Immune-related expression profiles of bisphosphonates-related osteonecrosis of the jaw in multiple myeloma.

Objective: To investigate the immune cellular and genomic profiles of bisphosphonates-related osteonecrosis (BRONJ) of the jaw and excavate potential small molecule drugs. Methods and materials: The genomic profile of a multiple myeloma (MM) patient with BRONJ was downloaded from Gene Expression Omnibus (GEO). The 22 immune cell subsets infiltration in the patient were predicted by cell-type identification by estimating relative subsets of RNA transcripts. In addition, the differently expressed immune-related genes (DEMGs) of BRONJ were identified, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for functional annotation. Then, potential drugs for BRONJ treatment were predicted by Connectivity Map (CMAP) based on DEGs. Results: High proportions of native CD4+T cells and M0 macrophages were observed while resting mast cells, NK cells, and eosinophils were downregulated in the BRONJ patient (P< 0.05). Resting dendritic cells and gamma delta T cells were positively correlated (r=0.93). Additionally, 36 DEMGs were screened from 336 DEGs in BRONJ expression profiles. GO enrichment analysis revealed that DEMGs were most associated with peptidyl-tyrosine modification, myeloid leukocyte migration, leukocyte chemotaxis and regulation of chemokine production(P<0.05). KEGG analysis indicated that DEMGs were mainly related to cytokine-cytokine receptor interaction, IL-17 signaling pathway and NF-Kappa B signaling pathway(P<0.05). Besides, 12 small molecule drugs were screened in MM patient with ONJ. Conclusion: The discovery of different composition of immune cell types and immune-related transcriptomes in BRONJ helps to explain the onset and development of MRONJ, which provides a novel target for BRONJ therapy.

THE CONTENT OF NK CELLS AND THEIR SUBTYPES IN THE CASE OF DRUG-INDUCED JAW OSTEONECROSIS.

The goal of the study is assessing the population structure of lymphocytes and the subpopulation composition of NK cells in the peripheral blood of humans in the case of drug-induced osteonecrosis of the jaw for improving the quality of diagnosis, treatment, and prevention of osteonecrosis of the jaw. Thirty patients were examined, including 15 patients with drug-induced osteonecrosis of the jaw (ONJ), 10 healthy individuals, and five patients with pyoinflammatory diseases of the maxillofacial region, aged 49 to 77. Every four weeks, the patients had been receiving 4 mg intravenous injections of bisphosphonates (the Zometa preparation (zoledronic acid)) for 1.5 - 3 years. The study was performed on a FACS Canto II flow cytometer manufactured by Becton Dickinson (BD), USA. In the patients with ONJ, an imbalance of the NK cell subtypes was observed. As to the common NK cells, the level of subtypes of cytolytic NK cells (CD3-CD16+(or hight)56dim) was elevated, and the level of cytokine-producing cells (СD3-CD16-(or low) 56bright) was reduced, compared to the healthy individuals (p<0.05). In the patients with ONJ, after the surgical treatment, the relative and absolute levels of lymphocytes and the total level of NK cells (CD3-CD16+/56+) normalized on the seventh day. The level of NK cells subtypes did not change after the treatment. This pathology is characterized by a low level of innate protection factors, as evidenced by the reduction of the total population of NK cells, and the imbalance of NK cells subtypes. The imbalance of NK cells (the natural killer cells) was an indicator of unfavorable prognosis for osteonecrosis treatment.

Monitoring indices of bone inflammatory activity of the jaw using SPECT bone scintigraphy: a study of ARONJ patients.

