Author response: Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using competing inert hydroxymethylene diphosphonate (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation The manuscript shows that bisphosphonate-related osteonecrosis of the jaw, a rare complication of osteoporosis treatment and bone marrow cancers, was prevented/alleviated in mice using a novel treatment which works by reversing the associated oral inflammation. The work in this manuscript is valuable and will be of significant interest to investigators in the bone and dental fields who conduct pre-clinical research. https://doi.org/10.7554/eLife.76207.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Nitrogen-containing bisphosphonates (N-BPs) are prototypical antiresorptive agents (Ebetino et al., 2011; Cremers et al., 2019; McKenna et al., 2020) initially marketed to prevent bone fractures and to treat osteopenia or osteoporosis (Fink et al., 2019; Black and Rosen, 2016). N-BPs have been widely prescribed for postmenopausal women who have a bone mineral density T score of −2.5 or less, a history of spine or hip fracture, or a Fracture Risk Assessment Tool score (Kanis et al., 2007) indicating increased fracture risk. N-BP treatment with an increased dose and frequency is also given to patients with multiple myeloma (Mhaskar et al., 2017) or metastatic cancers to bone (O’Carrigan et al., 2017; Goldvaser and Amir, 2019) to address tumor-induced osteolysis, hypercalcemia, and bone pain. N-BPs are well tolerated, exhibit few side-effects, and have established clinical benefits (Gordon, 2005). In the 2000’s, cases of osteonecrosis in the jawbone (ONJ) emerged among a minority of patients with a history of N-BP treatment, usually occurring at a dental infection site (Khan et al., 2015) or after dentoalveolar surgery such as a tooth extraction (Marx, 2003; Ruggiero, 2009). Originally designated as ‘bisphosphonate-related ONJ’ (BRONJ), this syndrome has also proven to be associated with non-bisphosphonate antiresorptive agents such as denosumab, a humanized anti-RANKL monoclonal antibody and anti-angiogenesis drug. As a result, the American Association for Oral & Maxillofacial Surgeons (AAOMS) has proposed the term ‘Medication-related ONJ’ (MRONJ) reflecting the association of ONJ with a multiplicity of antiresorptive agents (Ruggiero et al., 2014). Incidents of MRONJ reported to the United States Food and Drug Administration (FDA)’s Adverse Event Reporting System (FAERS) peaked from the first quarter of 2010 to the first quarter of 2014 with approximately 30,000 cases during this period, among which BRONJ represented the major fraction (Zhang et al., 2016). The FAERS database may underreport incidents because of variations in provider’s perceptions of how severe an event needs to be to warrant submission of the event report (Alatawi and Hansen, 2017). We present an alternative MRONJ estimation of approximately 23,511 new cases per year in the US (Table 1). Considering the long cure period, the cumulative MRONJ patient number may exceed this annual estimation; however, it is highly conceivable that MRONJ is a rare disease defined by the FDA’s orphan disease (Kempf et al., 2018). Table 1 Estimated MRONJ case numbers in the US based on MRONJ incidence for the major underlying diseases*. Underlying diseasesNew cases in the US (Year)MRONJ incidenceEstimated MRONJ cases (Year)*Multiple myeloma34,920†5.16% (Rugani et al., 2016)1,802Breast cancer330,840‡2.09% (Rugani et al., 2016)6,915Prostate cancer248,530†3.80% (Rugani et al., 2016)9,444Osteoporosis/Low bone mass53,500,000§0.01% (Khan et al., 2017)5,350Estimated annual incidents of MRONJ23,511 MRONJ: Medication-related osteonecrosis in the jawbone. * Based on an assumption that all these patients were treated by antiresorptive medications. † American Cancer Society. Cancer Facts & Figures 2021. Atlanta, Ga: American Cancer Society; 2021. ‡ Invasive breast cancer and ductal carcinoma: American Cancer Society. How Common Is Breast Cancer? Jan. 2020. Available at: https://www.cancer.org/cancer/breast-cancer/about/how-common-is-breast-cancer.html. § Center for Disease Control and Prevention at: https://www.cdc.gov/nchs/products/databriefs/db405htm. Typical disease symptoms include gingival dehiscence and exposure of necrotic jawbone (Ruggiero et al., 2014; Reid and Cornish, 2011; Otto et al., 2018), which often becomes infected, resulting in pain, erythema, and purulent drainage (Ruggiero, 2015). The extent of a BRONJ lesion has been measured for the absolute or relative open wound area and the time taken for mucosa to completely cover necrotic tissue and exposed bone (‘cure period’) (Beth-Tasdogan et al., 2017). A multicenter case registry