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The overactivation of the osteoclasts is a crucial pathological factor in the development of osteoporosis. MZF1, belonging to the scan-zinc finger family, plays a significant role in various processes associated with tumor malignant progression and acts as an essential transcription factor regulating osteoblast expression. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis. First, we established MZF1-deficient female mice and evaluated the femur bone phenotype by micro-computed tomography and histological staining. Our findings indicate that MZF1-/- mice exhibited a low bone mass osteoporosis phenotype. RANKL could independently induce the differentiation of RAW264.7 cells into osteoclasts, and we found that the expression level of MZF1 protein decreased gradually. Then, the CRISPR/Cas 9 gene-editing technique was used to build a RAW264.7 cell model with MZF1 knockout, and RANKL was used to independently induce MZF1-/- and wild-type cells to differentiate into mature osteoclasts. Tartrate-resistant acid phosphatase staining and F-actin fluorescence results showed that the MZF1-/- group produced more tartrate-resistant acid phosphatase-positive mature osteoclasts and larger actin rings. The expression of osteoclast-associated genes (including tartrate-resistant acid phosphatase, CTSK, c-Fos, and NFATc1) was evaluated by reverse transcription quantitative polymerase chain reaction and Western blot. The expression of key genes of osteoclast differentiation in the MZF1-/- group was significantly increased. Furthermore, we found that cell viability was increased in the early stages of RANKL-induced cell differentiation in the MZF1-/- group cells. We examined some prevalent ferroptosis markers, including malondialdehyde, glutathione, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis. The results suggest that MZF1 may be involved in osteoclast differentiation by regulating RANKL-induced ferroptosis of osteoclasts. Collectively, our findings shed light on the essential involvement of MZF1 in the regulation of osteoclastogenesis in osteoporosis and provide insights into its potential underlying mechanism., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The purpose of this study was to evaluate the effects of alendronate on orthodontic tooth movement (OTM) and bone modelling/remodelling in an osteogenesis imperfecta (OI) mice model., Materials and Methods: Ten-week-old male and female OI mice (Col1a2oim, n = 32) were divided into four groups: 1. Alendronate male (AM, n = 8), 2. Alendronate female (AF, n = 8), 3. saline male (SM, n = 8), and 4. saline female (SF, n = 8). The mice in all four groups received either Alendronate (0.05 mg/kg) or vehicle (saline 0.05 mg/kg) subcutaneously for 2 weeks prior to the placement of orthodontic spring. A nickel-titanium spring applying 3-5 cN of force was used to perform the OTM for 1 week. After 7 days of OTM, the OI mice were euthanized with CO2 inhalation and microfocus computed tomography and histological analyses were performed., Results: AM and AF mice showed a significant decrease (P < 0.05) in the rate of OTM compared with SM and SF mice, respectively. In addition, AM and AF mice showed a significant increase (P < 0.05) in the bone volume fraction (BVF) and tissue density (TD) compared with SM and SF mice. Histological analysis of haematoxylin-eosin staining revealed a hyalinization zone in AM and AF mice compared with SM and SF mice. Furthermore, tartrate-resistant acid phosphatase staining indicated decreased number of osteoclasts in AM and AF mice compared with SM and SF mice. Picrosirius red staining showed, Alendronate treatment led to thick uniform and smooth morphology of collagen fibres as compared with saline group. Similarly, second harmony generation images also revealed thicker collagen fibres at the periodontal ligament (PDL)-cementum entheses and PDL-alveolar bone entheses in AM and AF mice compared with SM and SF mice., Conclusions: Alendronate led to a decrease in the rate of OTM, increase in BVF and TD, decrease in the number of osteoclasts, and smooth and thick collagen fibres compared with saline in both male and female OI mice., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To investigate whether there is a difference in apical resorption activity during the development of roots in response to mechanical force in vivo., Methods: Maxillary first molars (M1) from postnatal day (PN) 21 and PN35 male rats were selected as representatives of the root-developing and root-completing groups, respectively. A mechanical force of 3 cN was applied to M1 on PN21 and PN35, and the maxilla was collected on PN28 and PN42. Odontoclastogenesis and root morphology were investigated using micro-focus X-ray computed tomography, followed by immunohistochemistry and quantitative real-time polymerase chain reaction to clarify root resorption activity., Results: Development of the mesiobuccal root (MBR) preceded the mesial root (MR). In the PN28 force application (FA) group, the dentine was bent, but the histology, including Hertwig's epithelial root sheath (HERS), was intact. No odontoclasts and resorption lacunae were found in the apical area of the MRs, and only lateral root resorption was observed. External apical root resorption (EARR) was observed in the MR of PN42 (FA) and in the MBR of both PN28 (FA) and PN42 (FA). The expression of osteopontin changed accordingly. No significant change occurred in osteoprotegerin or receptor activator of nuclear factor-κB ligand expression in the MRs of the PN28 (FA) group., Limitations: Our animal model did not adequately simulate the clinical process of tooth movement in humans., Conclusions: Force application delayed HERS dissociation on the compression side of the developing roots, leading to inhibitory effects on cementogenesis, which resulted in decreased odontoclast differentiation and prevention of EARR., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: The primary objective of this study was to quantify the orthodontic tooth movement (OTM) and orthodontically induced root resorption (OIRR) with differential force system in conjunction with minimal surgical insult., Material and Methods: 15-week-old, 48 male Wistar rats were used in the research and were randomly divided into six groups: 1. Group 1 (8 Wistar rats): OTM for 14 days with 8-g force; 2. Group 2 (8 Wistar rats): OTM for 14 days with 25-g force; 3. Group 3 (8 Wistar rats): OTM for 14 days with 100-g force; 4. Group 4 (8 Wistar rats): OTM for 14 days with 8-g force and alveolar decortications (ADs); 5. Group 5 (8 Wistar rats): OTM for 14 days with 25-g force and ADs; 6. Group 6 (8 Wistar rats): OTM for 14 days with 100-g force and ADs. A nickel-titanium spring was used to protract the molar mesially using maxillary incisors as an anchorage. ADs (minimal surgical insult) were done using a hand piece and a round bur, adjacent to the left first maxillary molar on the palatal alveolar bone. After 14 days of OTM, Wistar rats were killed and microfocus computed tomography and histological analysis were performed., Results: The 100-g group showed significant increase (P < 0.05) in OTM. However, with ADs, the OTM was significantly higher (P < 0.05) in 8 and 100 g. In addition, with ADs, there is significant increase (P < 0.05) in OIRR and significant decrease (P < 0.05) in bone volume fraction. Histological quantification of tartrate-resistant acid phosphatase indicated a significant increase (P < 0.05) in the number of osteoclasts with ADs when compared without ADs., Conclusions: Light force in conjunction with ADs are optimal to accelerate the OTM. Additionally, ADs increases the OIRR., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The vascular wall stores mesenchymal progenitor cells which are able to induce bone regeneration, via direct and paracrine mechanisms. Although much is known regarding perivascular cell regulation of osteoblasts, their regulation of osteoclasts, and by extension utility in states of high bone resorption, is not known. Here, human perivascular stem cells (PSCs) were used as a means to prevent autograft resorption in a gonadectomy-induced osteoporotic spine fusion model. Furthermore, the paracrine regulation by PSCs of osteoclast formation was evaluated, using coculture, conditioned medium, and purified extracellular vesicles. Results showed that PSCs when mixed with autograft bone induce an increase in osteoblast:osteoclast ratio, promote bone matrix formation, and prevent bone graft resorption. The confluence of these factors resulted in high rates of fusion in an ovariectomized rat lumbar spine fusion model. Application of PSCs was superior across metrics to either the use of unpurified, culture-defined adipose-derived stromal cells or autograft bone alone. Under coculture conditions, PSCs negatively regulated osteoclast formation and did so via secreted, nonvesicular paracrine factors. Total RNA sequencing identified secreted factors overexpressed by PSCs which may explain their negative regulation of graft resorption. In summary, PSCs reduce osteoclast formation and prevent bone graft resorption in high turnover states such as gonadectomy-induced osteoporosis., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis ( Pg ). We used a micro-osmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b + c-fms + Ly6C hi population is significantly elevated within the bone marrow, spleen and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b + c-fms + Ly6C hi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg -induced accumulation of CD11b + c-fms + Ly6C hi population from the bone marrow and periphery. Our results provide new insights into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases., Competing Interests: DISCLOSURE The authors declare no conflict of interest.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a microosmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b + c-fms + Ly6C hi population is significantly elevated within the bone marrow, spleen, and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b + c-fms + Ly6C hi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis, and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b + c-fms + Ly6C hi population from the bone marrow and periphery. Our results provide new insight into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases., (©2020 Society for Leukocyte Biology.)

