Inflammation Can Be a High-Risk Factor for Mucosal Nonunion of MRONJ by Regulating SIRT1 Signaling When Treated with an Oncologic Dose of Zoledronate.

Purpose: Zoledronate (ZA) stands as a highly effective antiresorptive agent known to trigger medication-related osteonecrosis of the jaw (MRONJ). Its clinical dosages primarily encompass those used for oncologic and osteoporosis treatments. While inflammation is recognized as a potential disruptor of mucosal healing processes associated with ZA, prior research has overlooked the influence of varying ZA dosages on tissue adaptability. Therefore, a deeper understanding of the specific mechanisms by which inflammation exacerbates ZA-induced MRONJ, particularly when inflammation acts as a risk factor, remains crucial. Methods: Cell proliferation and migration of human oral keratinocytes (HOK) was analyzed after treatment with different doses of ZA and/or lipopolysaccharide (LPS) to assess their possible effect on mucosal healing of extraction wounds. Mouse periodontitis models were established using LPS, and histological changes in extraction wounds were observed after the administration of oncologic dose ZA. Hematoxylin and eosin (HE) staining and immunofluorescence were used to evaluate mucosal healing. Results: In vitro, LPS did not exacerbate the effects of osteoporosis therapeutic dose of ZA on the proliferation and migration of HOK cells, while aggravated these with the oncologic dose of ZA treatment by inducing mitochondrial dysfunction and oxidative stress via regulating SIRT1 expression. Furthermore, SIRT1 overexpression can alleviate this process. In vivo, local injection of LPS increased the nonunion of mucous membranes in MRONJ and decreased the expression of SIRT1, PGC-1α, and MnSOD. Conclusion: Inflammation aggravates oncologic dose of ZA-induced mitochondrial dysfunction and oxidative stress via a SIRT1-dependent pathway, enhancing the risk of impaired mucosal healing in MRONJ. Our study implies that inflammation becomes a critical risk factor for MRONJ development at higher ZA concentrations. Elucidating the mechanisms of inflammation as a risk factor for mucosal non-healing in MRONJ could inform the development of SIRT1-targeted therapies.

Local E-rhBMP-2/ß-TCP Application Rescues Osteocyte Dendritic Integrity and Reduces Microstructural Damage in Alveolar Bone Post-Extraction in MRONJ-like Mouse Model.

The pathology of medication-related osteonecrosis of the jaw (MRONJ), often associated with antiresorptive therapy, is still not fully understood. Osteocyte networks are known to play a critical role in maintaining bone homeostasis and repair, but the exact condition of these networks in MRONJ is unknown. On the other hand, the local application of E-coli-derived Recombinant Human Bone Morphogenetic Protein 2/ß-Tricalcium phosphate (E-rhBMP-2/ß-TCP) has been shown to promote bone regeneration and mitigate osteonecrosis in MRONJ-like mouse models, indicating its potential therapeutic application for the treatment of MRONJ. However, the detailed effect of BMP-2 treatment on restoring bone integrity, including its osteocyte network, in an MRONJ condition remains unclear. Therefore, in the present study, by applying a scanning electron microscope (SEM) analysis and a 3D osteocyte network reconstruction workflow on the alveolar bone surrounding the tooth extraction socket of an MRONJ-like mouse model, we examined the effectiveness of BMP-2/ß-TCP therapy on the alleviation of MRONJ-related bone necrosis with a particular focus on the osteocyte network and alveolar bone microstructure (microcrack accumulation). The 3D osteocyte dendritic analysis showed a significant decrease in osteocyte dendritic parameters along with a delay in bone remodeling in the MRONJ group compared to the healthy counterpart. The SEM analysis also revealed a notable increase in the number of microcracks in the alveolar bone surface in the MRONJ group compared to the healthy group. In contrast, all of those parameters were restored in the E-rhBMP-2/ß-TCP-treated group to levels that were almost similar to those in the healthy group. In summary, our study reveals that MRONJ induces osteocyte network degradation and microcrack accumulation, while application of E-rhBMP-2/ß-TCP can restore a compromised osteocyte network and abrogate microcrack accumulation in MRONJ.

Photodynamic therapy repairs medication-related osteonecrosis of the jaw by reducing NF-kB protein in rats.

