Impaction of Mandibular First Molar by Tooth-Like Hard Tissue.

Impaction of permanent teeth during the mixed dentition stage is relatively common in clinical practice, but impaction of mandibular first molars is rare. This case report presents an impaction of the mandibular first molar due to a tooth-like hard tissue lesion. An 8-year-old girl was diagnosed with an impacted mandibular first molar. The roots of the impacted molars were almost completely developed. A spherical tooth-like hard tissue with a diameter of approximately 2 mm was observed at the alveolar crest between the impacted mandibular first and second molars. The lesion causing the impaction was excised, and the first molar was fenestrated and allowed to erupt naturally. We showed that even if the tooth root is almost complete, natural eruption can be expected if the lesion is removed and space for eruption is secured.

Involvement of oxidative stress in orofacial mechanical pain hypersensitivity following neonatal maternal separation in rats.

Patients with persistent pain have sometimes history of physical abuse or neglect during infancy. However, the pathogenic mechanisms underlying orofacial pain hypersensitivity associated with early-life stress remain unclear. The present study focused on oxidative stress and investigated its role in pain hypersensitivity in adulthood following early-life stress. To establish an early-life stress model, neonatal pups were separated with their mother in isolated cages for 2 weeks. The mechanical head-withdrawal threshold (MHWT) in the whisker pad skin of rats received maternal separation (MS) was lower than that of non-MS rats at postnatal week 7. In MS rats, the expression of 8-hydroxy-deoxyguanosine, a marker of DNA oxidative damage, was enhanced, and plasma antioxidant capacity, but not mitochondrial complex I activity, decreased compared with that in non-MS rats. Reactive oxygen species (ROS) inactivation and ROS-sensitive transient receptor potential ankyrin 1 (TRPA1) antagonism in the whisker pad skin at week 7 suppressed the decrease of MHWT. Corticosterone levels on day 14 increased in MS rats. Corticosterone receptor antagonism during MS periods suppressed the reduction in antioxidant capacity and MHWT. The findings suggest that early-life stress potentially induces orofacial mechanical pain hypersensitivity via peripheral nociceptor TRPA1 hyperactivation induced by oxidative stress in the orofacial region.

Alteration of monoaminergic systems in the caudal medulla and its possible link to diurnal increase of apnea in a mouse model of Rett syndrome.

PURPOSE: Methyl-CpG binding protein 2 (MeCP2)-deficient (Mecp2-/y) mice exhibit apneas that resemble respiratory abnormalities observed in Rett syndrome (RTT) patients. The present study aimed to clarify whether Mecp2-/y mice show diurnal variations in apnea as seen in RTT and how the MeCP2 deficiency affects monoaminergic systems that control breathing. METHODS: In 7-week-old Mecp2-/y mice, 24 h variation of apnea and effects of milnacipran, a serotonin/noradrenaline reuptake inhibitor, on the apnea were evaluated. The number of vesicular monoamine transporter 2 (VMAT2)-immunoreactive puncta in the caudal medulla was counted. Further, the effects of valproate (VPA) on the expression of tyrosine hydroxylase (TH) mRNA in the ventrolateral medulla of mice were assessed by RT-qPCR. RESULTS: Apnea occurred more frequently during the light phase under a 12:12 h light/dark environment in Mecp2-/y mice and milnacipran reduced apnea during the light phase but not during the dark phase. The number of VMAT2-immunoreactive puncta was reduced in Mecp2-/y mice. VPA treatment significantly increased TH mRNA expression in Mecp2-/y mice. CONCLUSION: Alteration of monoaminergic systems in the caudal medulla of Mecp2-/y mice is potentially relevant to the light-sensitive diurnal increase of apnea, and an improvement in monoaminergic neurotransmission can ameliorate the diurnal increase of apnea in Mecp2-/y mice.

A rare case of multiple brain abscesses caused by apical periodontitis of deciduous teeth in congenital heart disease: a case report.

