Association Between Baseline Anti-HLA (Class I and II) and Anti-MICA Antibodies and Inflammatory Cell Infiltrates in Grafted Bone After Maxillary Sinus Floor Augmentation: An Exploratory Secondary Histological Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present an article on the role of immunological factors in the patient's response to maxillary sinus surgery with different bone substitutes.

 

The article shares several similarities with another article by the same authors already published (reference 15).

 

Abstract

This section reports the main aspects of the summary of the paper

 

Introduction

The authors provide an update on bone regeneration in oral implantology.

They focus on immunological factors related to the use of different bone substitutes, highlighting their associated problems (HLA, MICA antibodies).

The authors also indicate the role of low-level laser therapy (LLLT) and photobiomodulation (PBM) in bone healing and the regulation of immune factors.

However, the authors do not include any content regarding the article's objectives in this section.

 

Material and methods

The authors should remove the article objectives paragraph (96-100 lines) from this section and move it to the end of the Introduction section.

The authors report the various procedures used to conduct the research.

However, the authors should include sufficient references to support the scientific content of each phase of the methodology, in order to improve the scientific quality of the article. This way, potential readers can assess whether the procedures are original to the authors or based on other scientific studies.

 

Results

The authors present the contents and tables of the most important findings of the research study.

The authors should express the results without resorting to another publication with a website. This approach is completely incorrect (lines 212-213).

At baseline, a positive anti-HLA class I result was found in 7/20 patients (35.0%), a positive anti-HLA class II result in 11/20 patients (55.0%), and a positive anti-MICA result in 9/20 patients (45.0%).

A positive statically significant correlation was observed between higher anti-HLA class I serum levels and higher ICI values

When PBM was taken into account, the relationship between immunization and ICI appeared stronger in the non-PBM group than in the PBM group

 

Discussion

The authors indicate the importance of immunological factors in bone regeneration. They compare these findings with other research studies.

The authors also discuss the role of photobiomodulation (PBM)in bone regeneration and the immune response.

The authors also present some limitations of the research, such as the small number of patients and the use of a cross-sectional, rather than longitudinal, histological evaluation.

Perhaps these aspects should be addressed in the discussion, with the authors suggesting possible future lines of research in this area.

It would be interesting if the authors presented some aspects related to the importance of the research study in relation to the clinical application of the results and its significance in the treatment of patients.

 

Conclusions

The conclusions are relevant and related to the objectives of the research study.

 

References

The authors should review this section.

 

There are some very old references on a topic that has a significant current impact. In fact, 13 (44.8%) of the 29 references are from the last 5 years.

 

Some references are missing their DOI.

 

Author Response

Response to Reviewer 1

Thank you very much for your thorough review and valuable comments. The authors sincerely appreciate the reviewer’s time and effort in evaluating the manuscript. We are confident that the manuscript has significantly improved thanks to all the reviewer’s suggestions and constructive remarks.

Introduction

“The authors provide an update on bone regeneration in oral implantology.

They focus on immunological factors related to the use of different bone substitutes, highlighting their associated problems (HLA, MICA antibodies).

The authors also indicate the role of low-level laser therapy (LLLT) and photobiomodulation (PBM) in bone healing and the regulation of immune factors.”

Thank you very much for these comments.

“However, the authors do not include any content regarding the article's objectives in this section.”

Thank you for this comment. We have added the objective of the study to the Introduction section.

 

Material and methods

“The authors should remove the article objectives paragraph (96-100 lines) from this section and move it to the end of the Introduction section.”

Thank you very much for this comment. The first paragraph of the Materials and Methods section has been substantially revised according to your suggestion, and the information concerning the objective of the study has been moved to the end of the Introduction section.

“The authors report the various procedures used to conduct the research.

However, the authors should include sufficient references to support the scientific content of each phase of the methodology, in order to improve the scientific quality of the article. This way, potential readers can assess whether the procedures are original to the authors or based on other scientific studies.”

Thank you for this valuable comment. We have added references where the methodological procedures are supported by the literature. In cases where the methodology represented our own original approach, this has been clarified in the manuscript and supported, where possible, by references to similar methodological approaches.