OBJECTIVE: To evaluate whether antimicrobial photodynamic therapy (aPDT) repairs bisphosphonate-related osteonecrosis of the jaw (BRONJ) modulated by the reduction of NF-kB protein in a murine model. METHODOLOGY: Male Wistar rats (N=30) were divided into the following groups (n=6/group): negative control (NC); experimental osteonecrosis (ONE); ONE + photosensitizer (PS); ONE + photobiomodulation (PBM); and ONE + aPDT. Over 8 weeks, ONE was induced by zoledronic acid 250 µg/kg injections, except in the NC group, which received sterile 0.9% saline, followed by extraction of the lower left first molar. Red light laser irradiation (wavelength ~660 nm, power 50 mW, energy of 2 J, energy dose of 66.67 J/cm2 for 40 s) was performed once a week for 4 weeks. Methylene blue 0.3% was used as PS. The animals were euthanized and examined macroscopically for the presence of exposed bone and epithelial repair and microscopically by histochemical (hematoxylin-eosin and Masson's trichrome staining) and immunohistochemical (anti-NF-kB) methods. Macroscopic and histomorphometric data were analyzed by one-way ANOVA and Tukey's post-test (p<0.05). RESULTS: Mucosal repair, viable osteocytes, and NF-kB immunostaining were observed in the NC, ONE+PS, ONE+PBM, and ONE+aPDT groups. The ONE group showed no mucosal repair, showing empty lacunae and multifocal immunostaining for NF-kB. The ONE+PBM and ONE+aPDT groups had greater deposition of extracellular matrix and less necrotic bone tissue (p<0.05). CONCLUSION: PBM and aPDT treatments for BRONJ were effective for bone and epithelial repair, in addition to reducing inflammation mediated by the decrease of NF-kB protein in the irradiated regions.

Novel Histopathological Findings of Micro Bone Fragments and Epithelial Response in the Oral Mucosa in Bisphosphonate-Related Osteonecrosis of the Jaw.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) occurs in the jawbone and interfacing oral mucosa of patients treated with bisphosphonates. Herein, we report novel histopathological findings in the oral mucosa of a surgical specimen obtained from a 61-year-old man with BRONJ. The resected jawbone and adjacent oral mucosa were separated for histological examination. The mucosal tissue was examined using Von Kossa staining and immunohistochemical (CK5/6, p63) staining of non-decalcified paraffin sections. Pseudoepitheliomatous hyperplasia (PEH), a microscopic feature of the mucosal epithelium in BRONJ, was observed in soft tissue specimens, concomitant with inflammatory cell infiltration. Von Kossa staining revealed small fragments of necrotic bone, tens to hundreds of micrometers in size, scattered within the connective tissues; the PEH forefront contacted some of the bone fragments. Immunohistochemical staining demonstrated that occasionally, the PEH not only contacted but also encompassed the bone fragments. To our knowledge, this is the first report of presence of micro bone fragments and their association with PEH in the oral mucosa in BRONJ.

Polysacharide of Agaricus blazei gel mitigates bone necrosis in model of the jaws related to bisphosphonate via Wnt signaling.

To investigate de effect of PAb gel on the bone tissue of rats submitted to Bisphosphonate-related osteonecrosis of the jaws (BRONJ). Initially, 54 animals were submitted to BRONJ model by Zoledronic Acid (ZA) (0.1 mg/kg 3x/wk for 9 wk, ip), followed by the 1st upper left molar extraction at the 8th wk. After tooth removal, the animals were divided into 3 groups, ZA that received placebo gel or PAb gel that received 1% PAb gel, inside the dental alveolus. The control Group (CONTROL) received 0.1 mg/kg of 0.9% saline and then placebo gel. Three weeks after tooth extraction, the animals were euthanized, and maxillae were colleted for macroscopic, radiographic, histological and Raman spectomery assays. Additionally, GSK3b, beta-catenin, and Runx2 mRNA expressions were determined. Blood samples were collected for the analysis of Bone-specific alkaline phosphatase (BALP) levels. PAb gel improved mucosal healing, increased the number of viable osteocytes, while it reduced the number of empty lacunae, as well as the amount of bone sequestration. Furthermore, PAb gel positively influenced the number and functionality of osteoblasts by stimulating Wnt signaling, thereby inducing bone remodeling. Additionally, PAb gel contributed to improved bone quality, as evidenced by an increase in bone mineral content, a decrease in bone solubility, and an enhancement in the quality of collagen, particularly type I collagen. PAb gel mitigated bone necrosis by stimulating of bone remodeling through Wnt signaling and concurrently improved bone quality. PAb gel emerges as a promising pharmacological tool for aiding in BRONJ therapy or potentially preventing the development of BRONJ.