study reported either resolution (35%) or improvement (10%) in 207 evaluable BRONJ patients within the study period of 2 years with conservative treatments such as irrigation and antibiotic medications (Schiodt et al., 2018). However, 37% of patients did not respond to the conventional treatment and exhibited either progression or stable conditions. Although the reported case incident is small, long-lasting severe clinical symptoms of MRONJ in some patients have created apprehension among patients and healthcare providers (Kelly et al., 2019; Jha et al., 2015). The National Institutes of Health (NIH) workshop on ‘Pathway to Prevention (P2P) for Osteoporotic Fracture’ in 2018 highlighted the acute need of research to mitigate serious adverse events such as MRONJ and atypical femur fracture to prevent the increasing threat of osteoporotic fractures (Siu et al., 2019). The P2P workshop particularly highlighted the markedly decreased acceptance of antiresorptive medications by osteoporosis patients, despite the significant benefit of these drugs for this indication (van de Laarschot et al., 2020). The recent decline in N-BP prescription, reflecting diminished patient acceptance of these drugs, has been linked to a statistical rise in bone-related complications (Williams et al., 2015). Due to the long-lasting half-life of N-BPs once chemisorbed to bone, the discontinuation of N-BPs and switching to other antiresorptive medications may still pose a risk of developing BRONJ (Anagnostis et al., 2017). It is therefore urgent to reduce the BRONJ risk associated with these otherwise effective medications for pathological osteolysis. The mechanism of BRONJ is not yet fully understood (Chang et al., 2018). BRONJ exclusively occurs in the jawbone, which uniquely associated with the gingival oral barrier tissue with one of the most active barrier immunities (Moutsopoulos and Konkel, 2018). The presence of N-BP on the jawbone is shared by all BRONJ patients (Lo Faro et al., 2019) and it is conceivable that jawbone N-BP may uniquely interact with oral barrier immunity. The major challenge has been the difficulty in separating the systemic and oral effects of N-BP therapy, which has severely hindered the elucidation of the mechanisms of BRONJ. N-BP chemisorption to bone results from its high affinity to hydroxyapatite (HAp), the mineral component of bone (Nancollas et al., 2006). Recent investigations in our laboratories revealed that pre-adsorbed N-BPs can be displaced from HAp by exposure to a second N-BP/BP in aqueous buffer both in vitro and in vivo (Hokugo et al., 2013; Hokugo et al., 2019). This led us to envision a new experimental model based on the selective removal of pharmacologically active legacy N-BP locally from jawbone by intra-oral application of a second BP with low pharmacological potency (lpBP). Here, we describe a liposome-based deformable nanoscale vesicle (DNV) formulation for topical trans-oral mucosa delivery of an lpBP to the underlying jawbone, and report its effect in attenuating N-BP-induced oral lesions using a murine BRONJ model and the elucidation of the mechanism involved in this pathology. Results Hydroxymethylene diphosphonate (HMDP, also known as hydroxymethylene bisphosphonate) was selected as the lpBP N-BPs such as zoledronate (ZOL) act on osteoclasts by inhibiting farnesyl diphosphate synthase (FPPS), thus interfering with the mevalonate pathway and protein prenylation (Russell, 2007). The nitrogen-containing side chain of N-BPs, an essential pharmacophore in all potent N-BP antiresorptive drugs, is absent in HMDP which, however, retains an α-OH group together with two phosphonate groups, conferring high affinity to HAp (Figure 1A). Thus, we postulated that HMDP in solution might be able to decrease the amount of legacy N-BP at the jaw bone surface by a competitive displacement mechanism as we reported (Hokugo et al., 2013; Hokugo et al., 2019). ZOL IV injection to mice increased femur trabecular bone volume over the saline vehicle solution injection control, whereas HMDP IV injection with the same dose did not alter the bone architecture (Figure 1B and C). The lack of pharmacological effect of HMDP in vivo was consistent with our previous study using a standard in vitro prenylation assay (Hokugo et al., 2019) and confirmed HMDP to be an lpBP. Figure 1 Download asset Open asset Competitive equilibrium-based dissociation of N-BP by low potency BP (lpBP). (A) Chemical structures (shown as the tetraacids) of N-BP, zoledronate (ZOL) and hydroxymethylene diphosphonate (HMDP). (B) ZOL but not HMDP affects mouse femur trabecular bone architecture. Mice received a bolus intravenous injection of 100 µl ZOL (40 nmol), HMDP (40 nmol) or vehicle saline (0.9% NaCl) solutions. Femurs (n=4 per group) were harvested 3 weeks after the IV injection and subjected to Micro-CT imaging. (C) HMDP did not affect the femur trabecular bone micro-architecture, whereas ZOL increased bone volume over total volume (BV/TV), connectivity density (ConnD) and trabecular thickness (Tb.Th). Trabecular number (Tb.N) and trabecular separation (Tb.Sp) measurements were not affected by ZOL. (Figure 1—source data 1) (D) In vitro demonstration of competitive displacement of legacy N-BP. Synthetic apatite-coated wells preincubated with 10 µM of 5-carboxyfluorescein-conjugated zoledronate (FAM-ZOL) were washed with MilliQ-treated pure water (MQW), then treated with 10 µM HMDP once (1×) or twice (2×) (n=3 per group). The FAM fluorescent signal measurement indicated significant reduction of the FAM-ZOL amount on the synthetic apatite. (Figure 1—source data 2) (E) Fluorescent signal measurement of the wash solutions from the in vitro experiment in (D) demonstrated removal of FAM-ZOL by the HMDP treatments (arrows). (Figure 1—source data 3) (F) In vitro osteoclastic pit formation assay. Synthetic apatite coated wells were preincubated with 10 µM ZOL followed by 10 µM HMDP treatment 1× or 2× (n=3 per group). RAW264.7 cells (2.5×104 cells per well) were then inoculated to each well in culture medium supplemented by mouse recombinant receptor activator of nuclear kappa-B ligand (RANKL). The areas of resorption pits generated by osteoclasts derived from RAW 264.7 cells were measured after 6 days of incubation and cell removal. Twice repeated HMDP treatments restored normal in vitro resorption pit formation. (Figure 1—source data 4) In (D) and (F), the graphs show the mean and SD (n=3 per group), and the Turkey test was used to analyze multiple samples. The statistical significance was determined to be at p<0.05. In (E), the graphs show the mean and SD (n=3 per group), and the Turkey test was used to analyze multiple samples within each time point. The statistical significance was determined to be at p<0.05. Different letters (e.g., a, b) are used to show statistically significant differences between multiple groups. Figure 1—source data 1 Source data of Figure 1C. https://cdn.elifesciences.org/articles/76207/elife-76207-fig1-data1-v3.pdf Download elife-76207-fig1-data1-v3.pdf Figure 1—source data 2 Source data of Figure 1D. https://cdn.elifesciences.org/articles/76207/elife-76207-fig1-data2-v3.pdf Download elife-76207-fig1-data2-v3.pdf Figure 1—source data 3 Source data of Figure 1E. https://cdn.elifesciences.org/articles/76207/elife-76207-fig1-data3-v3.pdf Download elife-76207-fig1-data3-v3.pdf Figure 1—source data 4 Source data of Figure 1F. https://cdn.elifesciences.org/articles/76207/elife-76207-fig1-data4-v3.pdf Download elife-76207-fig1-data4-v3.pdf Next, we tested the ability of HMDP to displace the pre-adsorbed N-BP from bone mineral surface. Synthetic apatite (carbonate apatite)-coated culture wells were pretreated with fluorescently tagged ZOL (FAM-ZOL: 10 µM). After thorough washing to remove non-chemisorbed FAM-ZOL, the wells were challenged by application of HMDP (10 µM in Milli-Q treated pure water: MQW). FAM-ZOL on the synthetic apatite was reduced by half (Figure 1D), and the wash solution contained a signal of dissociated FAM-ZOL (Figure 1E). After the second HMDP application, the amount of FAM-ZOL on the synthetic apatite was further reduced (Figure 1D). This experiment confirmed that a chemisorbed N-BP could be displaced by repeated applications of HMDP (Figure 1D and E). We further examined the effect of competitive removal of ZOL on osteoclastic bone resorption in vitro. ZOL chemisorbed on synthetic apatite inhibited osteoclastic bone resorption in vitro as expected, which was measured by the decreased resorption pit area created by RAW 264.7-derived osteoclasts (Figure 1F). A single application of HMDP increased the pit area, albeit at no statistical significance. When HMDP was applied twice, the resorption pit size significantly increased and reached the level of ZOL-untreated blank wells (Figure 1F). Although the FAM-ZOL experiment suggested that ZOL would not completely be removed after two applications of HMDP, osteoclastic activity was restored to a nearly normal level as ZOL-untreated control group (Figure 1F). We postulate that there may be a threshold bioavailable concentration of N-BP causing osteoclast abnormality. Therefore, our goal is not to be complete removal of legacy N-BP from the jawbone, but rather to decrease the local N-BP concentration below a BRONJ-triggering threshold. Preparation of the HMDP-deformable nanoscale vesicle (DNV) formulation In our previous proof-of-concept experiment, HMDP directly