue-resident macrophages) to be relevant in oral wound healing?

Lines 381–384: Microbiome-based therapeutics are highlighted for translational potential. Could the authors expand on current limitations, including strain stability, colonization persistence, safety, and regulatory considerations?

Lines 505–508: Clinical studies of probiotic interventions show variable outcomes. Which factors—such as delivery method, microbial strain specificity, host variability, or wound type—do the authors consider most critical for successful clinical translation?

 

Author Response

 Comment 1: Lines 23–31: The abstract emphasizes microbiome–macrophage crosstalk as a central determinant of oral wound healing. Could the authors clarify which conclusions are supported by direct experimental evidence in oral models versus extrapolated from cutaneous or gastrointestinal studies?

Response 1:

                 Thank you so much for the comment.  Microbiome -macrophage crosstalk is the central regulator of oral wound healing, direct experimental evidence from oral specific models are limited [1–3] and we have data on that which are unpublished. Most oral studies demonstrate associations between periodontal microbes and macrophage inflammatory responses, whereas detailed mechanistic insights—such as microbial metabolite–driven macrophage polarization, inflammasome regulation, and resolution of inflammation—are largely extrapolated from cutaneous and gastrointestinal wound-healing models [4–5].

Oral-specific models

1. Wang, Y., Mao, J., Wang, Y., Wang, R., Duan, D., Liu, Z., Hu, X., Yu, Z., & Shi, X. (2025). Macrophage-induced immunomodulation in oral tissue repair and regeneration: Recent advances and future perspectives. Journal of advanced research, S2090-1232(25)00954-3. Advance online publication. https://doi.org/10.1016/j.jare.2025.11.057.
2. Qian, J., Lu, E., Xiang, H., Ding, P., Wang, Z., Lin, Z., Pan, B., Zhang, C., & Zhao, Z. (2024). GelMA loaded with exosomes from human minor salivary gland organoids enhances wound healing by inducing macrophage polarization. Journal of nanobiotechnology, 22(1), 550. https://doi.org/10.1186/s12951-024-02811-y
3. Yang, L., Song, Y., Jiao, Y., Liu, S., Liu, Y., Guo, L., & Liu, Y. (2025). An innovative compound that promotes oral wound healing via mobilizing gingival mesenchymal stem cell homing. Biochemistry and biophysics reports, 43, 102154. https://doi.org/10.1016/j.bbrep.2025.102154Toma AI, Fuller JM, Willett NJ, Goudy SL. Oral wound healing models and emerging regenerative therapies. Transl Res. 2021 Oct;236:17-34. doi: 10.1016/j.trsl.2021.06.003. Epub 2021 Jun 20. PMID: 34161876; PMCID: PMC8380729.
4. Han, N., Jia, L., Guo, L., Su, Y., Luo, Z., Du, J., Mei, S., & Liu, Y. (2020). Balanced oral pathogenic bacteria and probiotics promoted wound healing via maintaining mesenchymal stem cell homeostasis. Stem cell research & therapy, 11(1), 61. https://doi.org/10.1186/s13287-020-1569-2

Extrapolated from cutaneous or gastrointestinal models
4. Chang PV, et al. Microbial metabolites regulate macrophage function and intestinal inflammation. Nature. 2014.5. Koh A, et al. From dietary fiber to host physiology: SCFAs as regulators of immunity. Cell. 2016.

5. Gao, X., Lu, C., Miao, Y., Ren, J., & Cai, X. (2023). Role of macrophage polarisation in skin wound healing. International wound journal, 20(7), 2551–2562. https://doi.org/10.1111/iwj.14119

Comment 2: Lines 48–52: The concepts of eubiosis and dysbiosis are introduced early in the manuscript. Please specify whether these states are defined primarily by microbial composition, functional output (e.g., metabolite profiles), or host immune responses in the context of oral wounds.

Response 2: In the context of oral wounds, the states of eubiosis and dysbiosis are not defined by a single metric but rather by a bidirectional and dynamic interplay between microbial composition, functional output, and the host immune response.

-             Microbial Composition: Eubiosis is a state of homeostasis characterized by a stable community of commensal bacteria that acts as a protective barrier [22]. Dysbiosis is a microbial shift towards disease [19]  often associated with community destabilization (loss of diversity/reduced community stability) and the overgrowth/expansion of harmful pathobionts, like P. gingivalis and F. nucleatum [16].

-             Host Immune Response: Eubiosis is maintained through a tolerogenic host–microbiome relationship in which the immune system recognizes commensals as non-pathogenic. Dysbiosis arises when microbial perturbations exceed immune regulatory capacity, triggering dysbiotic inflammatory networks and sustained pro-inflammatory signaling that interferes with tissue repair [16, 22].