The Preventive Effect of Preoperative and Postoperative Selenium on the Medication-Related Osteonecrosis of the Jaw: An Animal Study in Rats.

BACKGROUND: Medication-related osteonecrosis of the jaw (MRONJ) is a condition that can occur primarily in patients undergoing or have previously undergone therapy with bisphosphonates, particularly in the presence of risk factors, such as tooth extraction (TE). PURPOSE: This study aimed to evaluate the effect of selenium (SEL) administration on the prevention of osteonecrosis of the jaw in an MRONJ animal model. STUDY DESIGN, SETTING, AND SAMPLE: This study was a longitudinal in vivo animal study using a TE model in a sample of 48 Wistar rats. PREDICTOR VARIABLE: The predictor variables were SEL exposure, timing of SEL exposure, and zoledronic acid (ZOL) exposure. The animals were randomly assigned to 4 treatment groups (n = 12 per group): 1) saline (negative control), 2) ZOL (positive control), 3) SELpreop + ZOL, and 4) ZOL + SELpostop. The animals were administered saline (negative control) or ZOL (0.06 mg/kg, intraperitoneally) once a week for 5 weeks. All rats underwent TE at the end of the fifth week. SEL (0.3 mg/kg, intraperitoneally) was administered once daily for 15 days to the SELpreop + ZOL group before TE and to the ZOL + SELpostop group after TE. All animals were sacrificed at the end of the ninth week. MAIN OUTCOME VARIABLES: The primary outcome variables were new bone area, necrotic bone area, fibrosis, new connective tissue formation, and inflammatory cell infiltration in the histopathological analysis, as well as angiogenesis and percentage of osteoblasts in the immunohistochemical analysis. COVARIATES: There was none. ANALYSES: Statistical analysis was conducted using the Kruskal-Wallis test, followed by post hoc Bonferroni-corrected Mann-Whitney U tests, with a significance level of P ≤ .05. RESULTS: The new bone area was higher in the ZOL + SELpostop group (3.00 score) than in the saline group (0.58 ± 1.08 score, P < .001) and the ZOL group (0.82 ± 1.40 score, P = .001), while the necrotic bone area was lower in the ZOL + SELpostop group (0.08 ± 0.29 score) than in the ZOL group (2.82 ± 0.40 score, P < .001) and the SELpreop + ZOL group (1.67 ± 0.89 score, P = .007). The percentage of osteoblasts was higher in the ZOL + SELpostop group (18.73%) than in the saline group (8.63%, P < .001) and the ZOL group (0.07%, P < .001), and it was also higher in the SELpreop + ZOL group (18.49%) than in the ZOL group (0.07%, P < .001). CONCLUSION AND RELEVANCE: In conclusion SEL prevents MRONJ, with postoperative SEL demonstrating greater prevention effects. Given these findings, we hypothesize that SEL exposure may decrease the risk of MRONJ.

Comparative clinical study of phosphorous necrosis and medical-related osteonecrosis of the jaws.

BACKGROUND: Phosphorous necrosis of the jaw (PNJ) exhibits similar clinical and pathological features as medical-related osteonecrosis of the jaw (MRONJ). This study aims at comparing the similarities and differences between PNJ and MRONJ regarding pathological features and to provide a theoretical basis for the clinical diagnosis and management of PNJ. MATERIAL AND METHODS: A retrospective analysis was conducted to assess clinical differences among 38 PNJ patients and 31 MRONJ patients, who were diagnosed and treated between January 2009 and October 2022. Pathological alterations in bone tissue were evaluated using EDS, H&E, Masson, and TRAP staining on five specimens from both MRONJ and PNJ cases; furthermore, immunohistochemistry was used to determine the expression levels of OPG, RANKL, and Runx2. The mandibular coronoid process was removed from individuals with temporomandibular joint ankylosis to serve as a control. RESULTS: CBCT imaging demonstrated necrotic bone formation in block, strip, or plaque shapes. EDS analysis showed that the calcium/phosphorus ratio in the bone tissue of PNJ and MRONJ was significantly lower than that of the control group (P < 0.05). Additionally, staining indicated reduced osteoblast counts, disrupted bone trabecular structure, and decreased collagen fiber content in the bone tissues of PNJ and MRONJ. Immunohistochemistry demonstrated that RANKL expression was significantly lower in MRONJ compared to PNJ and control groups (P < 0.05). Conversely, Runx2 expression was significantly higher in PNJ than in MRONJ and control groups (P < 0.05), and there was no significant difference in OPG expression. CONCLUSION: PNJ and MRONJ demonstrate comparable clinical manifestations and pathological traits, although disparities may exist in their underlying exhibit comparable clinical manifestations, pathological traits, and molecular mechanisms.