 

Results

“The authors present the contents and tables of the most important findings of the research study.

The authors should express the results without resorting to another publication with a website. This approach is completely incorrect (lines 212-213).”

Thank you for this important comment. We have expanded the Results section by adding a new paragraph that presents the relevant findings directly in the manuscript, rather than referring the reader to another publication or website. Additional tables have also been added to improve clarity and completeness.

“At baseline, a positive anti-HLA class I result was found in 7/20 patients (35.0%), a positive anti-HLA class II result in 11/20 patients (55.0%), and a positive anti-MICA result in 9/20 patients (45.0%).

A positive statically significant correlation was observed between higher anti-HLA class I serum levels and higher ICI values.

When PBM was taken into account, the relationship between immunization and ICI appeared stronger in the non-PBM group than in the PBM group.”

Thank you for summarizing these findings. We have revised the Results section to present these outcomes more clearly and to ensure that the interpretation is consistent with the exploratory nature of the study.

 

Discussion

“The authors indicate the importance of immunological factors in bone regeneration. They compare these findings with other research studies.”

Thank you for this comment.

“The authors also discuss the role of photobiomodulation (PBM)in bone regeneration and the immune response.”

“The authors also present some limitations of the research, such as the small number of patients and the use of a cross-sectional, rather than longitudinal, histological evaluation.”

Thank you for these comments. We have revised the discussion of the study limitations to make these aspects clearer.

“Perhaps these aspects should be addressed in the discussion, with the authors suggesting possible future lines of research in this area.”

Thank you very much for this helpful suggestion. We have added a new paragraph to the Discussion section addressing potential future research directions.

“It would be interesting if the authors presented some aspects related to the importance of the research study in relation to the clinical application of the results and its significance in the treatment of patients.”

Thank you very much for this valuable suggestion. We have added a new paragraph discussing the potential clinical implications of our findings and their possible relevance for patient treatment.

 

Conclusions

The conclusions are relevant and related to the objectives of the research study.

Thank you very much for this positive comment.

 

References

“The authors should review this section.”

Thank you for this comment. The References section has been reviewed and corrected.

“There are some very old references on a topic that has a significant current impact. In fact, 13 (44.8%) of the 29 references are from the last 5 years.”

Thank you for this important comment. We agree that the reference list required updating due to the current relevance of the topic. The reference list has been revised and expanded. In the revised version, 20 of 35 references are from the last five years. In addition, several older references related to sinus augmentation and bone regeneration were replaced or supplemented with more recent clinical, histological, and methodological publications. Newly added references include recent studies on lateral window sinus floor elevation, histological and histomorphometric outcomes after sinus augmentation, photobiomodulation, Luminex-based antibody assessment, and platelet-concentrate adjuncts in regenerative dentistry.

“Some references are missing their DOI.”

Thank you for this comment. The reference list has been checked for DOI completeness and formatting consistency. Missing DOI information has been added where available.

   

Is attached

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an original and clinically relevant question by exploring the relationship between baseline anti-HLA/anti-MICA sensitization, local inflammatory infiltrates, graft material, and photobiomodulation after maxillary sinus floor augmentation; however, several issues should be addressed before it can be considered for acceptance. The authors should more clearly frame the study as an exploratory secondary analysis with a very small sample size, particularly because the four treatment subgroups include only five patients each and the histological outcome showed a very restricted distribution, with inflammatory infiltrates limited to scores of 0 or 1. The statistical interpretation should therefore be moderated, especially for marginal findings such as the association between anti-HLA class I reactivity and inflammatory infiltrates and the subgroup analysis suggesting a modifying effect of PBM. Effect sizes, confidence intervals, and a clearer discussion of multiple testing should be added. The methodology should be made more self-contained by providing essential details on randomization, allocation concealment, blinding, biomaterial characteristics, and the full PBM protocol, rather than relying mainly on the previous publication. The histological assessment also requires clarification, including the number of fields examined per specimen, examiner calibration, interobserver agreement, and blinding to treatment group and antibody status. Finally, the manuscript would benefit from substantial academic English editing to improve grammar, precision, and flow, as well as correction of formatting inconsistencies in the title, tables, abbreviations, and references. Overall, the work is promising and hypothesis-generating, but its conclusions should be substantially tempered and aligned with the exploratory nature and methodological limitations of the study.