Objectives: To investigate the role of non-receptor tyrosine kinases (NRTKs) in inflammation-induced osteoclastogenesis. Methods: Microarray analyses of global mRNA expression during receptor activator of NF-κB ligand (RANKL) and RANKL plus tumor necrosis factor (TNF)-α-induced osteoclast differentiation were performed. The inhibitory effect on TNF-α-induced osteoclast differentiation of A-419259, a potent inhibitor of hematopoietic cell kinase (Hck), was examined. The in vivo therapeutic effect of A-419259 treatment on lipopolysaccharide (LPS)-induced inflammatory bone destruction was evaluated. Results: We confirmed that Hck expression was selectively increased among the NRTKs during the osteoclast differentiation induced by RANKL and TNF-α, but not by RANKL alone. RANKL and TNF-α-induced osteoclast differentiation and they were dose-dependently inhibited by A-419259 treatment through inhibition of the expression of key regulators of osteoclastogenesis, including Prdm1 and Nfatc1 . Notably, LPS-induced inflammatory bone loss in murine calvarial bones was ameliorated by the administration of A-419259. Conclusions: Our results demonstrate that the administration of A-419259 is effective for the inhibition of osteoclast differentiation induced by TNF-α in the presence of RANKL. Therefore, an inhibitor of Hck may be useful as a potent anti-osteoclastogenic agent for the treatment of inflammatory bone destruction.

This study tested the hypothesis that mucosa associated lymphoid tissue 1 (Malt1) deficiency causes osteoporosis in mice by increasing osteoclastogenesis and osteoclast activity. A patient with combined immunodeficiency (CID) caused by MALT1 deficiency had low bone mineral density resulting in multiple low impact fractures that was corrected by hematopoietic stem cell transplant (HSCT). We have reported that Malt1 deficient Mϕs, another myeloid cell type, are hyper-responsive to inflammatory stimuli. Our objectives were to determine whether Malt1 deficient mice develop an osteoporosis-like phenotype and whether it was caused by Malt1 deficiency in osteoclasts. We found that Malt1 deficient mice had low bone volume by 12 weeks of age, which was primarily associated with reduced trabecular bone. Malt1 protein is expressed and active in osteoclasts and is induced by receptor activator of NF-κB ligand (RANKL) in preosteoclasts. Malt1 deficiency did not impact osteoclast differentiation or activity in vitro. However, Malt1 deficient (Malt1 -/- ) mice had more osteoclasts in vivo and had lower levels of serum osteoprotegerin (OPG), an endogenous inhibitor of osteoclastogenesis. Inhibition of Malt1 activity in Mϕs induced MCSF production, required for osteoclastogenesis, and decreased OPG production in response to inflammatory stimuli. In vitro, MCSF increased and OPG inhibited osteoclastogenesis, but effects were not enhanced in Malt1 deficient osteoclasts. These data support the hypothesis that Malt1 deficient mice develop an osteoporotic phenotype with increased osteoclastogenesis in vivo, but suggest that this is caused by inflammation rather than an effect of Malt1 deficiency in osteoclasts., (©2019 Society for Leukocyte Biology.)

Disturbed permanent tooth eruption is common in cleidocranial dysplasia (CCD), a skeletal disorder caused by heterozygous mutation of RUNX2, but the mechanism underlying is still unclear. As it is well known that dental follicle cells (DFCs) play a critical role in tooth eruption, the changed biological characteristics of DFCs might give rise to disturbance of permanent tooth eruption in CCD patients. Thus, primary DFCs from one CCD patient and normal controls were collected to investigate the effect of RUNX2 mutation on the bone remodeling activity of DFCs and explore the mechanism of impaired permanent tooth eruption in this disease. Conservation and secondary structure analysis revealed that the RUNX2 mutation (c.514delT, p.172fs) found in the present CCD patient was located in the highly conserved RUNT domain and converted the structure of RUNX2. After osteogenic induction, we found that the mineralised capacity of DFCs and the expression of osteoblast-related genes, including RUNX2, ALP, OSX, OCN and Col Iα1, in DFCs was severely interfered by the RUNX2 mutation found in CCD patients. To investigate whether the osteogenic deficiency of DFCs from the CCD patient can be rescued by RUNX2 restoration, we performed 'rescue' experiments. Surprisingly, the osteogenic deficiency and the abnormal expression of osteoblast-associated genes in DFCs from the CCD patient were almost rescued by overexpression of wild-type RUNX2 using lentivirus. All these findings indicate that RUNX2 mutation can reduce the osteogenic capacity of DFCs through inhibiting osteoblast-associated genes, thereby disturbing alveolar bone formation, which serves as a motive force for tooth eruption. This effect may provide valuable explanations and implications for the mechanism of delayed permanent tooth eruption in CCD patients.

Background: Bone impairment is a poorly described complication of nephropathic cystinosis (NC). The objectives of this study were to evaluate in vitro effects of cystinosin (CTNS) mutations on bone resorption and of cysteamine treatment on bone cells [namely human osteoclasts (OCs) and murine osteoblasts]., Methods: Human OCs were differentiated from peripheral blood mononuclear cells (PBMCs) of patients and healthy donors (HDs). Cells were treated with increasing doses of cysteamine in PBMCs or on mature OCs to evaluate its impact on differentiation and resorption, respectively. Similarly, cysteamine-treated osteoblasts derived from murine mesenchymal stem cells were assessed for differentiation and activity with toxicity and proliferation assays., Results: CTNS was expressed in human OCs derived from HDs; its expression was regulated during monocyte colony-stimulating factor- and receptor activator of nuclear factor-κB-dependent osteoclastogenesis and required for efficient bone resorption. Cysteamine had no impact on osteoclastogenesis but inhibited in vitro HD osteoclastic resorption; however, NC OC-mediated bone resorption was impaired only at high doses. Only low concentrations of cysteamine (50 μM) stimulated osteoblastic differentiation and maturation, while this effect was no longer observed at higher concentrations (200 µM)., Conclusion: CTNS is required for proper osteoclastic activity. In vitro low doses of cysteamine have beneficial antiresorptive effects on healthy human-derived OCs and may partly correct the CTNS-induced osteoclastic dysfunction in patients with NC. Moreover, in vitro low doses of cysteamine also stimulate osteoblastic differentiation and mineralization, with an inhibitory effect at higher doses, likely explaining, at least partly, the bone toxicity observed in patients receiving high doses of cysteamine.