-             Functional and metabolic Output: These states are further characterized by microbial activity, with eubiotic communities associated with homeostatic metabolic signaling, while dysbiotic communities produce virulence factors such as lipopolysaccharides and proteases that perpetuate inflammation and can delay wound healing [5, 12]. lipopolysaccharides and proteases that perpetuate inflammation and can delay wound healing [5, 12].

22. Rajasekaran, J.J., et al., Oral Microbiome: A Review of Its Impact on Oral and Systemic Health. Microorganisms, 2024. 12(9).

19.Zaura, E., et al., Acquiring and maintaining a normal oral microbiome: current perspective. Front Cell Infect Microbiol, 2014. 4: p. 85.

16. Juarez, V.M., A.N. Montalbine, and A. Singh, Microbiome as an immune regulator in health, disease, and therapeutics. Adv Drug Deliv Rev, 2022. 188: p. 114400.

 5.Zielinska, M., et al., Wound Microbiota and Its Impact on Wound Healing. Int J Mol Sci, 2023. 24(24).

12. Zenobia, C., K.L. Herpoldt, and M. Freire, Is the oral microbiome a source to enhance mucosal immunity against infectious diseases? NPJ Vaccines, 2021. 6(1): p. 80.

 

Comment 3: Lines 71–75: The manuscript states that the oral microbiome signals the transition from inflammation to regeneration. Are there specific microbial taxa or molecular mediators that have been causally linked to this transition in oral wound healing?

Response 3: Thank you for this insightful comment and the opportunity to clarify our wording. In the current literature, there is emerging mechanistic evidence that specific microbial taxa and their products can modulate the shift from pro‑inflammatory (M1) to pro‑regenerative (M2) macrophage phenotypes, which underpins the transition from inflammation to regeneration; however, direct causal demonstrations in human oral wound‑closure models remain limited.

Comment 4: Lines 77–83: Probiotic-based therapeutic approaches are presented as promising. Could the authors clarify whether the cited evidence derives predominantly from oral wound models or from extrapolation of skin and gut wound studies?

Response 4: Thank you for this thoughtful comment. The evidence base we cite for probiotic‑based therapeutic approaches is derived from a combination of oral, cutaneous, and gut/systemic wound or inflammation models, with a substantial proportion coming from non‑oral wound studies. In the revised manuscript, we now explicitly state that while cutaneous wound care has already incorporated microbiome‑based strategies such as probiotic therapies and microbial metabolite delivery, “similar strategies for oral wounds are underdeveloped,” to highlight that much of the interventional data are extrapolated from skin and gut contexts rather than from well‑controlled oral wound trials (Introduction, paragraph discussing therapeutic strategies).

Comment 5: Lines 134–136: Reduced immune cell infiltration is proposed as a mechanism underlying minimal scarring in oral wounds. Are there quantitative or comparative clinical data that directly support this hypothesis?

Response 5: Thank you for this important comment. The statement in our manuscript is primarily supported by quantitative comparative animal studies and translational human work, rather than by large, definitive clinical trials in patients. Comparative murine and porcine models show that oral mucosal wounds exhibit lower and more transient infiltration of neutrophils, macrophages, and other inflammatory cells than matched cutaneous wounds, which coincides with reduced profibrotic signaling and less fibrosis, providing quantitative support for this mechanism in preclinical systems. In humans, paired oral–skin wound studies demonstrate faster healing and a more rapidly resolving inflammatory gene signature in oral mucosa, consistent with a dampened immune response, although most of these studies focus on transcriptional profiles and healing kinetics rather than formal quantification of immune cell infiltrates in large clinical cohorts.

Comment 6: Lines 146–149: The authors suggest that microbiome composition directly influences macrophage polarization. How do they distinguish microbiome-driven effects from polarization mediated by host-derived cytokines and growth factors?

   Response 6: Thank you for this helpful comment. We have clarified that our conclusion is based on studies where microbial factors are experimentally controlled under defined cytokine conditions, showing that specific taxa or microbial products (e.g., LPS, OMVs, SCFAs, polysaccharide A) can shift macrophages toward M1 or M2 phenotypes independently of changes in the broader host cytokine milieu

Comment 7:Lines 152–155: Figure 1 summarizes the phases of oral wound healing. Could the authors more explicitly describe how oral-specific factors, such as saliva, mastication, and biofilm formation, mechanistically modify each phase compared with cutaneous wound healing?

Response 7: Thank you so much for suggestion. We have included 2-3 lines in each phase.(highlighted in red)

Comment 8:Lines 181–183: Dysbiosis is described as inducing dysfunctional neutrophil responses. Are there oral-specific studies demonstrating impaired neutrophil function, rather than simply enhanced inflammatory burden?

Response 8 :Thank you for this helpful comment. The manuscript cites oral-specific periodontitis studies where dysbiosis leads to neutrophil dysfunction—specifically, altered chemotaxis, excessive NET formation, and impaired bacterial killing—resulting in failed microbial control and tissue damage beyond mere increased inflammation.