Author Response

Response to Reviewer 2

Thank you very much for your thorough review and valuable comments. The authors sincerely appreciate the reviewer’s time and effort in evaluating the manuscript. We are confident that the manuscript has significantly improved thanks to all the reviewer’s suggestions and constructive remarks.

“The manuscript addresses an original and clinically relevant question by exploring the relationship between baseline anti-HLA/anti-MICA sensitization, local inflammatory infiltrates, graft material, and photobiomodulation after maxillary sinus floor augmentation; however, several issues should be addressed before it can be considered for acceptance.”

The authors would like to sincerely thank the Reviewer for this positive and constructive comment. We appreciate the recognition of the originality and clinical relevance of our study, and we have carefully revised the manuscript according to the Reviewer’s suggestions.

“The authors should more clearly frame the study as an exploratory secondary analysis with a very small sample size, particularly because the four treatment subgroups include only five patients each and the histological outcome showed a very restricted distribution, with inflammatory infiltrates limited to scores of 0 or 1.”

Thank you very much for this important comment. We agree that the exploratory nature of the study and the small sample size should be more clearly emphasized. Therefore, additional statistical analyses were performed, including Benjamini–Hochberg and Bonferroni corrections.

In the Statistical Analysis section, the following sentence was added:

“To address multiplicity, adjusted p-values using the Benjamini–Hochberg procedure and Bonferroni correction were additionally calculated for the main exploratory antibody–ICI correlation analyses. Given the small sample size, these adjusted analyses were interpreted as sensitivity analyses rather than confirmatory tests.”

In addition, the word “exploratory” was added to the title and to the Abstract. The conclusions were substantially tempered, and the Limitations section was expanded.

In the Results section, the previous statement “ A positive nominal association was observed between higher anti-HLA  and higher ICI values..” was revised to:

“A positive nominal association was observed between higher anti-HLA class I NBG ratio and higher ICI values (Spearman’s rho = 0.46; 95% CI: 0.03 to 0.75; nominal p = 0.048). However, after adjustment for the three exploratory antibody–ICI comparisons, this association did not remain statistically significant using either the Benjamini–Hochberg procedure or Bonferroni correction. Therefore, this finding should be interpreted as suggestive and hypothesis-generating rather than confirmatory.”

The following sentence “ For anti-HLA class II same direction of association was present.” was also revised:

“For anti-HLA class II and anti-MICA antibodies, the direction of association was also positive, but the confidence intervals included zero and the results did not reach statistical significance after adjustment for multiple testing.”

A new table, Table 4, was added to present the additional multiple-testing statistical analyses, and the previous Table 4 was renumbered as Table 5.

In the Discussion section, we added the following statement:

“Importantly, the association between anti-HLA class I reactivity and ICI was only nominally significant and did not remain significant after correction for multiple testing. Thus, this result should not be interpreted as definitive evidence of an antibody-driven inflammatory response, but rather as an exploratory signal that may justify further investigation in a larger prospectively powered cohort.”

“The statistical interpretation should therefore be moderated, especially for marginal findings such as the association between anti-HLA class I reactivity and inflammatory infiltrates and the subgroup analysis suggesting a modifying effect of PBM. Effect sizes, confidence intervals, and a clearer discussion of multiple testing should be added.”

Thank you very much for this important comment. We agree with the Reviewer’s suggestion. As mentioned above, additional statistical analyses were performed, including adjusted p-values using the Benjamini–Hochberg procedure and Bonferroni correction. Confidence intervals, and a clearer discussion of multiple testing have also been added. The interpretation of the findings has been moderated throughout the Results, Discussion, Limitations, and Conclusions sections.

“The methodology should be made more self-contained by providing essential details on randomization, allocation concealment, blinding, biomaterial characteristics, and the full PBM protocol, rather than relying mainly on the previous publication.”