Development of quantitative analysis software has enabled application of several standardised uptake values (SUV) for bone analysis in single photon emission computed tomography (SPECT). The present retrospective study aimed to develop a reliable method of monitoring bone inflammatory activity in antiresorptive agent-related osteonecrosis of the jaw (ARONJ) using SPECT quantitative analysis software. Fifteen ARONJ patients underwent SPECT before and after anti-inflammatory therapy. We calculated the mean maximum SUV (SUVmax) of the bilateral cranial bones using quantitative analysis software and used this as the control [C]. We attempted to adjust the SUVmax of the lesion [L] as follows: adjusted SUVmax (aSUVmax) = [L] - [C]. The optimum threshold to calculate the metabolic bone volume (MBV) (cm3) was [C] + 3. The threshold values obtained for each case were input to calculate MBV at each osteomyelitis site. Retrospectively, we compared aSUVmax and MBV of each patient's ARONJ before and after anti-inflammatory therapy. The patients' high aSUVmax or large MBV of the ARONJ reduced rapidly, reflecting individual clinical findings after treatment. Application of SPECT quantitative analysis software to monitor bone inflammatory activity in ARONJ could improve the prognosis-deciding abilities of clinicians and enable them to treat ARONJ effectively.

Current Understanding of the Pathophysiology of Osteonecrosis of the Jaw.

PURPOSE OF REVIEW: Osteonecrosis of the jaw (ONJ) is a rare and severe necrotic bone disease reflecting a compromise in the body's osseous healing mechanisms and unique to the craniofacial region. Antiresorptive and antiangiogenic medications have been suggested to be associated with the occurrence of ONJ; yet, the pathophysiology of this disease has not been fully elucidated. This article raises the current theories underlying the pathophysiology of ONJ. RECENT FINDINGS: The proposed mechanisms highlight the unique localization of ONJ. The evidence-based mechanisms of ONJ pathogenesis include disturbed bone remodeling, inflammation or infection, altered immunity, soft tissue toxicity, and angiogenesis inhibition. The role of dental infections and the oral microbiome is central to ONJ, and systemic conditions such as rheumatoid arthritis and diabetes mellitus contribute through their impact on immune resiliency. Current experimental studies on mechanisms of ONJ are summarized. The definitive pathophysiology is as yet unclear. Recent studies are beginning to clarify the relative importance of the proposed mechanisms. A better understanding of osteoimmunology and the relationship of angiogenesis to the development of ONJ is needed along with detailed studies of the impact of drug holidays on the clinical condition of ONJ.

Desarrollo de un modelo experimental de osteonecrosis de maxilar asociada al tratamiento crónico con aminobisfosfonatos / Experimental model of osteonecrosis of the jaw associated to chronic treatment with aminobisphosphonates

La osteonecrosis de maxilar asociada a aminobisfosfonatos (BRONJ) constituye un efecto secundario del tratamiento crónico con los más potentes. Un modelo experimental permitiría determinar la patogenia de dicha alteración. La oveja presenta características orales y del metabolismo óseo similar al humano y permite realizar manipulaciones bucales. Se evaluaron cambios clínicos, remodelación ósea y masa ósea maxilar en ovejas hembras adultas tratadas con zolendronato (ZOL), durante 22 meses y utilizando dosis equivalente al tratamiento de neoplasias. Seis ovariectomizadas (OVX) recibieron ZOL; 5 OVX y 4 SHAM (control) recibieron solución fisiológica. Al inicio, 4 y 22 meses se evaluó calcemia, fosfatemia, crosslaps (CTX) y fosfatasa alcalina ósea. Al final, se evaluó contenido mineral óseo de la hemimandíbula superior (CMO: mg/cm2). Al final del estudio, CTX disminuyó significativamente en ZOL (p<0,05) sin diferencias entre SHAM y OVX. En maxilar, los contenidos de Ca y P (g/g tejido) y CMO (g/cm2 ) disminuyeron en OVX vs. SHAM (p<0,05) y solo Ca y CMO respecto de ZOL (p<0,05). ZOL incrementó el contenido de Ca y CMO, mientras que el de P permaneció significativamente disminuido respecto de SHAM. La sobrevida en SHAM y OVX fue del 100% y en ZOL 77% (2 muertes); 2 ovejas del grupo ZOL presentaron necrosis de maxilar. Conclusiones: fue posible obtener desarrollo de BRONJ por tratamiento crónico con ZOL, el cual redujo notablemente la resorción y, según la relación Ca/P, posiblemente haya afectado la mineralización ósea. (AU)