BACKGROUND: A brain abscess is a focal infection in which abscesses form in the brain. A brain abscess is a rare but fatal disease when rupture occurs into the ventricles. We report a case of multiple brain abscesses caused by a hematogenous infection from the apical periodontitis of deciduous teeth. CASE PRESENTATION: The patient was a 7-years and 8-months-old male with congenital heart disease. The patient sought medical attention due to fever and headache, for which he was started on three antibiotics with a diagnosis of multiple brain abscesses. Given that apical periodontitis of deciduous teeth was strongly suspected as the source of the brain abscess, the deciduous teeth were extracted. Immediately after deciduous teeth extraction, the patient's headache and neurological symptoms disappeared. CONCLUSIONS: After teeth extraction, a clear shrinkage of the brain abscess was observed, and the patient was discharged from the hospital.

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.

Bone resorption improvement by conditioned medium of stem cells from human exfoliated deciduous teeth in ovariectomized mice.

Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells with multipotent differentiation potential present in the dental pulp tissue of the deciduous teeth. SHED produce secretions that have immunomodulatory and regenerative functions. In the present study, we investigated the effects of SHED-conditioned medium (SHED-CM) on osteopenia induced by the ovariectomy (OVX) phenotype and its corresponding immunological changes. Eleven-week-old female C3H/HeJ mice were subjected to OVX. SHED-CM was administered intraperitoneally in these mice for 4 weeks starting immediately after OVX. SHED-CM improved bone mass after OVX and elevated the polarization of M2 macrophages in the peritoneal cavity. SHED-CM also suppressed an OVX-induced increase in interferon-γ (INF-γ) and interleukin-17 (IL-17) concentrations in the peripheral blood. Inhibition of M2 macrophage polarization with neutralizing antibodies did not reduce the concentration of IFN-γ and IL-17 in peripheral blood, which were increased by OVX, and did not alleviate osteopenia induced by the OVX phenotype. Mechanistically, these findings suggest that SHED-CM alleviates bone resorption by suppressing the activation of IFN-γ and IL-17 cells by polarizing M2 macrophages. In conclusion, our data indicate that SHED-CM contains active secretions that may have promising efficacy to ameliorate OVX-induced osteopenia. We suggest that SHED-CM has the potential to be used as a novel therapeutic agent to inhibit osteoporosis.

Patients with SATB2-associated syndrome exhibiting multiple odontomas.

Special AT-rich sequence-binding protein 2 (SATB2)-associated syndrome (SAS) is characterized by alterations of SATB2. Its clinical features include intellectual disability and craniofacial abnormalities, such as cleft palate, dysmorphic features, and dental abnormalities. Here, we describe three previously undiagnosed, unrelated patients with SAS who exhibited dental abnormalities, including multiple odontomas. Although isolated odontomas are common, multiple odontomas are rare. Individuals in families 1 and 3 underwent whole-exome sequencing. Patient 2 and parents underwent targeted amplicon sequencing. On the basis of the hg19/GRCh37 reference and the RefSeq mRNA NM_001172517, respective heterozygous mutations were found and validated in Patients 1, 2, and 3: a splice-site mutation (chr2:g.200137396C > T, c.1741-1G > A), a nonsense mutation (chr2:g.200213750G > A, c.847C > T, p.R283*), and a frame-shift mutations (chr2:g.200188589_200188590del, c.1478_1479del, p.Q493Rfs*19). All mutations occurred de novo. The mutations in Patients 1 and 3 were novel; the mutation in Patient 2 has been described previously. Tooth mesenchymal cells derived from Patient 2 showed diminished SATB2 expression. Multiple odontomas were evident in the patients in this report; however, this has not been recognized previously as a SAS-associated phenotype. We propose that multiple odontomas be considered as an occasional manifestation of SAS.

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 24­well 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 volume­dependent manner. A co­culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate­resistant acid phosphatase (TRAP)­positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP­positive multinuclear cells (≥8 nuclei) was increased, in a media volume­dependent manner. There were also no differences in receptor activator of nuclear factor­κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume­dependent 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.