Thank you very much for this valuable observation. Following the Reviewer’s suggestion, the methodology has been expanded. We added more detailed information regarding randomization, allocation concealment, biomaterial characteristics, and the PBM protocol.

“The histological assessment also requires clarification, including the number of fields examined per specimen, examiner calibration, interobserver agreement, and blinding to treatment group and antibody status.”

Thank you very much for this helpful comment. We have added more detailed information on how the histological assessment was performed, including the number of representative fields examined per specimen and the blinding of the investigators. We also clarified how discrepant results were handled and how consensus was reached.

“Finally, the manuscript would benefit from substantial academic English editing to improve grammar, precision, and flow, as well as correction of formatting inconsistencies in the title, tables, abbreviations, and references.”

Thank you very much for this comment. The manuscript has been carefully checked again by an English-speaking reviewer, and additional corrections were made to improve grammar, precision, and flow. We also revised the title, tables, abbreviations, and references to improve formatting consistency.

“Overall, the work is promising and hypothesis-generating, but its conclusions should be substantially tempered and aligned with the exploratory nature and methodological limitations of the study.”

Thank you very much for recognizing the positive aspects of our work. We fully agree that the conclusions should be aligned with the exploratory nature of the study and its methodological limitations. Therefore, the conclusions have been substantially tempered, and the manuscript now presents the findings as hypothesis-generating rather than confirmatory.

   

Is attached

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have successfully implemented the modifications indicated by the reviewer in the text to improve the scientific quality of the article.

Author Response

We sincerely thank the Reviewer for the positive evaluation of our revised manuscript. We are grateful for the Reviewer’s constructive comments, which helped us improve the scientific quality, clarity, and overall presentation of the article. We appreciate the Reviewer’s confirmation that the requested modifications have been successfully implemented.

Reviewer 2 Report

Comments and Suggestions for Authors

Although it addresses a potentially relevant and underexplored question regarding the relationship between baseline anti-HLA/anti-MICA humoral sensitization and inflammatory cell infiltrates after maxillary sinus floor augmentation. The main limitations are methodological and interpretative rather than merely editorial: the study is a secondary exploratory analysis of a previously published randomized pilot cohort, and the authors must more clearly justify its scientific independence and added value beyond the original publication . In addition, the sample size is very small, with only 20 patients divided into four groups, which substantially limits statistical power and makes subgroup analyses, particularly those involving photobiomodulation, highly unstable . The histological outcome also requires stronger methodological support, as the inflammatory cell infiltrate score appears to be an exploratory semiquantitative scale rather than a validated endpoint, and this should be explicitly acknowledged and justified . Finally, the statistical interpretation should be substantially moderated, since the apparent association between anti-HLA class I reactivity and inflammatory infiltrates was only nominally significant and did not remain significant after correction for multiple comparisons, meaning that the findings should be presented strictly as hypothesis-generating rather than confirmatory . 

Author Response

Dear Reviewer,

We sincerely thank you for your careful, constructive, and insightful review. We appreciate the opportunity to improve the manuscript and believe that the revisions made in response to your comments have substantially strengthened the methodological framing, statistical interpretation and overall clarity of the paper.

Reviewer comment:

“Although it addresses a potentially relevant and underexplored question regarding the relationship between baseline anti-HLA/anti-MICA humoral sensitization and inflammatory cell infiltrates after maxillary sinus floor augmentation. The main limitations are methodological and interpretative rather than merely editorial: the study is a secondary exploratory analysis of a previously published randomized pilot cohort, and the authors must more clearly justify its scientific independence and added value beyond the original publication. In addition, the sample size is very small, with only 20 patients divided into four groups, which substantially limits statistical power and makes subgroup analyses, particularly those involving photobiomodulation, highly unstable. The histological outcome also requires stronger methodological support, as the inflammatory cell infiltrate score appears to be an exploratory semiquantitative scale rather than a validated endpoint, and this should be explicitly acknowledged and justified. Finally, the statistical interpretation should be substantially moderated, since the apparent association between anti-HLA class I reactivity and inflammatory infiltrates was only nominally significant and did not remain significant after correction for multiple comparisons, meaning that the findings should be presented strictly as hypothesis-generating rather than confirmatory.”