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a complication of chronic treatment with the most powerful aminobisphosphonates (BPs). An experimental animal model would allow to determine the pathogenesis of this complication. Ewes exhibit similar oral cavity characteristics and bone metabolism as humans, and they are suitable for oral cavity interventions. We examined herein the clinical manifestations, bone remodeling status, and maxillary bone mass in adult female ewes treated with zoledronate (ZOL) for 22 months. Six ovariectomized (OVX) ewes received ZOL; and 5 OVX and 4 SHAM animals received saline solution. At the start of the experiment, and at the 4 and 22 month-time points serum Ca, P, crosslaps (CTX), and bone alkaline phosphatase were measured. Bone mineral content (BMC) of the superior hemimandible was measured at the end of the experiment. At this time point, CTX was significantly decreased only in the ZOL group (p<0.05). Ca and P content (g/g tissue) and BMC in the mandible were significantly decreased in the OVX group compared to SHAM animals (p<0.05) and only Ca content and BMC were decreased when compared to ZOL (p<0.05). ZOL treatment increased the Ca content and BMC, whereas the P content remained low compared to the SHAM group (p<0.05). All ewes from the SHAM and OVX groups and 77% of the animals from the ZOL group survived until the end of the experiment, whereas two ewes of ZOL group exhibited BRONJ. Conclusion: under our experimental conditions, it was possible to induce BRONJ by the chronic ZOL administration, which in turn induced a high reduction in bone resorption as well as possibly impaired bone mineralization, based on the Ca/P ratio in the mandible. (AU)

Underlying Mechanisms and Therapeutic Strategies for Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ).

Bisphosphonates (BPs), with a non-hydrolysable P-C-P structure, are cytotoxic analogues of pyrophosphate, bind strongly to bone, are taken into osteoclasts during bone-resorption and exhibit long-acting anti-bone-resorptive effects. Among the BPs, nitrogen-containing BPs (N-BPs) have far stronger anti-bone-resorptive effects than non-N-BPs. In addition to their pyrogenic and digestive-organ-injuring side effects, BP-related osteonecrosis of jaws (BRONJ), mostly caused by N-BPs, has been a serious concern since 2003. The mechanism underlying BRONJ has proved difficult to unravel, and there are no solid strategies for treating and/or preventing BRONJ. Our mouse experiments have yielded the following results. (a) N-BPs, but not non-N-BPs, exhibit direct inflammatory and/or necrotic effects on soft tissues. (b) These effects are augmented by lipopolysaccharide, a bacterial-cell-wall component. (c) N-BPs are transported into cells via phosphate transporters. (d) The non-N-BPs etidronate (Eti) and clodronate (Clo) competitively inhibit this transportation (potencies, Clo>Eti) and reduce and/or prevent the N-BP-induced inflammation and/or necrosis. (e) Eti, but not Clo, can expel N-BPs that have accumulated within bones. (f) Eti and Clo each have an analgesic effect (potencies, Clo>Eti) via inhibition of phosphate transporters involved in pain transmission. From these findings, we propose that phosphate-transporter-mediated and inflammation/infection-promoted mechanisms underlie BRONJ. To treat and/or prevent BRONJ, we propose (i) Eti as a substitution drug for N-BPs and (ii) Clo as a combination drug with N-BPs while retaining their anti-bone-resorptive effects. Our clinical trials support this role for Eti (we cannot perform such trials using Clo because Clo is not clinically approved in Japan).

Plasticity of Myeloid Cells during Oral Barrier Wound Healing and the Development of Bisphosphonate-related Osteonecrosis of the Jaw.