Objectives: Alveolar decortication (AD) is a minimally invasive procedure that can be performed in the orthodontic office as an intervention to accelerate tooth movement. There is a gap in the literature evaluating the earlier and delayed responses after AD using lighter orthodontic forces in a rat model. Therefore, the aim of this study was to determine the effects of AD in the amount of orthodontic tooth movement and on alveolar bone remodelling in a rodent model, after 7 or 14 days., Materials and Methods: A total of 32 15-week-old male Wistar rats were used in four treatment groups: (1) orthodontic spring only (7 days), (2) orthodontic spring only + AD (7 days), (3) orthodontic spring only (14 days), and (4) orthodontic spring only + AD (14 days). A closed coil nickel-titanium spring delivering 8-10 g of force was used to move the molar mesially. Alveolar decortication was done using a high speed, quarter round bur adjacent to the left first maxillary molar, on the palatal alveolar bone. At each endpoint, rats were sacrificed and microfocus computed tomography and histological analysis were performed., Results: The spring + AD group presented with a significant increase in the rate of tooth movement when compared with spring only group, 7 and 14 days after the beginning of the experiments. In addition, the spring + AD group had a significant decrease in bone volume and tissue density and a significant increase in the trabecular spacing and the number of osteoclasts at 7 and 14 days. Furthermore, a fibrous tissue was found to replace the alveolar bone in the spring + AD group at day 14., Conclusion: Alveolar decortications enhanced bone remodelling around the tooth movement region and could be used as an adjunct surgical procedure to accelerate the rate of tooth movement.

Background: Inflammation and immune system alterations contribute to bone damage in many pathologies by inducing the differentiation of osteoclasts (OCs), the bone resorbing cells. This link is largely unexplored in chronic kidney disease (CKD) and haemodialysis (HD) patients, in which reduced renal function is accompanied by an increased inflammatory state and skeletal abnormality., Methods: We used ex vivo culture experiments to investigate the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) of CKD and HD patients, focusing on immune cell subsets and inflammatory cytokines such as LIGHT and receptor activator of nuclear factor κB ligand (RANKL)., Results: We observed spontaneous osteoclastogenesis with a significant increase in OC formation and bone resorbing activity in late-stage CKD and HD patients when compared with early-stage CKD patients and healthy donors, likely due to an increased expression of RANKL and LIGHT (homologous to Lymphotoxins exhibiting Inducible expression and competing with herpes simplex virus Glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) in PBMCs. Specific inhibition of these cytokines in PBMCs isolated from CKD stages 3b-5 and HD patients induced the reduction of OC formation in vitro. The phenotypic characterization of peripheral blood cells revealed a significant increase of OC precursors (CD14+CD11b+CD51/61+) and CD14+CD16+ monocytes in advanced CKD and HD patients compared with the control group., Conclusions: Our results suggest that circulating inflammatory monocytes from advanced CKD or HD patients trans differentiate into OCs in vitro and play a relevant role in mineral bone disorders and that LIGHT and RANKL represent new potential therapeutic targets in these settings., (© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objectives: The early phase of orthodontic tooth movement involves acute inflammatory response that may induce bone resorption. The aim of this study was to localize and quantify cells in the periodontium expressing proinflammatory mediators during orthodontically induced periapical root resorption of the rat mandibular molars., Materials and Methods: The levels of proinflammatory cytokines interleukin-1 (IL-1) α and β, tumor necrosis factor-α (TNF-α), inflammatory enzymes cyclooxygenase (COX) 1 and 2, and their product prostaglandin E2 (PGE2) in the root resorption site were compared to those in the corresponding area of the untreated periodontal ligament (PDL) of physiologically drifting teeth. Continuous heavy orthodontic force was applied to the mandibular first molar for 8 and 15 days while in occlusion to induce root resorption. Frozen sections including root resorption lacunae were analyzed for the activity of non-specific esterase (NSE) and tartrate-resistant acid phosphatase (TRAP) by enzyme histochemistry and for the expression of IL-1α, IL-1β, TNF-α, COX-1, COX-2, and PGE2 by immunohistochemistry., Results: The active root resorption lacunae had significantly more TRAP-positive multinucleated odontoclasts, whereas the number of NSE-positive cells of the monocyte-macrophage lineage did not differ from that in the control PDL. Several types of periodontal cells exhibited a significant increase in the expression of IL-1α, IL-1β, TNF-α, COX-2, and PGE2 in the root resorption zone, while COX-1 was rarely detected., Conclusions: These data suggest that proinflammatory mediators expressed in periodontal cells may synergistically promote apical root resorption in response to continuous heavy mechanical force applied to teeth., (© The Author 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Aim: The aim of this study is to evaluate and compare therapeutic effects of mesenchymal stem cell (MSCs) and osteoprotegerin (OPG) gene transfer applications on inhibition and/or repair of orthodontically induced inflammatory root resorption (OIIRR)., Materials and Methods: Thirty Wistar rats were divided into four groups as untreated group (negative control), treated with orthodontic appliance group (positive control), MSCs injection group, and OPG transfected MSCs [gene therapy (GT) group]. About 100g of orthodontic force was applied to upper first molar teeth of rats for 14 days. MSCs and transfected MSC injections were performed at 1st, 6th, and 11th days to the MSC and GT group rats. At the end of experiment, upper first molar teeth were prepared for genetical, scanning electron microscopy (SEM), fluorescent microscopy, and haematoxylin eosin-tartrate resistant acid phosphatase staining histological analyses. Number of total cells, number of osteoclastic cells, number of resorption lacunae, resorption area ratio, SEM resorption ratio, OPG, RANKL, Cox-2 gene expression levels at the periodontal ligament (PDL) were calculated. Paired t-test, Kruskal-Wallis, and chi-square tests were performed., Results: Transferred MSCs showed marked fluorescence in PDL. The results revealed that number of osteoclastic cells, resorption lacunae, resorption area ratio, RANKL, and Cox-2 were reduced after single MSC injections significantly (P < 0.05). GT group showed the lowest number of osteoclastic cells (P < 0.01), number of resorption lacunae, resorption area ratio, and highest OPG expression (P < 0.001)., Conclusions: Taken together all these results, MSCs and GT showed marked inhibition and/or repair effects on OIIRR during orthodontic treatment on rats., (© The Author 2016. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), and N -benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN 2 ) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K + multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC 50 = 0.3 μM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN 2 Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α; Tnfsf2 ) mRNA expression without affecting Il1b , Il6 , or oncostatin M ( Osm ) expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss., (© Society for Leukocyte Biology.)

Lutein, a member of the xanthophyll family of carotenoids, suppressed IL-1-induced osteoclast differentiation and bone resorption. The survival of mature osteoclasts was also suppressed by lutein in cultures. When lutein was added to the cultures of osteoblasts, lutein enhanced the formation of mineralized bone nodules by elevating BMP2 expression and inhibiting sclerostin expression. Lutein may be beneficial for bone health.

: Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue-which include mesenchymal, endothelial, and osteoclastic progenitors-enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells., Significance: This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals., (©AlphaMed Press.)

Osteoclasts are bone-resorbing cells and targets for treating bone diseases. Previously, we reported that distinct murine osteoclast precursor subsets, such as early blasts (CD31(hi) Ly-6C(-)), myeloid blasts (CD31(+) Ly-6C(+)), and monocytes (CD31(-) Ly-6C(hi)), respond differently to the osteoclastogenesis-inducing cytokines, macrophage colony-stimulating factor, and receptor activator for nuclear factor κB ligand. It is unknown, however, how these cell types respond to the osteoclast-stimulating inflammatory cytokine interleukin 1β. This study aims to investigate the effect of interleukin 1β on osteoclastogenesis derived from different mouse bone marrow precursors. Early blasts, myeloid blasts, and monocytes were sorted from mouse bone marrow cells using flow cytometry. Cells were cultured on plastic or on bone slices in the presence of macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand, without or with interleukin 1β (0.1-10 ng/ml). We found that interleukin 1β stimulated multinucleation and bone resorption of osteoclasts derived from the 3 precursors at different rates. The most large osteoclasts (>20 nuclei) and highest level of bone resorption (16.3%) was by myeloid blast-derived osteoclasts. Interleukin 1β particularly accelerated proliferation of early blasts and the most small osteoclasts (3-5 nuclei) formed on plastic. Life span varied among osteoclasts derived from different precursors: large osteoclasts (>2400 µm(2)) formed most rapidly (75 h) from myeloid blasts but had a short life span (30 h). Monocytes needed the longest time (95 h) for the generation of such large osteoclasts, but these cells had a longer life span (50 h). Our results indicate that the different bone marrow osteoclast precursors are differently stimulated by interleukin 1β with respect to proliferation, multinucleation, life span, and bone resorption., (© Society for Leukocyte Biology.)