Zoledronate treatment exerts sex-independent effects on bone and dental physicochemical properties in mice jaw necrosis.

INTRODUCTION: Bisphosphonate (BF) therapy is strongly related to the occurrence of medication-related osteonecrosis of the jaw (ONJ). However, no previous study has evaluated if there are sex-related differences on the ONJ establishment together with bone biomechanical alterations, and if they could have a synergy with the ZA treatment. MATERIALS AND METHODS: This study aimed to analyze the physicochemical properties of mineralized tissues in a zoledronate (ZA)-related osteonecrosis mouse model, by a 2 × 2-factorial design, considering sex (female/male) and treatment (ZA/Saline) factors (n = 8/group). After three ZA (1.0 mg/kg) or saline administrations (days 0, 7, 14), the lower left second molar was extracted (day 42). Further ZA administration (day 49) and euthanasia (day 70) were conducted. After confirmation of ZA-induced jaw necrosis (histologic and microtomographic analysis), spectroscopic and mechanical parameters were assessed. RESULTS: ZA-treated groups presented lower bone density due to impaired healing of tooth extraction socket. Sex-related alterations were also observed, with lower bone density in females. Regarding biomechanical parameters, sex and treatment exerted independent influences. ZA, although decreasing flexural modulus and yield stress, increases stiffness mainly due to a higher bone volume. Females show less resistance to higher loads compared to males (considering dimension-independent parameters). Additionally, ZA increases crystallinity in bone and dental structure (p < 0.05). In summary, although strongly related to osteonecrosis occurrence, ZA modifies bone and dental mineral matrix, improving bone mechanical properties. CONCLUSION: Despite sex-dependent differences in bone biomechanics and density, osteonecrosis was established with no sex influence. No synergistic association between sex and treatment factors was observed in this study.

Maximum and mean standardized uptake values of medication-related osteonecrosis of the jaw with bone SPECT/CT: comparison of mandibular pathologies, control and temporomandibular joints.

OBJECTIVE: Recently, single-photon emission CT/CT (SPECT/CT) plays an important role in assessing patients with medication-related osteonecrosis of the jaw (MRONJ). The aim of this study was to investigate maximum and mean standardized uptake values (SUVs) of MRONJ with bone SPECT/CT, especially comparison of mandibular pathologies, control and temporomandibular joints. METHODS: 61 mandibular patients with MRONJ who underwent bone SPECT/CT were included in this study. The maximum and mean SUVs of the lesion, right and left sides of the lesion, opposite side of the lesion as control, right and left temporomandibular joints were analyzed using a workstation and software. The SUVs of MRONJ were analyzed using one-way analysis of variance with Tukey's honestly significant difference test. Patient characteristics with MRONJ and SUVs were analyzed using Mann-Whitney U test. P-values less than 0.05 were considered to indicate statistical significance. RESULTS: The maximum and mean SUVs for opposite side of the lesions (4.4 ± 2.0 and 1.8 ± 0.7) were significantly lower than those for mandibular lesions (18.3 ± 8.1 and 6.3 ± 2.8), right side of the lesions (8.1 ± 3.9 and 2.9 ± 1.3) and left side of the lesions (8.1 ± 3.9 and 2.8 ± 1.4), respectively. The maximum and mean SUVs for right and left sides of the lesions, and opposite side of the lesions, right and left temporomandibular joints were not significant difference. Furthermore, the maximum SUVs of the mandibular lesions were a significant difference for age and staging. CONCLUSIONS: The maximum and mean SUVs with SPECT/CT can be useful in the quantitative management of MRONJ patients.

Radiologic findings of osteonecrosis, osteoradionecrosis, osteomyelitis and jaw metastatic disease with cone beam CT.