Response:

We thank the Reviewer very much for this careful and constructive assessment. In response, we revised the manuscript throughout to present the study more clearly as a secondary, exploratory, hypothesis-generating analysis and to avoid confirmatory wording.

First, we changed the title to better reflect the exploratory and secondary nature of the present analysis. The revised title now reads:

“Association between Baseline anti-HLA (class I and II) and anti-MICA antibodies and Inflammatory Cell Infiltrates in Grafted Bone after maxillary sinus floor augmentation: an exploratory secondary histological study.”

Second, we revised the Abstract to make the study design, statistical interpretation, and conclusions more cautious. In the Methods part of the Abstract, the study is now explicitly described as an exploratory secondary analysis:

“This exploratory secondary analysis included 20 adults undergoing lateral maxillary sinus lifting.”

In the Results part of the Abstract, we removed wording suggesting a statistically confirmed association or a PBM-related modifying effect. The previous wording:

“Higher anti-HLA class I reactivity was associated with higher ICI values, particularly in the non-PBM subgroup.”

was replaced with:

“A nominal positive correlation was observed between anti-HLA class I NBG ratio and ICI; however, this finding did not remain statistically significant after correction for multiple comparisons. Exploratory PBM subgroup estimates were directionally different but were based on very small subgroups and should not be interpreted as evidence of effect modification.”

The Conclusions part of the Abstract was also revised. The previous wording:

“Baseline humoral immunization did not appear to impair final histological bone quality, but it may be related to the inflammatory component of healing. PBM may modulate this relationship.”

was replaced with:

“The findings suggest a possible hypothesis-generating link between baseline humoral sensitization and mild local inflammatory infiltrates, which requires validation in larger, prospectively powered studies with predefined histological and immunological endpoints.”

Third, we clarified the scientific independence and added value of the present manuscript in relation to the previously published primary report. The revised Materials and Methods section now explicitly explains that the previous publication focused on regenerative outcomes, whereas the current manuscript addresses a separate immunological question:

“The scientific rationale and added value of the present report should be distinguished from the primary publication. The original study focused on clinical, radiological, and histological bone-regeneration outcomes after maxillary sinus augmentation with different biomaterials and adjunctive PBM. In contrast, the present secondary analysis addresses a separate immunological question: whether preoperative humoral sensitization, assessed by anti-HLA class I, anti-HLA class II, and anti-MICA antibody screening, is related to the local inflammatory component observed in bone-core biopsies after healing. Thus, the present manuscript does not aim to duplicate the primary regenerative outcome analysis, but to integrate baseline serological data with a local histological inflammatory endpoint that was not the central focus of the original report. Because this analysis was exploratory and based on the same randomized pilot cohort, its findings should be interpreted as hypothesis-generating.”

Fourth, in response to the Reviewer’s concern regarding the histological endpoint, we explicitly acknowledged that the inflammatory cell infiltrate score was study-specific and exploratory rather than a validated histological endpoint. At the same time, we clarified the rationale for assessing inflammatory infiltrates in relation to baseline immunological sensitization, referring to previous work in allogeneic intraoral bone grafting. The revised text now states:

“The ICI score used in this study was a study-specific exploratory semiquantitative score and should not be regarded as a previously validated histological endpoint. However, the rationale for evaluating local inflammatory cell infiltrates in relation to baseline immunological sensitization was informed by previous studies in allogeneic intraoral bone grafting, in which systemic immune reactivity was assessed together with local histological and immunohistochemical markers of inflammation, including pro-inflammatory cytokines and T-cell markers [21]. Therefore, in the present study, the ICI score was introduced as a structured descriptive tool to characterize the presence and intensity of inflammatory cell infiltrates in the available biopsy material.”