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVES: Characterize the cell profile and immunostaining of proinflammatory markers in an experimental model of bisphosphonate-related osteonecrosis of the jaw (BRONJ). MATERIALS AND METHODS: Male Wistar rats (n = 6-7) were treated chronically with saline solution or zoledronic acid (ZA) at 0.04, 0.20, and 1.00 mg kg(-1) (1.4 × 10(-7) , 6.9 × 10(-6) , and 3.4 × 10(-5)  mol kg(-1) ), and subsequently, the first left inferior molar was extracted. Were performed counting of viable and empty osteocyte lacunae, viable and apoptotic osteoclasts, polymorphonuclear neutrophil, mast cells (toluidine blue), and the positive presence cells for CD68, tumor necrosis factor-alpha (TNF-α), IL (interleukin)-1ß, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-kB) and IL-18 binding protein (IL-18 bp). RESULTS: BRONJ was showed in ZA treated with 0.20 and 1.00 mg kg(-1) . There is a dose dependent increase in percentage of empty osteocyte lacunae (P < 0.001) and apoptotic osteoclasts (P < 0.001), counting of total osteoclasts (P = 0.003), polymorphonuclear neutrophil cells (P = 0.009), cytoplasmic-positive cells of CD68 (P < 0.001), TNF-α (P = 0.001), IL-1ß (P = 0.001), iNOS (P < 0.001), NF-kB (P = 0.006), and nuclear-positive cells of NF-kB (P = 0.011). Consequently, there is no difference in mast cells (P = 0.957), and IL-18 bp immunostaining decreases dose dependently (P = 0.005). CONCLUSIONS: BRONJ is characterized by increases in immunostaining for proinflammatory markers and NF-kB and inversely associated with cells exhibiting IL-18 bp.

Effect of bisphosphonates on macrophagic THP-1 cell survival in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non-nitrogen-containing) clodronate on macrophagic THP-1 cells. STUDY DESIGN: Activated THP-1 cells were exposed to .5 to 50 µM of nitrogen-containing bisphosphonates and .5 to 500 µM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining. RESULTS: All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 µM and 500 µM; P < .05). Low concentrations (0.5 µM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations. CONCLUSIONS: Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. MATERIALS AND METHODS: THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50 µM) and clodronate (125, 250 and 500 µM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72 h with zoledronate, ibandronate, alendronate and clodronate. RESULTS: Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5 µM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. CONCLUSIONS: All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. CLINICAL RELEVANCE: These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Systemic immunity shapes the oral microbiome and susceptibility to bisphosphonate-associated osteonecrosis of the jaw.

BACKGROUND: Osteonecrosis of the jaw (ONJ) is a rare but serious adverse drug effect linked to long-term and/or high-dose exposure to nitrogen-bisphosphonates (N-BP), the standard of care for the treatment of bone fragility disorders. The mechanism leading to bisphosphonate-associated ONJ (BAONJ) is unclear and optimal treatment strategies are lacking. Recent evidence suggests that BAONJ may be linked to drug-induced immune dysfunction, possibly associated with increased susceptibility to infections in the oral cavity. The objective of this investigation was to comprehensively assess the relationship linking immune function, N-BP exposure, the oral microbiome and ONJ susceptibility. METHODS: Leukocyte gene expression of factors important for immunity, wound healing and barrier function were assessed by real-time quantitative PCR and the oral microbiome was characterized by 454 pyrosequencing of the 16S rRNA gene in 93 subjects stratified by N-BP exposure and a history of ONJ. RESULTS: There were marked differences in the systemic expression of genes regulating immune and barrier functions including RANK (p = 0.007), aryl hydrocarbon receptor (AHR, p < 0.001), and FGF9 (p < 0.001), which were collectively up-regulated in individuals exposed to N-BP without ONJ relative to treatment controls. In contrast, the expression levels of these same genes were significantly down-regulated in those who had experienced BAONJ. Surprisingly, the oral microbiome composition was not directly linked to either BAONJ or N-BP exposure, rather the systemic leukocyte expression levels of RANK, TNFA and AHR each explained 9% (p = 0.04), 12% (p = 0.01), and 7% (p = 0.03) of the oral bacterial beta diversity. CONCLUSIONS: The oral microbiome is unlikely causative of ONJ, rather individuals with BAONJ lacked immune resiliency which impaired their capacity to respond adequately to the immunological stress of N-BP treatment. This may be the common factor linking N-BP and anti-RANK agents to ONJ in at-risk individuals. Preventive and/or therapeutic strategies should target the wound healing deficits present in those with ONJ.

Osteonecrosis of the Jaw Developed in Mice: DISEASE VARIANTS REGULATED BY γδ T CELLS IN ORAL MUCOSAL BARRIER IMMUNITY.