Objectives: An anti-osteoporotic herbal formula ELP containing Epimedii Herba (E), Ligustri Lucidi Fructus (L) and Psoraleae Fructus (P) was studied to investigate the herb-herb interaction (or the possible synergistic effect) among each component and to identify the principal herbs in different modes of action., Methods: Rat osteoblast-like UMR-106 cells proliferation, rat MSCs-derived osteoblastogenesis and RANKL-induced RAW 264.7 osteoclastogenesis were adopted to investigate the bone-forming activity and bone-degrading activity of the herbal extracts. In the statistical aspect, a modified Tallarida's approach was employed to assess the synergistic effects in herbal combinations., Key Findings: Psoraleae Fructus is the active herb for stimulating osteoblast proliferation, and mild synergy was detected in the pairwise combinations EL, LP and formula ELP. In osteoblastogenesis assay, E and L are the principal herbs for promoting osteoblast differentiation and significant synergy was detected in the pairwise combination EL. For inhibiting osteoclast formation, L is the active herb and significant synergy was detected in the 3-way combination ELP., Conclusions: The presence of E, L and P is essential for ELP formula as a whole to act against osteoporosis via enhancing bone formation and reducing bone reabsorption. An optimal dosage at 150 μg/ml was proposed for ELP based on our findings., (© 2016 Royal Pharmaceutical Society.)

Vitamin B12 deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B12 deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B12 deficiency suggests that vitamin B12 plays an important role in Pi homeostasis.

Background: Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment., Methods: We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided., Results: High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation., Conclusions: Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: Histone deacetylase 1 (HDAC1) is highly expressed in the synovium of RA patients. Thus we aimed to investigate a novel HDAC inhibitor (HDACi), NW-21, designed to target HDAC1. The effect of NW-21 on osteoclast formation and activity, cytokine and chemokine expression in vitro and arthritis in mice was assessed., Methods: The effects on human osteoclast formation and activity derived from human blood monocytes stimulated with receptor activator of nuclear factor κB ligand (RANKL) and M-CSF were assessed. The anti-inflammatory activity of NW-21 was assessed using human monocytes stimulated with either TNF-α or lipopolysaccharide for 24 h. mRNA expression of monocyte chemotactic protein 1 (MCP-1), TNF-α, macrophage inflammatory protein 1α (MIP-1α), IL-1 and RANTES (regulated on activation, normal T cell expressed and secreted) was assessed. The effect of NW-21 in the collagen antibody-induced arthritis model was assessed following daily oral administration at 5 mg/kg/day. The HDAC1 inhibitors NW-21 and MS-275 were compared with a broad-acting HDACi, 1179.4b. Effects on inflammation and bone were assessed using paw inflammation scoring, histology and live animal micro-CT., Results: NW-21 suppressed osteoclast formation and activity as well as significantly reducing mRNA expression of MCP-1 and MIP-1α in monocytes stimulated by lipopolysaccharide or TNF-α (P < 0.05) in vitro. Only inhibitors that targeted HDAC1 (NW-21 and MS-275) reduced inflammation and bone loss in the arthritis model., Conclusion: The results indicate that inhibitors targeting HDAC1, such as NW-21 and MS-275, may be useful for treating RA, as such drugs can simultaneously target both inflammation and bone resorption., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: We earlier found that TNFα but not interleukin (IL)-17 is indispensable in the pathogenesis of spontaneously occurring rheumatoid arthritis (RA)-like disease in our newly established FcγRIIB-deficient C57BL/6 (B6) mouse model, designated KO1. Here, we examined the role of IL-6 in the pathogenesis of RA features in KO1, with particular reference to cartilage and bone destruction in arthritic joints., Methods: To evaluate the preventive effect of MR16-1, a rat anti-mouse IL-6 receptor (IL-6R) mAb, 4-month-old preclinical KO1 mice were divided into three groups: the first treated with MR16-1 for 6 months, the second treated with normal rat IgG, as a control, and the third left untreated. The incidence and severity of arthritis, immunological abnormalities, and transcription levels of receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), and inflammatory cytokines/chemokines in ankle joint tissues were compared among the three groups. The therapeutic effect of MR16-1 was examined by treating 7-month-old KO1 mice in the early stages of arthritis for 2 months., Results: Compared with the findings in the KO1 mice left untreated or treated with normal rat IgG, the development of arthritis was markedly suppressed in mice with MR16-1 treatment started from preclinical stages. The suppression was associated with the decrease in production of autoantibodies, rheumatoid factors (RF), and anti-cyclic citrullinated peptide (CCP). Histologically, marked synovitis, pannus formation, and cartilage and bone destruction associated with the increase in tartrate-resistant acid phosphatase (TRAP)-positive osteoclast generation were evident in the two control groups; however, these findings were virtually absent in MR16-1-treated mice. Real-time PCR analysis revealed that the up-regulated expression levels of MCP-1, IL-6, and TNFα, and the aberrantly high RANKL/OPG expression ratio in synovial joint tissues from the two control groups of mice with overt arthritis were significantly suppressed in MR16-1-treated mice. In mice with therapeutic MR16-1 treatment, there was no progression in arthritis score and the RANKL/OPG ratio in joint tissues was significantly suppressed., Conclusions: Administration of an anti-IL-6R mAb ameliorated spontaneously occurring RA-like disease features, indicating that IL-6, as well as TNFα, plays a pivotal role in the pathogenesis of RA in KO1 mice. Current studies showed that, in addition to the role in enhancing autoantibody production, IL-6 promotes synovial tissue inflammation and osteoclastogenesis, leading to the severe synovitis with pannus formation and the progressive cartilage and bone destruction in multiple joints.

Summary Objectives: This study evaluated the effect of low-level laser therapy (LLLT) on the tendency of rat molars to relapse following orthodontic tooth movement (OTM)., Material and Methods: Maxillary rat molars were moved mesially for 10 days. Animals were randomly assigned to group I (non-irradiated) or II (irradiation with LLLT). Appliances were removed, and the molars allowed to relapse for 1, 3, 5, 7, 14, or 21 days; rats in group II received LLLT according to a protocol. Bone density of periapical alveolar bone was measured using radiographs and Digora software. Dental supporting structures were examined histologically with haematoxylin and eosin and tartrate-resistant acid phosphatase., Results: In both groups, first molar relapse was rapid 1 day after the end of active treatment; by 21 days percentage relapse was measured as 86.11 per cent in group I, and 72.22 per cent in group II. Osteoclast number was highest at the end of active OTM, and thereafter successively decreased during the relapse phase in both groups. Decrease in number, and redistribution of osteoclasts occurred more rapidly in the non-irradiated than the LLLT group. Whilst molar relapse was generally less and osteoclast numbers generally higher in group II compared to group I, the differences were not significant. There was no significant difference in bone density between the two groups., Conclusions: These results indicate that LLLT may reduce the relapse tendency, possibly due in part to bone formation in previous tension areas, and to redistribution of osteoclasts following removal of orthodontic force. The role of LLLT in the prevention of orthodontic relapse requires further study., (© The Author 2014. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We examined the effects of heptamethoxyflavone (HMF), a citrus flavonoid on inflammatory bone resorption. HMF suppressed the osteoclast formation and PGE2 production induced by IL-1. In mouse calvarial organ cultures, HMF attenuated the bone resorption elicited by LPS. HMF suppressed bone resorption in the mandibular alveolar bone. HMF may protect against inflammatory bone loss such as periodontal disease.