PURPOSE: The purpose of this study was to assess CBCT scans of patients with medication related osteonecrosis of the jaws (MRONJ), osteoradionecrosis (ORN), osteomyelitis (OM) and jaw metastatic disease (JM), evaluate the presence and extent of radiologic findings, identify radiologic parameters that may distinguish the four entities and last, introduce a new modified radiographic index (CRIm), in order to contribute to the diagnosis of these conditions. METHODS: Τwo major databases were retrospectively searched for fully documented and diagnosed CBCT scans of MRONJ, ORN, OM and JM from 2006 to 2019. 335 CBCT scans met the inclusion criteria and were assessed under standardized viewing conditions blindly by 2 observers. The CRIm index proposed in this study evaluates: lytic changes, sclerosis, periosteal bone formation, sequestration, non-healing extraction sockets and other findings which included: sinus implication, inferior alveolar canal implication and jaw fracture. Lytic changes, sclerosis, periosteal bone formation, sequestration and non-healing extraction sockets were scored as: absent (0), localized/single (1) and extensive/multiple (2). Each one of other findings were scored individually as: absent (0) and present (1). For statistical analysis t-test, Pearson's r correlation coefficient, one-way ANOVA and Bonferonni were performed. RESULTS: Extensive lytic changes were the most common finding, especially for ORN, where it occurred in all CBCT scans (100%). The mean value of the CRIm index differs significantly between CBCT scans with MRONJ and JM, as well as between those with OM and JM (Bonferroni p < 0.001). CONCLUSIONS: The new modified Composite Radiographic Index introduced in this study, appears to have improved an objective approach to the previously used Composite Radiographic Index by means of cumulative radiologic features. Τhe predominance of certain radiologic features in one or more of these entities may lead the diagnostician towards the correct diagnosis.

Alterations in macrophage polarization in the craniofacial and extracranial skeleton after zoledronate application and surgical interventions - an in vivo experiment.

Purpose: Medication-related osteonecrosis occurs exclusively in the jaw bones. However, the exact pathogenesis of medication-related osteonecrosis of the jaw (MRONJ) and the unique predisposition of the jaw bones have not been elucidated, making its treatment a challenge. Recent evidence indicates that macrophages might play a pivotal role in MRONJ pathogenesis. The aim of the present study was to compare the macrophage populations between the craniofacial and extracranial skeleton and to investigate the changes induced by zoledronate (Zol) application and surgical interventions. Materials and methods: An in vivo experiment was performed. 120 wistar rats were randomized to 4 groups (G1, G2, G3, G4). G1 served as an untreated control group. G2 and G4 received Zol injections for 8 weeks. Afterwards, the right lower molar of the animals from G3 and G4 was extracted and the right tibia osteotomized followed by osteosynthesis. Tissue samples were taken from the extraction socket and the tibia fracture at fixed time points. Immunohistochemistry was conducted to determine the labeling indexes of CD68+ and CD163+ macrophages. Results: Comparing the mandible and the tibia, we observed a significantly higher number of macrophages and a heightened pro-inflammatory environment in the mandible compared to the tibia. Tooth extraction caused an increase of the overall number of macrophages and a shift toward a more pro-inflammatory microenvironment in the mandible. Zol application amplified this effect. Conclusion: Our results indicate fundamental immunological differences between the jaw bone and the tibia, which might be a reason for the unique predisposition for MRONJ in the jaw bones. The more pro-inflammatory environment after Zol application and tooth extraction might contribute to the pathogenesis of MRONJ. Targeting macrophages might represent an attractive strategy to prevent MRONJ and improve therapy. In addition, our results support the hypothesis of an anti-tumoral and anti-metastatic effect induced by BPs. However, further studies are needed to delineate the mechanisms and specify the contributions of the various macrophage phenotypes.

Dietary nitrate improves jaw bone remodelling in zoledronate-treated mice.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication that occurs in patients with osteoporosis or metastatic bone cancer treated with bisphosphonate. There is still no effective treatment and prevention strategy for BRONJ. Inorganic nitrate, which is abundant in green vegetables, has been reported to be protective in multiple diseases. To investigate the effects of dietary nitrate on BRONJ-like lesions in mice, we utilized a well-established mouse BRONJ model, in which tooth extraction was performed. Specifically, 4 mM sodium nitrate was administered in advance through drinking water to assess the short- and long-term effects on BRONJ. Zoledronate injection could induce severe healing inhibition of the tooth extraction socket, while addition of pretreating dietary nitrate could alleviate the inhibition by reducing monocyte necrosis and inflammatory cytokines production. Mechanistically, nitrate intake increased plasma nitric oxide levels, which attenuated necroptosis of monocytes by downregulating lipid and lipid-like molecule metabolism via a RIPK3 dependent pathway. Our findings revealed that dietary nitrate could inhibit monocyte necroptosis in BRONJ, regulate the bone immune microenvironment and promote bone remodelling after injury. This study contributes to the understanding of the immunopathogenesis of zoledronate and supports the feasibility of dietary nitrate for the clinical prevention of BRONJ.