Osteonecrosis of the jaw (ONJ), an uncommon co-morbidity in patients treated with bisphosphonates (BP), occurs in the segment of jawbone interfacing oral mucosa. This study aimed to investigate a role of oral mucosal barrier γδ T cells in the pathogenesis of ONJ. Female C57Bl/6J (B6) mice received a bolus zoledronate intravenous injection (ZOL, 540 µg/kg), and their maxillary left first molars were extracted 1 week later. ZOL-treated mice (WT ZOL) delayed oral wound healing with patent open wounds 4 weeks after tooth extraction with characteristic oral epithelial hyperplasia. γδ T cells appeared within the tooth extraction site and hyperplastic epithelium in WT ZOL mice. In ZOL-treated γδ T cell null (Tcrd(-/-) ZOL) mice, the tooth extraction open wound progressively closed; however, histological ONJ-like lesions were identified in 75 and 60% of WT ZOL and Tcrd(-/-) ZOL mice, respectively. Although the bone exposure phenotype of ONJ was predominantly observed in WT ZOL mice, Tcrd(-/-) ZOL mice developed the pustule/fistula disease phenotype. We further addressed the role of γδ T cells from human peripheral blood (h-γδ T cells). When co-cultured with ZOL-pretreated human osteoclasts in vitro, h-γδ T cells exhibited rapid expansion and robust IFN-γ secretion. When h-γδ T cells were injected into ZOL-treated immunodeficient (Rag2(-/-) ZOL) mice, the oral epithelial hyperplasia developed. However, Rag2(-/-) ZOL mice did not develop osteonecrosis. The results indicate that γδ T cells are unlikely to influence the core osteonecrosis mechanism; however, they may serve as a critical modifier contributing to the different oral mucosal disease variations of ONJ.

Diagnosis and management of osteonecrosis of the jaw: a systematic review and international consensus.

This work provides a systematic review of the literature from January 2003 to April 2014 pertaining to the incidence, pathophysiology, diagnosis, and treatment of osteonecrosis of the jaw (ONJ), and offers recommendations for its management based on multidisciplinary international consensus. ONJ is associated with oncology-dose parenteral antiresorptive therapy of bisphosphonates (BP) and denosumab (Dmab). The incidence of ONJ is greatest in the oncology patient population (1% to 15%), where high doses of these medications are used at frequent intervals. In the osteoporosis patient population, the incidence of ONJ is estimated at 0.001% to 0.01%, marginally higher than the incidence in the general population (<0.001%). New insights into the pathophysiology of ONJ include antiresorptive effects of BPs and Dmab, effects of BPs on gamma delta T-cells and on monocyte and macrophage function, as well as the role of local bacterial infection, inflammation, and necrosis. Advances in imaging include the use of cone beam computerized tomography assessing cortical and cancellous architecture with lower radiation exposure, magnetic resonance imaging, bone scanning, and positron emission tomography, although plain films often suffice. Other risk factors for ONJ include glucocorticoid use, maxillary or mandibular bone surgery, poor oral hygiene, chronic inflammation, diabetes mellitus, ill-fitting dentures, as well as other drugs, including antiangiogenic agents. Prevention strategies for ONJ include elimination or stabilization of oral disease prior to initiation of antiresorptive agents, as well as maintenance of good oral hygiene. In those patients at high risk for the development of ONJ, including cancer patients receiving high-dose BP or Dmab therapy, consideration should be given to withholding antiresorptive therapy following extensive oral surgery until the surgical site heals with mature mucosal coverage. Management of ONJ is based on the stage of the disease, size of the lesions, and the presence of contributing drug therapy and comorbidity. Conservative therapy includes topical antibiotic oral rinses and systemic antibiotic therapy. Localized surgical debridement is indicated in advanced nonresponsive disease and has been successful. Early data have suggested enhanced osseous wound healing with teriparatide in those without contraindications for its use. Experimental therapy includes bone marrow stem cell intralesional transplantation, low-level laser therapy, local platelet-derived growth factor application, hyperbaric oxygen, and tissue grafting.