GNF-2, a tyrosine kinase inhibitor, was developed to overcome imatinib-resistant mutations found in CML patients. Osteoclasts are the principal bone-resorbing cells that are responsible for bone diseases, such as osteoporosis, tumor-induced osteolysis, and metastatic cancers. In this study, we investigated the effect of GNF-2 on osteoclast development induced by RANKL and M-CSF. We found that GNF-2 inhibited osteoclast differentiation from BMMs. GNF-2 suppressed RANKL-induced NF-κB transcriptional activity and the induction of c-Fos and NFATc1, which are two key transcription factors in osteoclastogenesis. We also observed that GNF-2 dose-dependently inhibited the proliferation of osteoclast precursors through the suppression of the M-CSFR c-Fms. In addition, GNF-2 accelerated osteoclast apoptosis by inducing caspase-3 and Bim expression. Furthermore, GNF-2 interfered with actin cytoskeletal organization and subsequently blocked the bone-resorbing activity of mature osteoclasts. In agreement with its in vitro effects, GNF-2 reduced osteoclast number and bone loss in a mouse model of LPS-induced bone destruction. Taken together, our data reveal that GNF-2 possesses anti-bone-resorptive properties, suggesting that GNF-2 may have therapeutic value for the treatment of bone-destructive disorders that can occur as a result of excessive osteoclastic bone resorption.

Glucocorticoids (GCs) are potent drugs to treat rheumatoid arthritis but exert adverse skeletal effects. Compound A (CpdA) is a selective GC receptor modulator with an improved risk/benefit profile in mouse models of inflammation and bone loss. Here we tested whether CpdA also exerts bone-sparing effects under proinflammatory circumstances using the collagen-induced arthritis model, a murine model of rheumatoid arthritis. CpdA decreased disease activity, paw swelling, and the paw temperature by 43%, 12%, and 7%, respectively, but was less potent than dexamethasone (DEX), which reduced these parameters by 72%, 22%, and 10%, respectively. Moreover, T cells isolated from CpdA- and DEX-treated animals were less active based on proliferation rates after challenge with type II collagen and produced smaller amounts of interferon-γ and TNF as compared with T cells from PBS-treated mice. Histological assessment of the joints confirmed the weaker potency of CpdA as compared with DEX in preventing infiltration of inflammatory cells, induction of osteoclastogenesis, and destruction of articular cartilage. Due to the lack of GC-susceptible arthritis models, we were not able to fully address the bone-sparing potential of CpdA in inflammatory conditions. Nevertheless, the bone formation marker procollagen type 1 N-terminal peptide, a surrogate marker for GC-mediated suppression of bone formation, was significantly decreased by DEX in arthritic mice but not by CpdA. Our data indicate that CpdA moderately suppresses inflammation, whereas the concurrent effects on bone remain unknown. In light of its narrow therapeutic range, CpdA may be more useful as a molecular tool for dissecting GC actions rather than a therapeutic agent.

Systematic studies of autosomal dominant osteopetrosis (ADO) were followed by the identification of underlying mutations giving unique possibilities to perform translational studies. What was previously designated ADO1 turned out to be a high bone mass phenotype caused by a missense mutation in the first propeller of LRP5, a region of importance for binding inhibitory proteins. Thereby, ADO1 cannot be regarded as a classical form of osteopetrosis but must now be considered a disease of LRP5 activation. ADO (Albers-Schönberg disease, or previously ADO2) is characterized by increased number of osteoclasts and a defect in the chloride transport system (ClC-7) of importance for acidification of the resorption lacuna (a form of Chloride Channel 7 Deficiency Osteopetrosis). Ex vivo studies of osteoclasts from ADO have shown that cells do form normally but have reduced resorption capacity and an expanded life span. Bone formation seems normal despite decreased osteoclast function. Uncoupling of formation from resorption makes ADO of interest for new strategies for treatment of osteoporosis. Recent studies have integrated bone metabolism in whole-body energy homeostasis. Patients with ADO may have decreased insulin levels indicating importance beyond bone metabolism. There seems to be a paradigm shift in the treatment of osteoporosis. Targeting ClC-7 might introduce a new principle of dual action. Drugs affecting ClC-7 could be antiresorptive, still allowing ongoing bone formation. Inversely, drugs affecting the inhibitory site of LRP5 might stimulate bone formation and inhibit resorption. Thereby, these studies have highlighted several intriguing treatment possibilities, employing novel modes of action, which could provide benefits to the treatment of osteoporosis.

Objectives: MicroRNAs, a class of noncoding RNAs, play roles in human diseases. MicroRNA-223 (miR-223) is reported to play critical roles in osteoclastogenesis. The purpose of this study was to analyze the expression pattern of miR-223 in rheumatoid arthritis (RA) synovium and examine the suppression of osteoclastogenesis from human peripheral blood mononuclear cells (PBMC) by overexpression of miR-223., Methods: Expression of miR-223 in synovium from RA patients was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) and section in situ hybridization. MiR-223 was overexpressed in an osteoclastogenesis coculture system with PBMC and RA synovial fibroblast. At 3 weeks after transfection of double-stranded miR-223, the formation of tartrate-resistant acid phosphatase (TRAP)-stained multinucleated cells was analyzed to evaluate the inhibitory effect of miR-223 on osteoclastogenesis., Results: MiR-223 was more highly expressed in RA synovium than in osteoarthritis (OA) synovium due to the increased number of miR-223-positive cells in RA synovium. MiR-223 was expressed in the superficial and sublining layers, and macrophages, monocytes, and CD4 T cells also expressed miR-223. The number of TRAP-positive multinucleated cells was significantly decreased by overexpression of miR-223 in a dose-dependent manner. The expression of osteoclastogenesis marker genes was significantly down-regulated by miR-223 overexpression., Conclusion: MiR-223 is intensely expressed in RA synovium, and overexpression of miR-223 suppresses osteoclastogenesis in vitro. This study demonstrates the possibility of gene therapy with miR-223 to treat bone destruction in RA patients.

Objective: Decoy receptor 3 (DCR3) has been known to modulate immune functions of monocyte or macrophage. In the present study, we investigated the mechanism and the effect of DCR3 on RANK ligand (RANKL)-induced osteoclastogenesis., Methods: We treated cells with DCR3 in RANKL-induced osteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed by pit formation assay. The mechanism of inhibition was studied by biochemical analysis such as RT-PCR and immunoblotting. In addition, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis and apoptosis signalling were evaluated by immunoblotting and using flow cytometry., Results: DCR3 inhibited RANKL-induced TRAP(+) multinucleated cells and inhibited RANKL-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) nuclear translocation in RAW264.7 cells. Also, DCR3 significantly inhibited the bone-resorbing activity of mature osteoclasts. Moreover, DCR3 enhanced RANKL-induced cell apoptosis and enhanced RANKL-induced Fas ligand expression. The mechanisms were mediated via the intrinsic cytochrome c and activated caspase 9 apoptosis pathway., Conclusion: We postulated that the inhibitory activity of DCR3 on osteoclastogenesis occurs via down-regulation of RANKL-induced NFATc1 expression and induction of cell apoptosis. Our results postulated DCR3 as a possible new remedy against inflammatory bone destruction.