Fifth, we revised the PBM subgroup interpretation to avoid suggesting a demonstrated modifying effect of photobiomodulation. The revised Results section now presents these findings as descriptive subgroup estimates only:

“When PBM status was considered descriptively, the correlation estimate between anti-HLA class I NBG ratio and ICI was numerically higher in the non-PBM subgroup than in the PBM subgroup. In the non-PBM subgroup, rho was 0.65 (95% CI: 0.03 to 0.91; nominal p = 0.044), whereas in the PBM subgroup, rho was 0.29 (95% CI: −0.42 to 0.78; nominal p = 0.425). Given the very small subgroup size, wide confidence intervals, and absence of formal interaction testing, these results do not provide evidence that PBM modifies the relationship between baseline immunization and ICI. They should be interpreted strictly as exploratory, hypothesis-generating observations.”

Sixth, we revised the Discussion to remove statements that could imply a confirmed antibody-driven inflammatory response or a proven immunomodulatory effect of PBM. The revised paragraph now reads:

“The nominal association between anti-HLA class I reactivity and ICI should be interpreted with particular caution. Although the direction of the association was positive, this observation did not remain statistically significant after correction for multiple comparisons, and the ICI outcome had a restricted distribution limited to low-grade scores. Therefore, this result should not be interpreted as evidence of an antibody-driven inflammatory response. Rather, it represents an exploratory signal that may help formulate hypotheses for future studies. Similarly, the PBM subgroup findings should not be interpreted as demonstrating an immunomodulatory effect of PBM. The subgroup estimates were based on very small numbers, had wide confidence intervals, and were not powered to test interaction or effect modification. Therefore, these findings should be considered only as descriptive observations requiring prospective validation.”

Seventh, we expanded the Limitations section to directly address the Reviewer’s concerns regarding the secondary nature of the analysis, small sample size, instability of subgroup analyses, non-validated ICI score, restricted ICI distribution, lack of functional antibody testing, and the fact that the nominal anti-HLA class I association did not remain statistically significant after correction for multiple comparisons. The revised limitations now state:

“The present findings should be interpreted in light of several important limitations. First, this study represents a secondary exploratory analysis of a previously published randomized pilot cohort and was not prospectively powered to assess immunological–histological associations. The total sample size was small and the original four treatment subgroups included only five patients each. Therefore, no confirmatory inference can be made from subgroup comparisons and the PBM-related findings should be interpreted only as descriptive observations rather than evidence of effect modification. Second, the ICI score was a study-specific exploratory semiquantitative scale and should not be regarded as a validated histological endpoint. In addition, all observed ICI values were restricted to scores of 0 or 1, indicating a narrow outcome distribution and limited discriminatory capacity. Third, antibody assessment was based on screening assay readouts and did not include functional antibody testing, local HLA/MICA expression analysis, cytokine profiling, or immunohistochemical characterization of the inflammatory infiltrates. Finally, the nominal association between anti-HLA class I reactivity and ICI did not remain statistically significant after correction for multiple comparisons. For these reasons, the present results should be treated strictly as preliminary, descriptive, and hypothesis-generating, requiring confirmation in larger prospectively powered studies.”

Finally, we revised the main Conclusions of the manuscript to maintain the same cautious interpretation as in the Abstract. The revised conclusions now emphasize that all ICI scores remained low, that the anti-HLA class I finding was nominal and did not remain statistically significant after correction for multiple comparisons, and that larger prospective studies are needed before any biological or clinical conclusions can be drawn.

We believe that these revisions directly address the Reviewer’s concerns by clarifying the independent scientific rationale of the present analysis, explicitly acknowledging the exploratory nature of the ICI score, adding effect estimates and multiplicity-adjusted p-values, moderating the interpretation of PBM subgroup findings, and consistently presenting the results as preliminary and hypothesis-generating rather than confirmatory.

We thank the Reviewer again for the constructive comments, which helped us substantially improve the manuscript.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Acceptable for publication, provided the manuscript remains framed strictly as an exploratory, hypothesis-generating secondary analysis. The study addresses a biologically plausible and underexplored question, uses a clearly described clinical cohort, includes appropriate ethical and methodological reporting, and interprets the findings cautiously. Although the sample size is small and the histological endpoint is not validated, these limitations are transparently acknowledged and do not undermine the manuscript’s value as a preliminary contribution. Therefore, no major scientific obstacle prevents acceptance.