Immunohistological expression of human ß-defensin-1 and human ß-defensin-2 in exacerbation of acute and secondary chronic osteomyelitis of the mandible.

BACKGROUND: The majority of patients diagnosed with osteomyelitis of the jaw have severe complaints. Unfortunately, the pathogenesis still remains unclear. Human ß-defensins expressed in epithelial and bone tissues as a part of the innate immunity may be involved in disease development. In this study, we hypothesize that expression levels of human ß-defensin-1 and -2 in the acute and secondary chronic osteomyelitis may be altered in comparison with healthy bone and with bisphosphonate-associated necrosis as well as irradiation from a previous study. METHODS: Bone samples were collected during surgical debridement in a total of eight patients suffering from acute or secondary chronic osteomyelitis of the jaw. Expression levels of hBD-1 and -2 were quantified and related to non-stained cells. Ratios were compared by one-way ANOVA and multiple tests by Holm-Bonferroni. RESULTS: Multiple testing revealed no significant differences for expression levels of human ß-defensin-1 between all groups, whereas labeling index of human ß-defensin-2 was significantly different between specimens of bisphosphonate-associated osteonecrosis of the jaws and all other groups. No significant difference occurred between samples of floride osteomyelitis and healthy bone for expression of hBD-1 and -2. CONCLUSIONS: Although the affected patients showed all clinical signs of acute inflammation, expression levels in acute and secondary chronic osteomyelitis in the jaws did not reveal statistically significant differences compared with healthy bone samples. The weak immunological host response in terms of a putative genetically predisposition should be further discussed as pathogenesis factor for osteomyelitis in the future.

Macrophages and bisphosphonate-related osteonecrosis of the jaw (BRONJ): evidence of local immunosuppression of macrophages in contrast to other infectious jaw diseases.

OBJECTIVES: Bisphosphonates (BIP) are well established in bone diseases. A serious side effect is the bisphosphonate-related osteonecrosis of the jaw (BRONJ). Among different aetiology factors, local suppression of immune functions is gaining interest. The aim of this study was to analyze the function of macrophages in BRONJ in contrast to patients with osteoradionecrosis (ORN) and secondary chronic osteomyelitis (OM) of the jaws. Samples were also taken from patients with bisphosphonate medication (BP) without signs of infection, radiation therapy (RA), and osteoporosis (OP) as controls. MATERIAL AND METHODS: One hundred five patients with surgery to the jaw were included in this study: 33 patients with BRONJ, 17 with ORN, 11 with secondary chronic OM, 8 with RA, 25 with BP medication and 11 with OP. Samples were histologically analysed and monocytes/macrophages stained using CD14 and CD68. The number of positively marked cells was counted per view (pv), and the CD68/CD14 ratio was calculated. Statistically, the Naïve-Bayes and decision-tree classifier were used. RESULTS: The number of CD14 positive cells was 10.3 cells/pv in the BRONJ-group in as compared to 5 in the ORN- and 3.8 in the OM-group respectively. The number of CD68 positive cells was 11.4/pv (BRONJ-group) as compared to 14/pv (ORN-group) and 12.7/pv (OM-group). With 0.89, the BRONJ-group showed a statistically different CD68/CD14 ratio than ORN-group with 3.39 and OM-group with 3.03. CONCLUSIONS: Our results indicate a different expression of CD14 and CD68 markers of monocytes/macrophages in BRONJ as compared to other jaw infections. This could be a sign of macrophage immunosuppression by BPs. In contrast, patients receiving BP medication without BRONJ showed no differences to other controls. CLINICAL RELEVANCE: This is the first study that clinically indicates a compromised macrophage function at BRONJ sites in contrast to ORN or secondary OM sites. The BRONJ itself could be forwarded by this effect.

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw.

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

Evaluation of serum biomarkers IL-17 and CTX for BRONJ: a pilot clinical case-control study.

A serious complication of bisphosphonate (BP) therapy is BP-related osteonecrosis of the jaw (BRONJ). Currently, no biomarkers exist to identify patients at risk. We evaluated whether interleukin-17 and C-telopeptide correlate with BRONJ development. We conducted a case-control study using patients with a history of BP therapy. Quantitative enzyme-linked immunosorbent assay and Student's t-test were done. Both markers were significantly higher in BRONJ, suggesting altered immune responses and bone remodeling may play roles in BRONJ development.