We examined the effects of β-cryptoxanthin, a typical carotenoid, on inflammatory periodontitis. β-Cryptoxanthin suppressed lipopolysaccharide (LPS)-induced osteoclast formation in co-cultures of bone marrow cells and osteoblasts. In a mouse model of periodontitis, it suppressed bone resorption in the mandibular alveolar bone in vitro and restored alveolar bone loss induced by LPS in vivo. β-Cryptoxanthin might protect against periodontal disease.

Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAK(Δmyeloid)), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAK(Δmyeloid) mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells.

Background: Staphylococcus aureus arthritis causes severe and rapid joint damage despite antibiotics. Thus, there is a need to identify new treatment targets in addition to antibiotics. Lately, interleukin 15 (IL-15) has been implicated both in osteoclastogenesis and in bacterial clearance-2 important issues in S. aureus-induced joint destruction. This has prompted us to investigate the importance of IL-15 in S. aureus-induced arthritis., Methods: Toxic shock syndrome toxin-1 producing S. aureus was intravenously inoculated in IL-15 knockout and wildtype mice and in wildtype mice treated with anti-IL-15 antibodies (aIL-15ab) or isotype control antibody., Results: Absence of IL-15, either in knockout mice or after treatment with aIL-15ab, significantly reduced weight loss compared with controls during the infection. The severity of synovitis and joint destruction was significantly decreased in IL-15 knockout and aIL-15ab treated mice compared with controls. In IL-15 knockout mice there was a reduced number of osteoclasts in the joints. The host's ability to clear bacteria was not influenced in the IL-15 knockout mice, but significantly increased after treatment with aIL-15ab., Conclusions: IL-15 is a mediator of joint destruction in S. aureus-induced arthritis and contributes to general morbidity, which makes this cytokine an interesting treatment target in addition to conventional antibiotics.

Noggin is an antagonist of bone morphogenetic proteins (BMP), and its overexpression causes suppressed osteoblastogenesis and osteopenia. Global inactivation of Noggin results in severe developmental defects and prenatal lethality, but the consequences of the conditional inactivation of Noggin on the postnatal skeleton are not known. To study the function of noggin in osteoblasts, we generated tissue-specific null Noggin mice by mating Noggin conditional mice, where the Noggin allele is flanked by loxP sequences, with mice expressing the Cre recombinase under the control of the osteocalcin promoter (Oc-Cre). Noggin conditional null mice exhibited decreased weight, shortened femoral length, and generalized osteopenia. Bone histomorphometric and microarchitectural analyses of distal femurs revealed decreased bone volume due to a reduced number of trabeculae in 1- and 3-month-old Noggin conditional null mice. Vertebral microarchitecture confirmed the osteopenia observed in Noggin conditional null mice. Osteoclast number was increased in 1-month-old male Noggin conditional null mice, and bone formation was increased in 3-month-old mice, but female mice did not exhibit increased bone remodeling. In conclusion, Noggin inactivation causes osteopenia, suggesting that BMP in excess have a detrimental effect on bone or that noggin has a BMP-independent role in skeletal homeostasis.

Osteoclasts are key players in the maintenance of bone, which is an endocrine target and organ. Bisphosphonates, used for the management of metastatic bone diseases and osteoporosis, suppress osteoclasts. However, the impact of continuously suppressed osteoclasts is unknown. In this study, mice received zoledronic acid (ZA) for 13 months, nearly half the lifespan of mice, and the effects of continual osteoclast suppression on the bone environment and oral wound healing were determined. ZA therapy suppressed osteoclasts, resulting in significantly more bone mass compared with control. Despite continuous and intense suppression of bone loss in mice receiving ZA, serum calcium levels were maintained in the normal range. No differences were noted in serum tartrate-resistant acid phosphatase (TRAP) 5b levels between ZA-treated and control mice. Histomorphometric analyses of bones revealed that ZA therapy significantly decreased osteoclasts on the bone surface but, instead, substantially increased TRAP(+) mononuclear cells and osteoclasts that were not on the bone surface. When oral trauma was induced, such TRAP(+) mononuclear and nonattached osteoclasts increased considerably with increased inflammatory cell infiltration in the wounds. As a result, oral wound healing was hindered at the connective tissue level. Healing of the epithelium was unaffected. These findings indicate that the continual suppression of osteoclasts does not affect serum calcium levels and that long-term ZA therapy stimulates nonattached osteoclast and TRAP(+) mononuclear cell formation that are expanded rapidly in response to oral trauma. Caution should be exercised when using the serum TRAcP5b to estimate the efficacy of antiresorptive therapy.

5-Lipoxygenase catalyzes leukotriene generation from arachidonic acid. The gene that encodes 5-lipoxygenase, Alox5, has been identified in genome-wide association and mouse Quantitative Trait Locus studies as a candidate gene for obesity and low bone mass. Thus, we tested the hypothesis that Alox5(-/-) mice would exhibit metabolic and skeletal changes when challenged by a high-fat diet (HFD). On a regular diet, Alox5(-/-) mice did not differ in total body weight, percent fat mass, or bone mineral density compared with wild-type (WT) controls (P < 0.05). However, when placed on a HFD, Alox5(-/-) gained more fat mass and lost greater areal bone mass vs. WT (P < 0.05). Microarchitectural analyses revealed that on a HFD, WT showed increases in cortical area (P < 0.01) and trabecular thickness (P < 0.01), whereas Alox5(-/-) showed no change in cortical parameters but a decrease in trabecular number (P < 0.05) and bone volume fraction compared with WT controls (P < 0.05). By histomorphometry, a HFD did not change bone formation rates of either strain but produced an increase in osteoclast number per bone perimeter in Alox5(-/-) mice (P < 0.03). In vitro, osteoclastogenesis of marrow stromal cells was enhanced in mutant but not WT mice fed a HFD. Gene expression for Rankl, Pparg, and Cox-2 was greater in the femur of Alox5(-/-) than WT mice on a HFD (P < 0.01), but these increases were suppressed in the Alox5(-/-) mice after 8 wk of treatment with celecoxib, a cyclooxygenase-2 inhibitor. In sum, there is a strong gene by environmental interaction for bone mass when mice lacking the Alox5 gene are fed a HFD.

The differentiation and functions of osteoclasts are regulated by receptor activator of nuclear factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL) system that stimulates osteoclasts formation. Macrophage colony-stimulating factor (M-CSF) is also essential for osteoclastogenesis. A recent immunocytochemical study reported that RANKL/RANK and M-CSF/c-fms were localized in the periodontal ligament of rat molars during experimental orthodontic tooth movement. The present study focused on the expressions of RANKL/RANK and M-CSF/c-fms in root resorption area during experimental tooth movement in rats. Forty 6-week-old male Wistar rats were subjected to an orthodontic force of 10 or 50 g with a closed coil spring (wire size: 0.005 inch, diameter: 1/12 inch) ligated to the maxillary first molar cleat by a 0.008 inch stainless steel ligature wire to induce a mesial tipping movement of the upper first molars. Experimental tooth movement was undertaken for 10 days. Each sample was sliced into 6 μm continuous sections in a horizontal direction and prepared for haematoxylin and eosin (H and E) and immunohistochemistry staining for tartrate-resistant acid phosphatase (TRAP), RANK, RANKL M-CSF, and c-fms in root resorption area. Statistical analysis was carried out using a Mann-Whitney U-test with a significance level of P<0.01. On days 7 and 10, immunoreactivity for RANKL/RANK and M-CSF/c-fms was detected in odontoclasts with an orthodontic force of 50 g, but not 10 g. Therefore, RANKL/RANK and M-CSF/c-fms systems may be involved in the process of root resorption by heavy orthodontic force.