Effect of tyrosine kinase inhibitor sunitinib on tissue repair at tooth extraction sites.

BACKGROUND: Both zoledronic acid, a potent bisphosphonate, and the antiangiogenic drug sunitinib are included in anticancer protocols and have also been associated with jaw osteonecrosis. Our aim was to compare the effect of these drugs on tissue repair at tooth extraction sites. METHODS: Wistar rats were allocated into four groups: (1) sunitinib; (2) sunitinib/zoledronic acid; (3) zoledronic acid; (4) control group. The animals underwent tooth extractions and maxillae were macro- and microscopically analyzed. RESULTS: On macroscopic evaluation, the zoledronic acid group showed a significantly higher frequency of oral mucosal lesion; lesions in the sunitinib/zoledronic acid group were larger, albeit not significantly so. The sunitinib/zoledronic acid group had significantly less epithelium than the zoledronic acid and control group, but showed no significant difference compared to the sunitinib group. The sunitinib/zoledronic acid and zoledronic acid groups did not differ from each other, but had significantly less connective tissue and more non-vital bone and microbial colonies than sunitinib and control groups, whereas these latter two groups did not significantly differ from each other. Vital bone and inflammatory infiltrate did not significantly differ between groups. CONCLUSION: Sunitinib alone is not associated with non-vital bone, whereas the sunitinib/zoledronic acid combination and zoledronic acid alone are.

Significance of bisphosphonates on angiogenesis in vivo and their effect under geranyl-geraniol addition - could it alter the treatment of bisphosphonate-associated necrosis of the jaw?

PURPOSE: The aim of this study was to contribute to the understanding of the inhibitory effects of bisphosphonates on tissues, with a special focus on angiogenesis. Referring to bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ), it should be shown that the local addition of the isoprenoid geranyl-geraniol (GGOH) prevents vascularization processes. METHODS: A mouse model with n = 24 animals which received an injection of a collagen matrix was used. In 4 subgroups (n = 6), we examined the effect of zoledronate on the sprouting of capillary-like structures into the matrix, with and without the presence of geranyl-geraniol, as well as testing against control groups with PBS injections or collagen matrix containing PBS instead of GGOH. This was followed by a histological evaluation of the capillary-like structures. RESULTS: Zoledronate inhibits the sprouting of blood vessels into a collagen matrix in vivo; in the presence of GGOH this effect is significantly weakened by a factor of 3.9 (p = 0.00068). CONCLUSION: This work commits to the investigation of the pathophysiology of BP-ONJ and shows a possible causal therapeutic path via the topical application of GGOH.

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

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.

Porphyromonas gingivalis Induces Bisphosphonate-Related Osteonecrosis of the Femur in Mice.

One of the most prominent characteristics of bisphosphonate-related osteonecrosis of the jaw(BRONJ) is its site-specificity. Osteonecrosis tends to occur specifically in maxillofacial bones, in spite of a systemic administration of the medicine. Previous studies suggested rich blood supply and fast bone turnover might be reasons for BRONJ. Yet, a sound scientific basis explaining its occurrence is still lacking. The present study aimed to explore the role of Porphyromonas gingivalis (P. gingivalis), an important oral pathogen, on the site-specificity of bisphosphonate-induced osteonecrosis and to elucidate its underlying mechanism. Mice were intraperitoneally injected with zoledronic acid (ZA) or saline for 3 weeks. In the third week, the right mandibular first molars were extracted and circular bone defects with a diameter of 1 mm were created in right femurs. After the operation, drug administration was continued, and P. gingivalis suspension was applied to the oral cavities and femur defects. The mice were killed after four or eight weeks postoperatively. The right mandibles and femurs were harvested for micro-CT and histological analyses. A poor healing of bone defects of both jaws and femurs was noted in mice injected with both ZA and P. gingivalis. Micro-CT analysis showed a decreased bone volume, and histological staining showed an increased number of empty osteocyte lacunae, a decreased collagen regeneration, an increased inflammatory infiltration and a decreased number of osteoclasts. In addition, the left femurs were collected for isolation of osteoclast precursors (OCPs). The osteoclastogenesis potential of OCPs was analyzed in vitro. OCPs extracted from mice of ZA-treated groups were shown to have a lower osteoclast differentiation potential and the expression level of related genes and proteins was declined. In conclusion, we established a mouse model of bisphosphonate-related osteonecrosis of both the jaw and femur. P. gingivalis could inhibit the healing of femur defects under the administration of ZA. These findings suggest that P. gingivalis in the oral cavity might be one of the steering compounds for BRONJ to occur.