IL-17-mediated M1/M2 macrophage alteration contributes to pathogenesis of bisphosphonate-related osteonecrosis of the jaws.

PURPOSE: Osteonecrosis of the jaw (ONJ) is emerging as one of the important complications in cancer patients treated with antiresorptive agents. This study explored the potential role of interleukin (IL)-17-mediated M1/M2 macrophage alterations in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). EXPERIMENTAL DESIGN: The expression of IL-17 and M1 and M2 macrophage markers at the local mucosal site of human BRONJ lesions was examined by immunofluorescence studies. BRONJ-like disease was induced in C57BL/6 mice and multiple myeloma-burdened mice by intravenous injection of zoledronate to evaluate the correlation of elevated IL-17 levels with changes in M1 and M2 macrophage phenotypes and the therapeutic effects of blocking IL-17 on pathogenesis of BRONJ-like disease. RESULTS: Increased T-helper (TH)17 cells and IL-17 cytokine correlate with an increase in M1/M2 macrophages ratio at the local mucosal site of both murine and human BRONJ lesion. Convincingly, in mice burdened with multiple myeloma, a combination of elevated suprabasal level and drug-induced IL-17 activity augmented the incidence of BRONJ; both systemic increase of IL-17 and disease severity could be reversed by adoptive transfer of ex vivo expanded M2 macrophages. Targeting IL-17 via specific neutralizing antibodies or a small inhibitory molecule, laquinimod, significantly decreased M1/M2 ratio and concomitantly suppressed BRONJ-like condition in mice. Mechanistically, IL-17 enhanced IFN-γ-induced M1 polarization through augmenting STAT-1 phosphorylation while suppressing IL-4-mediated M2 conversion via inhibiting STAT-6 activation. CONCLUSIONS: These findings have established a compelling linkage between activated IL-17-mediated polarization of M1 macrophages and the development of BRONJ-like conditions in both human disease and murine models.

Can peripheral blood γδ T cells predict osteonecrosis of the jaw? An immunological perspective on the adverse drug effects of aminobisphosphonate therapy.

Nitrogen-bisphosphonates (n-BP), often referred to as aminobisphosphonates, are the most commonly prescribed drugs for the treatment of disorders of bone fragility. However, long-term continuous treatment predisposes certain individuals to serious rare side effects, such as bisphosphonate-associated osteonecrosis of the jaw (BAONJ). n-BP use is known to unintentionally activate a subset of innate T cells called Vγ9Vδ2 T cells, but the consequence of this chronic immune stimulation has remained unexplored. The primary objectives of this study were to 1) determine the fate of Vγ9Vδ2 T cells in osteoporotic patients on n-BP therapy as a function of time and type of therapy; 2) evaluate the proportion of Vγ9Vδ2 T cells in patients who had recently experienced n-BP-associated ONJ. We found there is a notable loss of Vγ9Vδ2 T cells over time in osteoporotic patients on n-BP therapy, particularly those on intravenous (iv) therapy (Spearman r = -0.55, p < 0.0001 iv; r = -0.3, p < 0.03 oral) (n = 68); no difference was observed in total T cells, monocytes, or granulocytes. Importantly, the observed negative effect on Vγ9Vδ2 T cells coincides with the reported route of administration and timing of the rare occurrence of BAONJ. Patients (n = 6) who had experienced BAONJ were all found to be significantly deficient in Vγ9Vδ2 T cells (median = 0.07%) in comparison to age- and sex-matched treatment-naïve controls (N = 11; median = 2.40%), U = 0, p = 0.001; this was the only consistent difference in the leukocytes assessed. All BAONJ cases had an underlying condition that further contributed to impaired immunity. We propose Vγ9Vδ2 T cells show a strong potential to serve as harbingers of possible adverse immune effects of n-BP therapy, particularly in those patients already having a compromised immune system as they may be most vulnerable to the development of conditions such as BAONJ.