Osteoclasts have traditionally been associated exclusively with catabolic functions that are a prerequisite for bone resorption. However, emerging data suggest that osteoclasts also carry out functions that are important for optimal bone formation and bone quality. Moreover, recent findings indicate that osteoclasts have different subtypes depending on their location, genotype, and possibly in response to drug intervention. The aim of the current review is to describe the subtypes of osteoclasts in four different settings: 1) physiological, in relation to turnover of different bone types; 2) pathological, as exemplified by monogenomic disorders; 3) pathological, as identified by different disorders; and 4) in drug-induced situations. The profiles of these subtypes strongly suggest that these osteoclasts belong to a heterogeneous cell population, namely, a diverse macrophage-associated cell type with bone catabolic and anabolic functions that are dependent on both local and systemic parameters. Further insight into these osteoclast subtypes may be important for understanding cell-cell communication in the bone microenvironment, treatment effects, and ultimately bone quality.

Local release of TGF-beta during times of high bone turnover leads to elevated levels within the bone microenvironment, and we have shown that TGF-beta suppresses osteoclast apoptosis. Therefore, understanding the influences of TGF-beta on bone resorbing osteoclasts is critical to the design of therapies to reduce excess bone loss. Here we investigated the mechanisms by which TGF-beta sustains suppression of osteoclast apoptosis. We found TGF-beta rapidly increased leukemia inhibitory factor (LIF) expression and secretion by phosphorylated mothers against decapentaplegic-dependent and -independent signaling pathways. TGF-beta also induced suppressor of cytokine signaling 3 (SOCS3) expression, which was required for TGF-beta or LIF to promote osteoclast survival by. Blocking LIF or SOCS3 blocked TGF-beta promotion of osteoclast survival, confirming that LIF and SOCS3 expression are necessary for TGF-beta-mediated suppression of osteoclast apoptosis. Investigation of the mechanisms by which LIF promotes osteoclast survival revealed that LIF-induced expression of Bcl-X(L) and repressed Bcl-2 interacting domain expression by activating MAPK kinase, AKT, and nuclear factor-kappaB pathways. Suppression of Janus kinase/signal transducer and activator of transcription signaling further increased Bcl-X(L) expression and enhanced osteoclast survival, supporting that this pathway is not involved in prosurvival effects of TGF-beta and LIF. These data show that TGF-beta coordinately induces LIF and SOCS3 to promote prosurvival signaling. This alters the ratio of prosurvival Bcl2 family member Bcl-X(L) to proapoptotic family member Bcl-2 interacting domain, leading to prolonged osteoclast survival.

The purpose of the study was to investigate the relationship between root resorption and repair in human premolars that had been orthodontically intruded. The objective was to examine these processes related to time and root development. Seventy-six premolars were divided into subgroups: 33 teeth were intruded and then extracted (G1); 25 teeth were intruded and then left in situ for varying periods before extraction (G2); 18 teeth served as the controls (G3). All teeth were examined by light microscopy. Using non-parametric statistical analysis, differences between the groups were examined with the Pearson chi-square test. Teeth in G1 and G2 had significantly more resorptive lesions, 55 and 64 per cent, respectively, than the controls of 11 per cent. Resorption was observed over the whole root surface and increased with time. The occurrence increased to 100 per cent in both experimental groups after 36 days of intrusion. The appearance of lesions in relation to root development showed no differences between G1 and G2. In the apical part of the root, total resorption of the dentine was sometimes observed, but no resorptions extended into the predentine. Resorptive lesions undergoing repair were seen in both groups, with significantly more repair in G2 (58 per cent) than in G1 (32 per cent). Active resorption and repair were sometimes seen at the same resorption site. Deposition of cellular and acellular cementum was found to the same extent over the whole root when repair took place. With time, resorption appeared over the whole root surface. In some teeth, resorptive activity continued up to 10 days after removal of forces but on the other hand, repair of the resorbed area sometimes started during active movement. The individual variation in repair was much wider compared with resorption. The predentine layer in the apical area appeared not to be affected by the resorptive process.

Context: Children with 21-hydroxylase deficiency (21-OHD) need chronic glucocorticoid (cGC) therapy to replace congenital deficit of cortisol synthesis. cGC therapy is the most frequent and severe form of drug-induced osteoporosis, and different mechanisms have been proposed to explain its pathogenesis., Objective: We investigated the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) from 18 children with 21-OHD on cGC therapy and 25 controls who never received GCs. We also evaluated the presence of circulating osteoclast precursors (OCPs) and the role of T cells in osteoclast formation., Results: Spontaneous osteoclastogenesis, without adding macrophage-colony stimulating factor and receptor activator of nuclear factor-kappaB ligand (RANKL), and significantly higher osteoclasts resorption activity occurred in 21-OHD patients. Conversely, macrophage-colony stimulating factor and RANKL were essential to trigger and sustain osteoclastogenesis in controls. Furthermore, in 21-OHD patients, we identified a significant percentage of CD11b-CD51/CD61 and CD51/61-RANK-positive cells, which are OCPs strongly committed. Additionally, we demonstrated a T cell-dependent osteoclastogenesis from 21-OHD patients' PBMCs. T cells from patients expressed high levels of RANKL and low levels of osteoprotegerin (OPG) with respect to controls. Moreover, 21-OHD patients had higher soluble RANKL and lower OPG serum levels compared with controls; thus, soluble RANKL to OPG ratio was significantly higher in patients than controls., Conclusions: The present study showed for the first time a high osteoclastogenic potential of PBMCs from 21-OHD patients on cGC therapy. This spontaneous osteoclastogenesis seems to be supported by both the presence of circulating OCPs and factors released by T cells.

Bone disease is a hallmark of multiple myeloma (MM). Occurring in the majority of MM patients, it is associated with bone pain, fractures, and hypercalcemia and has major impacts on quality of life. Furthermore, bone resorption activity has been shown to be an independent risk factor for overall survival in patients with symptomatic MM. Myeloma is characterized by a unique form of bone disease with lytic bone destruction that is not followed by reactive bone formation (uncoupling). This review focuses on recent advances in our understanding of the biology of osteoclast activation and osteoblast inhibition in MM, diagnostic standards, and recent progress in treatment options for myeloma bone disease. Translational research has enabled a rapid transfer of mechanistic insights from the bench to the bedside and will hopefully result in better treatment options and outcome for patients in near future.

Myeloma bone disease (MBD) leads to progressive destruction of the skeleton and is the most severe cause of morbidity in multiple myeloma. Its pathogenetic mechanisms are not fully understood, though the current evidence points to osteoclast (OC) hyperactivity coupled with defective osteoblast function unable to counteract bone resorption. OCs are generated in bone marrow by myeloid progenitors through increased levels of receptor activator of nuclear factor kappaB ligand and M-CSF, whose intracellular pathways propagate signals that activate sequential transcription factors, resulting in the production of major OC enzymes that drive specific functions such as acidification and degradation of the bone matrix. Osteolytic lesions, however, are not characterized by massive OC content, whereas malignant plasma cells, which are usually present in a high number, may occur as large multinucleated cells. The possibility that myeloma cells fuse and generate polykaryons in vivo is suggested by the in vitro formation of multinuclear cells that express tartrate-resistant acid phosphatase and produce pits and erosive lacunae on experimental osteologic substrates. Further, the detection in vivo of polykaryons with chromosome translocations typical of myeloma cells lends support to the view that myeloma polykaryons may act as functional OCs and participate in the skeletal destruction by resorbing bone.