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 tartrate­resistant 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 transcription­quantitative 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 osteoclast­specific genes, TRAP, RANK, matrix metalloproteinase­9, cathepsin­K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell­specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC­STAMP) 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.

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 osteoclast­associated 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.

Hydrogen sulfide maintains mesenchymal stem cell function and bone homeostasis via regulation of Ca(2+) channel sulfhydration.

Gaseous signaling molecules such as hydrogen sulfide (H2S) are produced endogenously and mediate effects through diverse mechanisms. H2S is one such gasotransmitters that 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 in order to regulate their self-renewal and osteogenic differentiation, and H2S deficiency results in defects in BMMSC differentiation. H2S deficiency causes aberrant intracellular Ca(2+) influx because of reduced sulfhydration of cysteine residues on multiple Ca(2+) TRP channels. This decreased Ca(2+) flux downregulates PKC/Erk-mediated Wnt/ß-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H2S-deficient mice display an osteoporotic phenotype that can be rescued by small molecules that release H2S. These results demonstrate that H2S regulates BMMSCs and that restoring H2S levels via nontoxic donors may provide treatments for diseases such as osteoporosis that can arise from H2S deficiencies.

Short-term mechanical stress inhibits osteoclastogenesis via suppression of DC-STAMP in RAW264.7 cells.

Mechanical stress is an important factor in bone homeostasis, which is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. However, little is known about the effects of mechanical stress on osteoclast differentiation. In this study, we examined the effects of short-term mechanical stress on osteoclastogenesis by applying tensile force to RAW264.7 cells stimulated with receptor activator of nuclear factor-κB ligand (RANKL) using a Flexercell tension system. We counted the number of osteoclasts that were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (two or more nuclei) with or without application of mechanical stress for 24 h. Osteoclast number was lower after mechanical stress compared with no mechanical stress. Furthermore, mechanical stress for up to 24 h caused downregulation of osteoclast-specific gene expression and fusion-related molecule [dendritic cell specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), E-cadherin, Integrin αV and Integrin ß3] mRNA levels. Protein expression of DC-STAMP decreased with mechanical stress for 24 h compared to the control without mechanical stress, whereas the expression of E-cadherin, Integrin αV and Integrin ß3 was slightly decreased. Nuclear factor of activated T cells c1 (NFATc1) mRNA levels were decreased at 6 h and increased at 12 and 24 h compared with the control. The levels of NFATc2, NFATc3 mRNA did not change compared with the control group. By contrast, mechanical stress for 24 h significantly enhanced NFAT transcriptional activity compared with the control, despite a decrease in DC-STAMP mRNA and protein levels. These results suggest that short-term mechanical stress strongly inhibits osteoclastogenesis through the downregulation of DC-STAMP and other fusion-related molecules and that short-term mechanical stress induces a negative regulatory mechanism that cancels the enhancement of NFAT transcriptional activity.

Stage-dependent suppression of the formation of dentin-resorbing multinuclear cells with migration inhibitory factor in vitro.

The macrophage migration inhibitory factor (MIF) is a crucial mediator of immune responses and is known to play a pivotal role in cell proliferation and differentiation. In this study, we assessed whether MIF exerts regulatory effects on osteoclast formation in bone marrow cells and, if so, by what mechanism. Bone marrow cells were either co-cultured with MC3T3-E1 cells or cultured with macrophage-colony stimulating factor (M-CSF) and the soluble form of the receptor activator of the nuclear factor-κB ligand (RANKL). Under the influence of MIF, the formation of osteoclastic multinuclear cells was examined. The number of multinuclear TRAP-positive cells formed in the co-culture was significantly reduced when MIF (≥0.1 µg/ml) was exogenously applied during the third and fourth days of the 6-day cultivation period. MIF affected neither the number of mononuclear TRAP-positive cells induced with M-CSF and RANKL, nor the expression of RANKL and osteoprotegerin in MC3T3-E1 cells. TRAP-positive cells cultured on dentin slices with MIF showed lower dentin-resorbing activity than those cultured without MIF. These results suggest that MIF has no regulatory roles in the differentiation of bone marrow cells to mononuclear TRAP-positive cells, but has inhibitory effects on the formation of mature osteoclasts by preventing cell fusion, which may eventually interfere with the osteoclast-mediated dentin resorption.

Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells.

Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.

Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-α.

Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. Here we show that proinflammatory T cells inhibit the ability of exogenously added bone marrow mesenchymal stem cells (BMMSCs) to mediate bone repair. This inhibition is due to interferon γ (IFN-γ)-induced downregulation of the runt-related transcription factor 2 (Runx-2) pathway and enhancement of tumor necrosis factor α (TNF-α) signaling in the stem cells. We also found that, through inhibition of nuclear factor κB (NF-κB), TNF-α converts the signaling of the IFN-γ-activated, nonapoptotic form of TNF receptor superfamily member 6 (Fas) in BMMSCs to a caspase 3- and caspase 8-associated proapoptotic cascade, resulting in the apoptosis of these cells. Conversely, reduction of IFN-γ and TNF-α concentrations by systemic infusion of Foxp3(+) regulatory T cells, or by local administration of aspirin, markedly improved BMMSC-based bone regeneration and calvarial defect repair in C57BL/6 mice. These data collectively show a previously unrecognized role of recipient T cells in BMMSC-based tissue engineering.

Platinum nanoparticles suppress osteoclastogenesis through scavenging of reactive oxygen species produced in RAW264.7 cells.

Recent research has shown that platinum nanoparticles (nano-Pt) efficiently quench reactive oxygen species (ROS) as a reducing catalyst. ROS have been suggested to regulate receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation. In the present study, we examined the direct effects of platinum nano-Pt on RANKL-induced osteoclast differentiation of murine pre-osteoclastic RAW 264.7 cells. The effect of the nano-Pt on the number of osteoclasts was measured and their effect on the mRNA expression for osteoclast differentiation was assayed using real-time PCR. Nano-Pt appeared to have a ROS-scavenging activity. Nano-Pt decreased the number of osteoclasts (2+ nuclei) and large osteoclasts (8+ nuclei) in a dose-dependent manner without affecting cell viability. In addition, this agent significantly blocked RANKL-induced mRNA expression of osteoclastic differentiation genes such as c-fms, NFATc1, NFATc2, and DC-STAMP as well as that of osteoclast-specific marker genes including MMP-9, Cath-K, CLC7, ATP6i, CTR, and TRAP. Although nano-Pt attenuated expression of the ROS-producing NOX-family oxidases, Nox1 and Nox4, they up-regulated expression of Nox2, the major Nox enzyme in macrophages. These findings suggest that the nano-Pt inhibit RANKL-stimulated osteoclast differentiation via their ROS scavenging property. The use of nano-Pt as scavengers of ROS that is generated by RANKL may be a novel and innovative therapy for bone diseases.

Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cells.

The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.

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.

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.

Matrix metalloproteinases are involved in mechanical stretch-induced activation of skeletal muscle satellite cells.

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor. Experiments reported herein provide new evidence that matrix metalloproteinases (MMPs) are involved in the NO-dependent release of HGF in vitro. When rat satellite cells were treated with 10 ng/ml recombinant tissue inhibitor-1 of MMPs (TIMP-1) and subjected to treatments that induce activation in vitro, i.e., sodium nitroprusside (SNP) of an NO donor or mechanical cyclic stretch, the activation response was inhibited. In addition, conditioned medium generated by cultures treated with TIMP-1 plus SNP or mechanical stretch failed to activate cultured satellite cells and did not contain HGF. Moreover, NO(x) assay demonstrated that TIMP-1 does not impair NO synthase activity of stretched satellite cell cultures. Therefore, results from these experiments provide strong evidence that MMPs mediate HGF release from the matrix and that this step in the pathway is downstream from NO synthesis.