injected into mouse palatal gingiva in ZOL-pretreated mice prior to the maxillary first molar extraction was shown to prevent the development of BRONJ (Hokugo et al., 2019). BRONJ lesions exhibit ulcerative gingival tissue exposing necrotic jawbone. Because injection into pliable oral tissue will be challenging in some cases, and is a more focal delivery, we designed a formulation of HMDP enabling the compound to penetrate through the oral mucosa epithelial layer, making topical delivery to the jawbone possible. Liposomes have often been used as a drug carrier for controlled delivery to enhance drug concentrations in targeted tissues and to achieve therapeutic effects using minimum drug doses (Abu Lila and Ishida, 2017). DNV comprises a modified liposome drug carrier (Figure 2A) synthesized by a controlled microfluidics system, which due to nanovesicle deformability allows penetration through the keratinized epithelial layer of skin (Subbiah et al., 2017). Figure 2 Download asset Open asset Manufacturing and trans-oral mucosal penetration evaluation of hydroxymethylene diphosphonate (HMDP)-DNV and AF647-ZOL-DNV. (A) Diagram of deformable nanoscale vesicles (DNV), a liposome derivative. (B) Flow diagram of micro-fluidics based DNV synthesis. (C) Confocal laser scanning microscopy of AF647-ZOL-DNV. Approximately 100–200 nm DNV particles exhibited an AF647 signal. (D) Characterization of DNV formulations. (E) Protocol for intra-oral topical application to mouse palatal tissue. The reconstituted DNV solution in MQW (3 µl) was topically applied to the palatal gingiva between maxillary molar teeth and covered by a custom-made oral appliance fabricated with auto-polymerizing dental resin. After 1 hr, the oral appliance was removed. (F) After topical application of AF647-ZOL-DNV, mouse maxillary bones were harvested and AF647 fluorescence was measured. The AF647 fluorescent signal from the maxillary bone region of interest (ROI) increased with up to 75 µM AF7647-ZOL in DNV applied and then reached a plateau. (Figure 2—source data 1) (G) AF647-ZOL-DNV and AF647-ZOL in non-deformable formulation (AF647-ZOL-nDNV) were reconstituted in either MQW or 20% polyethylene glycol (PEG). AF647-ZOL-DNV in MQW most efficiently delivered the drug to the maxillary bone through trans-oral mucosal route. (Figure 2—source data 2) In (F) and (G), the graphs show the mean and SD (n=6 per group), and the Turkey test was used to analyze multiple samples. The statistical significance was determined to be at p<0.05. Different letters (e.g., a, b) are used to show statistically significant differences between multiple groups. Figure 2—source data 1 Source data of Figure 2F. https://cdn.elifesciences.org/articles/76207/elife-76207-fig2-data1-v3.pdf Download elife-76207-fig2-data1-v3.pdf Figure 2—source data 2 Source data of Figure 2G. Download HMDP-DNV HMDP in the aqueous and by a of and (Figure is a that DNV HMDP-DNV was through a for followed by and (Figure DNV a ZOL et al., was (Figure to trans-oral mucosa drug delivery in We also synthesized nanoscale vesicle for as a The BP DNV was by size and surface potential (Figure 1D), which are reported to in liposome drug delivery et al., et al., 2018). The size for this has been shown to be between 100 nm and nm (Subbiah et al., 2017). After through the gingival and oral mucosa epithelial DNV is to HMDP to the bone mineral which is in solution The of clinical N-BPs to HAp and effect at the HAp surface on the potential have been (Nancollas et al., et al., 2017). the potential of HAp is and by ZOL. However, HAp which is in to HMDP and HMDP, a side a potential surface Therefore, DNV with a surface potential was selected to jawbone to the The drug delivery through mouse palatal gingiva was designed to HMDP-DNV for 1 application, the mouse was covered by a custom-made oral appliance using dental to from and (Figure In the AF647-ZOL-DNV was in MQW at concentrations 75 and and 3 µl of the AF647-ZOL-DNV solution was applied to the mouse palatal gingival tissue. mouse the palatal bone were examined for fluorescent signal (Figure AF647 fluorescent signal at the maxillary bone increased with up to an applied concentration of 75 µM of AF647-ZOL and then reached a plateau. We then AF647-ZOL-DNV and which were reconstituted in MQW or 20% polyethylene glycol (PEG). The AF647-ZOL-DNV reconstituted in MQW revealed the fluorescent indicating AF647-ZOL delivery to the jawbone in vivo (Figure This study also demonstrated that was effective in trans-oral mucosa delivery of AF647-ZOL to the jawbone. of a new mouse HMDP-DNV applications prior to dentoalveolar in mice mice weeks were treated by a bolus ZOL intravenous injection from the Control mice received a vehicle solution