Bisphosphonates Cause Osteonecrosis of the Jaw-Like Disease in Mice

Bisphosphonate-associated osteonecrosis of the jaw (BONJ) is a morbid bone disease linked to long-term bisphosphonate use. Despite its broad health impact, mechanistic study is lacking. In this study, we have established a mouse model of BONJ-like disease based on the equivalent clinical regimen in myeloma patients, a group associated with high risk of BONJ. We demonstrate that the murine BONJ-like disease recapitulates major clinical and radiographical manifestations of the human disease, including characteristic features of osseous sclerosis, sequestra, avascular, and radiopaque alveolar bone in the jaw that persists beyond a normal course of wound healing following tooth extraction. We find that long-term administration of bisphosphonates results in an increase in the size and number of osteoclasts and the formation of giant osteoclast-like cells within the alveolar bone. We show that the development of necrotic bone and impaired soft tissue healing in our mouse model is dependent on long-term use of high-dose bisphosphonates, immunosuppressive and chemotherapy drugs, as well as mechanical trauma. Most importantly, we demonstrate that bisphosphonate is the major cause of BONJ-like disease in mice, mediated in part by its ability to suppress osseous angiogenesis and bone remodeling. The availability of this novel mouse model of BONJ-like disease will help elucidate the pathophysiology of BONJ and ultimately develop novel approaches for prevention and treatment of human BONJ. Copyright © American Society for Investigative Pathology

Cell-based Immunotherapy with Mesenchymal Stem Cells Cures Bisphosphonate-related Osteonecrosis of the Jaw-like Disease in Mice

Patients on high-dose bisphosphonate and immunosuppressive therapy have an increased risk of bisphosphonate-related osteonecrosis of the jaw (BRONJ); despite the disease severity, its pathophysiology remains unknown, and appropriate therapy is not established. Here we have developed a mouse model of BRONJ-like disease that recapitulates major clinical and radiographic manifestations of the human disease, including characteristic features of an open alveolar socket, exposed necrotic bone or sequestra, increased inflammatory infiltrates, osseous sclerosis, and radiopaque alveolar bone. We show that administration of zoledronate, a potent aminobisphosphonate, and dexamethasone, an immunosuppressant drug, causes BRONJ-like disease in mice in part by suppressing the adaptive regulatory T cells, Tregs, and activating the inflammatory T-helper-producing interleukin 17 cells, Th17. Most interestingly, we demonstrate that systemic infusion with mesenchymal stem cells (MSCs) prevents and cures BRONJ-like disease possibly via induction of peripheral tolerance, shown as an inhibition of Th17 and increase in Treg cells. The suppressed Tregs/Th17 ratio in zoledronate- and dexamethasone-treated mice is restored in mice undergoing salvage therapy with Tregs. These findings provide evidence of an immunity-based mechanism of BRONJ-like disease and support the rationale for in vivo immunomodulatory therapy using Tregs or MSCs to treat BRONJ. © 2010 American Society for Bone and Mineral Research

Hydrogen Sulfide Maintains Mesenchymal Stem Cell Function and Bone Homeostasis via Regulation of Ca2+ Channel Sulfhydration

Gaseous signaling molecules such as hydrogen sulfide (H2S) are produced endogenously and mediate effects through diverse mechanisms. H2S is one such gasotrasmitter which regulates multiple signaling pathways in mammalian cells, and abnormal H2S metabolism has been linked to defects in bone homeostasis. Here, we demonstrate that bone marrow mesenchymal stem cells (BMMSCs) produce H2S to regulate their self-renewal and osteogenic differentiation, and H2S deficiency results in defects in BMMSC differentiation. H2S deficiency causes aberrant intracellular Ca2+ influx, due to reduced sulfhydration of cysteine residues on multiple Ca2+ TRP channels. This decreased Ca2+ flux downregulates PKC/Erk-mediated Wnt/β-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H2S-deficient mice display an osteoporotic phenotype, which can be rescued by small molecules which release H2S. These results demonstrate H2S regulates BMMSCs, and restoring H2S levels via non-toxic donors may provide treatments for diseases such as osteoporosis which can arise from H2S deficiencies

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24-well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume-dependent manner. A co-culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (>= 2 nuclei), whereas the formation of large osteoclastic TRAP-positive multinuclear cells (>= 8 nuclei) was increased, in a media volume-dependent manner. There were also no differences in receptor activator of nuclear factor-kappa B ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume-dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems

Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast-associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied

Release from optimal compressive force suppresses osteoclast differentiation

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate-resistant acid phosphatase (TRAP) -positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription-quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast-specific genes, TRAP, RANK, matrix metalloproteinase-9, cathepsin-K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell-specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC-STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments

Anti-RANKL Inhibits Thymic Function and Causes DRONJ in Mice

Background. Denosumab, a human monoclonal antibody against receptor activator of nuclear factor-kappa B ligand (RANKL), is a novel bone antiresorptive agent used in patients with osteoporosis or metastatic bone cancer. Denosumab-related osteonecrosis of the jaw (DRONJ) has been recently reported in patients using denosumab. However, the mechanisms of DRONJ are not fully understood. Appropriate pathogenic mechanisms of DRONJ have yet to be established. Therefore, we investigated the pathogenesis of DRONJ in mice. Methods. Anti-mouse RANKL monoclonal antibody and melphalan were performed to create a mouse model of DRONJ-like lesions in female C57BL/6J mice. We examined the development of DRONJ-like lesions and immune function. Results. We showed that administration of anti-mouse RANKL monoclonal antibody and melphalan caused DRONJ-like lesions that recapitulated major clinical manifestations of the human disease, including the characteristic features of an open alveolar socket and exposed necrotic bone. In the analysis using a mouse model of DRONJ-like lesion, it was revealed that anti-mouse RANKL monoclonal antibody and melphalan suppress autoimmune regulator (AIRE) expression in the thymus and imbalanced T cell populations. Conclusion. This study suggests evidence of an immunity-based mechanism of DRONJ-like disease. This work may contribute to a better understanding of the pathogenesis of human DRONJ