Comment 9:Lines 204–208: Beneficial commensals such as Streptococcus salivarius are reported to promote M2 macrophage polarization. Is this effect supported by in vivo oral wound models, or is it primarily inferred from epithelial or in vitro immune studies?

Response 9 :Thank you for this clarifying question. The manuscript's statement regarding S. salivarius and A. naeslundii promoting M2 polarization is primarily supported by mechanistic studies demonstrating their effects on epithelial barrier integrity and immune signaling pathways, rather than direct in vivo oral wound closure models. We have revised the text to specify that "these commensals enhance epithelial barrier function and activate M2-favoring signaling pathways" (supported by refs 45, 49), 

Comment10:Lines 226–229: Microbial metabolites, including short-chain fatty acids, are discussed as immunomodulators. Given their relatively low concentrations in the oral cavity, how physiologically relevant are these metabolites to oral wound healing?

Response10: Thank you for raising this important point regarding metabolite concentrations. While SCFAs are present at lower levels in saliva compared to the gut, their local production by oral biofilms and uptake by mucosal immune cells can still achieve physiologically relevant concentrations sufficient to modulate TLR/NF-κB signaling and macrophage polarization, as demonstrated in the cited mechanistic studies.

Comment11:Lines 255–264: Dysbiosis is proposed to impair fibroblast activity and extracellular matrix remodeling. Could the authors clarify whether these effects are mediated directly by microbial factors or indirectly via macrophage-derived cytokines?

Response11: Thank you for this insightful question. The manuscript describes dysbiosis impairing fibroblast function and ECM remodeling primarily through indirect mechanisms, where dysbiotic taxa sustain M1 macrophage polarization and prolonged pro-inflammatory cytokine release (e.g., IL-1, TNF-α, IL-6), which in turn disrupts fibroblast activation, collagen deposition, and matrix maturation. While direct microbial effects on fibroblasts (e.g., via LPS sensing) are possible, the cited evidence emphasizes macrophage-mediated pathways as the dominant mechanism in oral wounds. 

Comment 12:Lines 269–276: Remodeling is attributed to balanced MMP and TIMP activity. Are there oral wound–specific data linking microbiome composition to regulation of MMP/TIMP expression during the remodeling phase?

Response 12: Thank you for this insightful question. we cited oral dysbiosis disrupting proliferative phase fibroblast function via sustained M1 macrophage cytokines (which indirectly dysregulate MMPs/TIMPs), direct oral wound-specific data linking microbiome composition to MMP/TIMP expression during remodeling remain limited. 

Comment 13;Lines 303–312: The manuscript compares microbiome–immune interactions across gut, skin, and oral tissues. How do the authors justify these cross-tissue generalizations given the unique mechanical, salivary, and microbial dynamics of the oral cavity?

Response13: Thank you for this point. Cross-tissue comparisons leverage conserved TLR signaling and macrophage polarization mechanisms while acknowledging oral-specific salivary and mechanical influences; 

Comment 14: Lines 329–359: Macrophage polarization is discussed primarily within the M1/M2 paradigm. Do the authors consider emerging macrophage subpopulations (e.g., M2b, tissue-resident macrophages) to be relevant in oral wound healing?

Response14:Thank you for the question.The M1/M2 framework is justified as it captures the core inflammation-to-resolution transition central to the manuscript's focus, while emerging subpopulations like M2b (regulatory) and tissue-resident macrophages contribute nuanced functions that align with this spectrum. 

Comment15: Lines 381–384: Microbiome-based therapeutics are highlighted for translational potential. Could the authors expand on current limitations, including strain stability, colonization persistence, safety, and regulatory considerations?

Response15:Thank you for this key question. The manuscript notes "limited clinical translation" due to strain stability challenges from salivary flow, poor colonization against established biofilms, safety concerns in immunocompromised patients, and regulatory hurdles requiring strain characterization—now explicitly detailed in a new limitations paragraph(line 406-414)

Comment16: Lines 505–508: Clinical studies of probiotic interventions show variable outcomes. Which factors—such as delivery method, microbial strain specificity, host variability, or wound type—do the authors consider most critical for successful clinical translation?

Response16: Streptococcus salivarius (especially K12 strain), Lactobacillus reuteri, and Bifidobacterium breve emerge as the most consistently effective probiotic strains across preclinical and limited clinical studies cited in the manuscript.​

Key findings from Table 2 & Table 4:

• Oral microbiota eubiosis: S. salivarius , L. reuteri listed as primary beneficial strains vs pathogens.​
• Clinical trial evidence: L. reuteri lozenges reduced post-surgical swelling/pain (though no significant wound healing acceleration).​
• Preclinical wound models: B. breve promoted oral mucosal healing via IL-10; L. reuteri neutralized LPS and enhanced phagocytosis.​

The manuscript emphasizes strain-specific effects rather than ranking efficacy, noting variable clinical translation due to delivery and colonization challenges.