IV after the ZOL or vehicle the maxillary first molar was (Figure wound healing of the control group was and the open gingival wound was 1 after the tooth the tooth extraction wound of the group was which was by a gingival open wound 4 weeks (Figure Figure 3 with 2 all Download asset Open asset Disease of mouse and human BRONJ (A) for a bisphosphonate-related osteonecrosis of the jaw (BRONJ) lesion in Mice received a bolus IV injection of zoledronate (ZOL) (40 or vehicle saline solution were subjected to maxillary first molar (B) that mouse pretreated with ZOL IV injection exhibited delayed wound healing with open wound and gingival (C) BRONJ lesions with open tooth extraction wound and gingival (D) Micro-CT of mouse that delayed bone in the and palatal extraction in ZOL-pretreated a of BRONJ (E) demonstration of tooth extraction of human BRONJ. (F) evaluation of mouse BRONJ lesion with osteonecrosis gingival inflammation and epithelial to the necrotic bone (G) A of human BRONJ lesion with osteonecrosis and gingival epithelial A time experimental diagram of hydroxymethylene diphosphonate nanoscale vesicles (DNV) mice received ZOL IV HMDP in MQW 3 of or HMDP-DNV in MQW 3 of was topically applied to the palatal gingiva prior to the maxillary first molar Micro-CT evaluation of tooth extraction which in the treated with or HMDP topical applications of HMDP-DNV prior to the tooth extraction significantly increased tooth extraction socket bone to and HMDP (Figure data 1) topical applications of HMDP-DNV prior to the tooth extraction reduced the development of osteonecrosis to the treatment of and HMDP which to exhibit BRONJ (Figure data 2) In and the graphs show the mean and SD (n=3 per control group and per experimental group), and the Turkey test was used to analyze multiple samples. The statistical significance was determined to be at p<0.05. Different letters (e.g., a, b) are used to show statistically significant differences between multiple groups. Figure data 1 Source data of Figure Download Figure data 2 Source data of Figure Download The clinical of BRONJ is an oral wound with the exposed jawbone or the development of to the jawbone surface (Figure defined MRONJ BRONJ as the oral wound for weeks in human patients (Ruggiero et al., to the normal oral wound healing such as tooth extraction, which would be within this The tooth extraction wound in mice in 3 which is et al., et al., 2015). However, we that the conventional mouse often delayed the tooth extraction wound healing due to in the extraction socket (Hokugo et al., 2019). the mouse tooth extraction model by after tooth extraction for 1 we that the tooth extraction wound was as as 1 (Hokugo et al., 2019). this mouse tooth extraction the BRONJ is as open oral wound for 1 or after tooth The clinical is by using oral The of open oral wound is as the of with wound in each group at a given healing After tooth extraction, the socket a wound healing resulting in bone Micro-CT is a method for We have established a method to the bone volume in the mouse tooth extraction socket using Micro-CT (Figure 1). BRONJ delayed the bone process and often represented as tooth extraction socket in ZOL pretreated mice (Figure A recent reported the of for BRONJ et al., which exhibited with extraction and lesion (Figure The development of osteonecrosis is the of BRONJ In the mouse the of harvested maxillary tissues was The tooth extraction site of mice exhibited a area of osteonecrosis defined by a of The gingival tissue to the necrotic bone cell associated with epithelial (Figure The bone from human BRONJ patients demonstrated the as the of osteonecrosis and associated with epithelial (Figure reported as et al., 2014). the mouse BRONJ the effect of topical application of HMDP-DNV was examined to it would the BRONJ In this HMDP in MQW or HMDP-DNV in MQW were applied topically to the palatal tissue of mice pretreated with ZOL IV injection prior to the maxillary first molar extraction (Figure time application of HMDP and HMDP-DNV did not affect the delayed tooth extraction wound healing in Micro-CT and (Figure Therefore, we increased the HMDP dose by twice topical applications prior to the tooth After 2 topical the HMDP-DNV treated group increased bone at the level of the no ZOL-pretreated control whereas applications of and HMDP no significant effect (Figure of the 2 group normal extraction wound The 2 and 2 revealed bone of a BRONJ lesion (Figure The area of osteonecrosis in the maxillary bone in the 2 application group was significantly reduced (Figure such as tooth extraction have been reported as a risk to ONJ in patients with a history of N-BP (Ruggiero et al., 2014). The results that the twice repeated applications of