Osteoporosis constitutes a major worldwide public health burden characterized by enhanced skeletal fragility. Bone metabolism is the combination of bone resorption by osteoclasts and bone formation by osteoblasts. Whereas increase in bone resorption is considered as the main contributor of bone loss that may lead to osteoporosis, this loss is accompanied by increased bone marrow adiposity. Osteoblasts and adipocytes share the same precursor cell and an inverse relationship exists between the two lineages. Therefore, identifying signaling pathways that stimulate mesenchymal stem cells osteogenesis at the expense of adipogenesis is of major importance for developing new therapeutic treatments. For this purpose, we identified by transcriptomic analysis the oxytocin receptor pathway as a potential regulator of the osteoblast/adipocyte balance of human multipotent adipose-derived stem (hMADS) cells. Both oxytocin (OT) and carbetocin (a stable OT analogue) negatively modulate adipogenesis while promoting osteogenesis in both hMADS cells and human bone marrow mesenchymal stromal cells. Consistent with these observations, ovariectomized (OVX) mice and rats, which become osteoporotic and exhibit disequilibrium of this balance, have significant decreased OT levels compared to sham-operated controls. Subcutaneous OT injection reverses bone loss in OVX mice and reduces marrow adiposity. Clinically, plasma OT levels are significantly lower in postmenopausal women developing osteoporosis than in their healthy counterparts. Taken together, these results suggest that plasma OT levels represent a novel diagnostic marker for osteoporosis and that OT administration holds promise as a potential therapy for this disease.

Objectives: The relationship between inflammation, destruction and new tissue formation leading to ankylosis, determines the severity and prognosis of patients with SpA. Recent data in mice and men suggest that new cartilage and bone formation and subsequent ankylosis are uncoupled from chronic inflammation. These data challenge the hypothesis that inflammation and tissue damage trigger an excessive repair response in SpA. We tested whether inhibition of bone erosion by targeting osteoclasts, would prevent or influence joint ankylosis in a mouse model., Methods: Male DBA/1 mice from different litters were caged together at the age of 8 weeks. Treatment with zoledronic acid (ZA) (100 ng/g) or placebo was started at the age of 10 weeks and administered every 2 weeks. Clinical incidence and severity of arthritis were evaluated twice a week until the age of 26 weeks. At this point, bone density measurements were performed, mice were sacrificed and severity of arthritis was evaluated by histology., Results: Treatment with ZA did not affect incidence or clinical severity of arthritis in male DBA/1 mice. ZA treatment significantly increased bone mineral density and content as demonstrated by dual X-ray densitometry and peripheral quantitative CT. However, the treatment did not affect histomorphological appearance of arthritis or ankylosis., Conclusions: These data suggest that bone erosion at the enthesis does not necessarily precede entheseal ankylosis. Therefore, these observations further support the concept that inflammation and new tissue formation in SpA are at least partially uncoupled events and may be different therapeutic targets.

Objective: Most breast cancer patients with bone metastases will receive bisphosphonate treatment. This post hoc analysis investigated whether early normalization of urinary N-telopeptide of type I collagen (NTX) levels during bisphosphonate therapy correlates with a long-term reduction in skeletal-related event (SRE) risk and mortality in patients with breast cancer., Methods: This was a retrospective subset analysis of a phase III randomized trial comparing i.v. zoledronic acid with pamidronate treatment in patients with bone metastases from breast cancer or multiple myeloma. Patients with breast cancer who had NTX assessments at baseline and at months 1 and 3 (n = 342) were classified as normal (NTX < 64 nmol/mmol creatinine) or elevated (NTX > or = 64 nmol/mmol creatinine). The relative risk for an SRE or death was estimated with Cox regression models., Results: Among the 328 evaluable patients treated with zoledronic acid, 196 patients (59.7%) had elevated baseline NTX, with 149 of those patients (76.0%) having normalized NTX levels and 31 patients (15.8%) having persistently elevated NTX levels at 3 months. The normalized NTX group had significantly lower risks for a first SRE, a first fracture or surgery to bone, or death than the group that had persistently elevated NTX levels., Conclusions: These results suggest that early normalization of elevated baseline NTX while receiving zoledronic acid is associated with longer event-free and overall survival times compared with persistently elevated NTX. Further analyses in cancer patients with elevated marker levels are warranted to confirm the implications of these findings.

Objectives: Periarticular osteoporosis and joint destruction are major complications in rheumatoid arthritis (RA), caused by osteoclast-mediated bone resorption. However, the mechanisms of monocyte/osteoclast maturation and role of RA endothelial cells (RAECs) in the control of osteoclastogenesis remain unclear. The present study was designed to determine the most important factors that influence monocyte accumulation and osteoclast formation among the many factors produced by RAEC., Methods: We analysed the expression profiles of various genes in human endothelial cells from various organs (RA synovium, umbilical vein, skin, liver sinusoid, renal glomerulus and brain) using oligonucleotide microarrays. Specifically, up-regulated gene in RAECs was assessed by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunostaining of RA synovia. Migration of monocytes was assessed by the chemotactic chamber EZ-TAXIScan. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation was observed by microscopy., Results: Among many epithelial-expressed factors, macrophage colony-stimulating factor (M-CSF) gene was abundantly expressed specifically in RAECs. Genes of fibroblast growth factor-2, interleukin-6 and osteoprotegerin were also overexpressed in RAECs. Migration of monocytes and osteoclast formation in co-cultures promoted by culture supernatants of RAECs were inhibited by M-CSF neutralizing antibody., Conclusions: M-CSF produced by RAECs is involved in osteoclastogenesis from monocytes, migration and TRAP-positive MNC formation.

Objectives: Receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been demonstrated to be critical regulators of osteoclast generation and activity. In addition, RANKL has been implicated as an important mediator of bone erosion in rheumatoid arthritis (RA). However, the expression of RANKL and OPG at sites of pannus invasion into bone has not been examined. The present study was undertaken to further elucidate the contribution of this cytokine system to osteoclastogenesis and subsequent bone erosion in RA by examining the pattern of protein expression for RANKL, OPG and the receptor activator of NF-kappaB (RANK) in RA at sites of articular bone erosion., Methods: Tissues from 20 surgical procedures from 17 patients with RA were collected as discarded materials. Six samples contained only synovium or tenosynovium remote from bone, four samples contained pannus-bone interface with adjacent synovium and 10 samples contained both synovium remote from bone and pannus-bone interface with adjacent synovium. Immunohistochemistry was used to characterize the cellular pattern of RANKL, RANK and OPG protein expression immediately adjacent to and remote from sites of bone erosion., Results: Cellular expression of RANKL protein was relatively restricted in the bone microenvironment; staining was focal and confined largely to sites of osteoclast-mediated erosion at the pannus-bone interface and at sites of subchondral bone erosion. RANK-expressing osteoclast precursor cells were also present in these sites. OPG protein expression was observed in numerous cells in synovium remote from bone but was more limited at sites of bone erosion, especially in regions associated with RANKL expression., Conclusions: The pattern of RANKL and OPG expression and the presence of RANK-expressing osteoclast precursor cells at sites of bone erosion in RA contributes to the generation of a local microenvironment that favours osteoclast differentiation and activity. These data provide further evidence implicating RANKL in the pathogenesis of arthritis-induced joint destruction.

It has not yet been clarified how sex hormones affect craniofacial bone development immediately after birth. The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial bone development immediately after birth, in terms of the internal structure of the mandible in newborn mice with orchiectomy (ORX) and ovariectomy (OVX). ORX, OVX and a sham-operation were performed on 40 five-day-old C57BL/6J mice. Eight weeks after surgery, each mandible was subjected to histomorphometric analysis of trabecular (Tr) and cortical (Ct) bone mineral density (BMD) by peripheral quantitative computed tomography (pQCT). In the experimental groups, a significant reduction in BMD was found in comparison with the control groups. In histomorphometric analysis, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the condyle and the thickness of the condylar cartilage layer was significantly greater in the experimental mice than in the controls. Trabecular bone volume of the condyle measured on azocarmine-aniline blue (AZAN) sections was significantly less in the experimental mice than in the controls. These results indicate that mandibular growth is inhibited by sex hormone disturbances and the relevant internal structures changed. The findings show that sex hormones are one of the key determinants of mandibular growth and development immediately after birth.