Lansoprazole and zoledronate delays hard tissue healing of tooth extraction sockets in dexamethasone-treated mice.

Proton pump inhibitors (PPIs) are among the most commonly prescribed medicines for the management of acid-related gastrointestinal diseases. Osteonecrosis of the jaw (ONJ) is a serious adverse event that is associated with the use of antiresorptive and antiangiogenic agents. According to previous clinical reports, the use of PPIs contributes to the pathogenesis of severe ONJ that requires surgery. Here, we investigated the effects of lansoprazole (LP) or LP in combination with zoledronate (ZOL) on ONJ development in mice. C57BL/6J mice were administered ZOL (125 µg/kg intravenously, twice weekly) and/or LP (10 mg/kg intraperitoneally; 3 weeks of 3 consecutive days followed by 1 day off). One week after initiation of the study, the first molar was atraumatically extracted. Concurrently with ZOL administration, dexamethasone (Dex) was administered (5 mg/kg intraperitoneally, twice weekly). Micro-computed tomography and histological evaluation were performed to characterize femoral structures, tooth extraction sockets, and osteonecrosis areas. The results showed that ZOL/Dex significantly increased bone mass compared to saline/Dex, while the simultaneous administration of LP and ZOL/Dex diminished the ZOL-induced enhancement of bone mass. In the alveolar bone around the tooth extraction socket, necrotic bone was significantly increased in the LP/Dex group compared to the saline/Dex group. However, no signs of more severe ONJ-like lesions were observed following combined administration of LP and ZOL/Dex, other than an increase in the number of non-attached TRAP-positive cells. Our findings in a mouse model suggest that LP use can be a risk factor for the development of ONJ.

Are Bacteria Just Bystanders in the Pathogenesis of Inflammatory Jaw Conditions?

PURPOSE: It is unclear whether certain bacteria initiate the development of inflammatory jaw conditions, or whether these diseases create a milieu for dysbiosis and secondary colonization of indigenous flora. At present, there are no comparative studies on the types of bacteria that colonize different inflammatory jaw conditions. Accordingly, this study aims to identify and compare the types of bacteria isolated in osteomyelitis, osteoradionecrosis, and MRONJ. METHODS: This is a retrospective cohort study of patients diagnosed with inflammatory jaw conditions. The predictor variables were classification of bacteria as oral flora, categorized herein as resident bacteria, non-resident bacteria, or opportunistic organisms. The outcome variables were a diagnosis of osteomyelitis, osteoradionecrosis, and MRONJ. Covariates were age, sex, penicillin allergy, a diagnosis of diabetes and a history of smoking. Data analysis was performed using ANOVA and chi-squared tests. RESULTS: A total of 105 patients with inflammatory jaw conditions were enrolled. The final sample size was 69 subjects of which 16 were diagnosed with osteomyelitis, 20 with osteoradionecrosis, and 33 with MRONJ. There was no difference in the frequency that resident bacteria were isolated. Non-resident bacteria, which included Staphylococcus and Enterococcus among others, were isolated more frequently at 75% in osteomyelitis compared to 60% in osteoradionecrosis and 48% in MRONJ cases. There is weak evidence of significant difference when comparing osteomyelitis and MRONJ cases (P = .08). Opportunistic organisms, which included Mycobacterium and Candida, were isolated more frequently in osteoradionecrosis at 30% compared to 12.5% in osteomyelitis and 12.12% in MRONJ cases. There is weak evidence of significant difference when comparing osteoradionecrosis and MRONJ cases (P = .1). CONCLUSION: Non-resident bacteria including Staphylococcus and Enterococcus may be more frequently isolated in patients with osteomyelitis, while opportunistic organisms like Mycobacterium and Candida may be more frequently found in patients diagnosed with osteoradionecrosis.