Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using competing inert hydroxymethylene diphosphonate
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Evaluation Summary:
The manuscript shows that bisphosphonate-related osteonecrosis of the jaw, a rare complication of osteoporosis treatment, was prevented in mice using a novel treatment which works by reversing the associated oral inflammation. The work in this manuscript has the potential to be impactful if limitations are addressed. It will be of interest to investigators in the bone and dental fields who conduct pre-clinical research.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this antiresorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in liposome-based deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesions. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved proinflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology is related to N-BP levels in jawbones and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.
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Evaluation Summary:
The manuscript shows that bisphosphonate-related osteonecrosis of the jaw, a rare complication of osteoporosis treatment, was prevented in mice using a novel treatment which works by reversing the associated oral inflammation. The work in this manuscript has the potential to be impactful if limitations are addressed. It will be of interest to investigators in the bone and dental fields who conduct pre-clinical research.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Okawa et al show that topical oral application of an agent used in SPECT imaging, hydroxymethylene diphosphonate (HMDP-DNV), displaces pre-existing nitrogen-containing bisphosphonate (N-BP) from the jawbone of mice and prevents the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ), a devastating complication that rarely occurs after invasive dental procedures in N-BP treated patients. They further demonstrate pro-inflammatory genomic signaling in gingival cells of N-BP treated mice, which reverses with HMDP-DNV. The methods are well-described overall and the results are potentially important. However, limitations include the short study period and the lack of multiple time points. Additional data to address these limitations would help to strengthen the authors' conclusions. If these …
Reviewer #1 (Public Review):
Okawa et al show that topical oral application of an agent used in SPECT imaging, hydroxymethylene diphosphonate (HMDP-DNV), displaces pre-existing nitrogen-containing bisphosphonate (N-BP) from the jawbone of mice and prevents the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ), a devastating complication that rarely occurs after invasive dental procedures in N-BP treated patients. They further demonstrate pro-inflammatory genomic signaling in gingival cells of N-BP treated mice, which reverses with HMDP-DNV. The methods are well-described overall and the results are potentially important. However, limitations include the short study period and the lack of multiple time points. Additional data to address these limitations would help to strengthen the authors' conclusions. If these results are added, this work could have a high impact in the field and the data could set the stage for further testing. The significance lies in the unmet need for therapeutic options to prevent this complication, which is widely dreaded and impedes the use of often needed bisphosphonate therapy.
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Reviewer #2 (Public Review):
Okawa et al. investigated the effects of the selective removal of zoledronic acid (ZA) from the jawbone using an intra-oral application of hydroxy methylene diphosphonate (HMDP) formulated in deformable nanoscale vesicles (DNV) using a mouse model of bisphosphonate-related osteonecrosis of the jaw (BRONJ). After maxillary tooth extraction, control ZA-treated mice had delayed gingival wound closure, healing inhibition of the tooth extraction socket, and alveolar bone osteonecrosis consistent with human BRONJ. Furthermore, single-cell RNA sequencing of gingival cells showed oral barrier immune dysregulation and an unresolved pro-inflammatory reaction. In contrast, the application of HMDP-DNV to mice previous to the tooth extraction resulted in accelerated gingival wound closure, socket healing, and attenuation …
Reviewer #2 (Public Review):
Okawa et al. investigated the effects of the selective removal of zoledronic acid (ZA) from the jawbone using an intra-oral application of hydroxy methylene diphosphonate (HMDP) formulated in deformable nanoscale vesicles (DNV) using a mouse model of bisphosphonate-related osteonecrosis of the jaw (BRONJ). After maxillary tooth extraction, control ZA-treated mice had delayed gingival wound closure, healing inhibition of the tooth extraction socket, and alveolar bone osteonecrosis consistent with human BRONJ. Furthermore, single-cell RNA sequencing of gingival cells showed oral barrier immune dysregulation and an unresolved pro-inflammatory reaction. In contrast, the application of HMDP-DNV to mice previous to the tooth extraction resulted in accelerated gingival wound closure, socket healing, and attenuation of the osteonecrosis lesion. In addition, the gingival single-cell RNA sequencing demonstrated the resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. Based on these results, the authors claimed that BRONJ is predominantly induced by the oral N-BP and suggested that HMDP-DNV is a possible effective therapy for BRONJ.
The authors show data supporting a mechanism of how ZA induces BRONJ and an approach for preventing BRONJ by applying an inactive N-BP that replaces an active N-BP (ZA). Overall the study is interesting and novel. However, it also presents some weaknesses and unclear elements that raise questions.
• A major weakness of this study is that it only investigates the effects of treatments 2 wks post-extraction. The ONJ-related exposed bone is a chronic manifestation of antiresorptive medication. In clinical studies, as the authors mentioned, the efficacy of treatments to treat BRONJ related oral wounds takes a long time, even years.
• The authors justified the evaluation of the HMDP-DNV treatment for only 2 wks based on the experience that providing a soft diet post-extraction contributes to the oral wound's healing process; however, as it is well-established, the uneventful wound healing (without infection) of the extraction socket in a rodent could take several weeks and even months to complete if bone healing is taken into account. Moreover, if we consider that N-BPs delay healing, an experiment that only investigates the model for two weeks is insufficient; therefore, it is likely that the model is investigating the effects on early healing but not BRONJ.
• Another limitation of the study is that it only tested a one-time point. If the treatment effect of HMDP-DNV, as the authors implied, were not to revitalize the necrotic bone but to remove the affected bone by normalizing the osteoclastic activity and halting further necrosis, earlier time points would be needed to substantiate these statements. In addition, to investigate BRONJ development and progression later time points would have been important.
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Author Response
Reviewer #1 (Public Review):
Okawa et al show that topical oral application of an agent used in SPECT imaging, hydroxymethylene diphosphonate (HMDP-DNV), displaces pre-existing nitrogen-containing bisphosphonate (N-BP) from the jawbone of mice and prevents the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ), a devastating complication that rarely occurs after invasive dental procedures in N-BP treated patients. They further demonstrate pro-inflammatory genomic signaling in gingival cells of N-BP treated mice, which reverses with HMDP-DNV. The methods are well-described overall and the results are potentially important. However, limitations include the short study period and the lack of multiple time points. Additional data to address these limitations would help to strengthen the authors' …
Author Response
Reviewer #1 (Public Review):
Okawa et al show that topical oral application of an agent used in SPECT imaging, hydroxymethylene diphosphonate (HMDP-DNV), displaces pre-existing nitrogen-containing bisphosphonate (N-BP) from the jawbone of mice and prevents the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ), a devastating complication that rarely occurs after invasive dental procedures in N-BP treated patients. They further demonstrate pro-inflammatory genomic signaling in gingival cells of N-BP treated mice, which reverses with HMDP-DNV. The methods are well-described overall and the results are potentially important. However, limitations include the short study period and the lack of multiple time points. Additional data to address these limitations would help to strengthen the authors' conclusions. If these results are added, this work could have a high impact in the field and the data could set the stage for further testing. The significance lies in the unmet need for therapeutic options to prevent this complication, which is widely dreaded and impedes the use of often needed bisphosphonate therapy.
We agree with this comment and performed an additional experiment to investigate a long-term healing of HMDP-DNV treatment. We performed an additional experiment, in which the outcome of HMDP-DNV topical treatment to the tooth extraction wound of ZOL-injected mice was obtained after 4 weeks of the tooth extraction. In addition to the reported effect of HMDP-DNV at 2 weeks after tooth extraction, this additional experiment addressed: (1) the longer study period and (2) more than one time point for treatment assessment.
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