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Osteoclastic Bone Resorption (osteoclastic + bone_resorption)
Selected AbstractsDirect and Indirect Actions of Fibroblast Growth Factor 2 on Osteoclastic Bone Resorption in CulturesJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2000Hiroshi Kawaguchi M.D., Ph.D. Abstract Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10,9 M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10,8 M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (,10,12 M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10,11 M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10,9 M , 10,8 M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (,10,12 M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (,10,9 M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently. [source] Osteoclastic bone resorption induced by innate immune responsesPERIODONTOLOGY 2000, Issue 1 2010Masanori Koide First page of article [source] Pharmacologic profile of zoledronic acid: A highly potent inhibitor of bone resorptionDRUG DEVELOPMENT RESEARCH, Issue 4 2002Jonathan R. Green Abstract Bisphosphonates are effective in treating benign and malignant skeletal diseases characterized by enhanced osteoclastic bone resorption (i.e., osteoporosis, Paget's disease, tumor-induced osteolysis). The nitrogen-containing bisphosphonate pamidronate is currently the standard treatment for hypercalcemia of malignancy (HCM) and skeletal complications of bone metastases. Zoledronic acid, a novel nitrogen-containing bisphosphonate with an imidazole substituent, has demonstrated more potent inhibition of osteoclast-mediated bone resorption than all other bisphosphonates, including pamidronate, in both in vitro and in vivo preclinical models. Zoledronic acid inhibited ovariectomy-induced bone loss in adult monkeys and rats, and long-term treatment prevented skeletal turnover and subsequent bone loss, reduced cortical porosity, and increased mechanical strength. Zoledronic acid also significantly inhibited bone loss associated with arthritis, bone metastases, and prosthesis loosening. The increased potency of zoledronic acid vs. pamidronate has been demonstrated clinically: zoledronic acid (4 or 8 mg iv) was superior to pamidronate (90 mg iv) in normalizing corrected serum calcium in patients with HCM. In patients with bone metastases, low doses of zoledronic acid (, 2 mg) suppressed bone resorption markers , 50% below baseline, whereas pamidronate 90 mg yielded only 20 to 30% suppression. Importantly, the increased potency of zoledronic acid is not associated with an increased incidence of local (bone) or systemic adverse events. Zoledronic acid does not impair bone mineralization and, compared with pamidronate, has a greater renal and intestinal tolerability therapeutic index. Thus, based on preclinical assays and clinical data, zoledronic acid is the most potent bisphosphonate tested to date. Given its potency and excellent safety profile, zoledronic acid is now poised to become the new standard of treatment for HCM and metastatic bone disease. Drug Dev. Res. 55:210,224, 2002. © 2002 Wiley-Liss, Inc. [source] Identification of a novel mutation of SH3BP2 in cherubism and demonstration that SH3BP2 mutations lead to increased NFAT activation ,,HUMAN MUTATION, Issue 7 2006Steven A. Lietman Abstract We describe a novel missense mutation (Aspartic acid to Asparagine, p.D419N (g.1371G>A, c.1255G>A) within exon 9 of SH3BP2 in a patient with cherubism, an autosomal dominant syndrome characterized by excessive osteoclastic bone resorption of the jaw. Two siblings and the father were carriers but lacked phenotypic features. Transient expression of p.D419N (c.1255G>A), as well as three previously described exon 9 mutations from cherubism patients (p.R415Q (c.1244G>A), p.D420E (c.1259G>A), and p.P418R (c.1253C>G)) increased activity of NFAT (nuclear factor of activated T-cells), an osteoclastogenic mediator, indicating that cherubism results from gain of function mutations in SH3BP2. Published 2006 Wiley-Liss, Inc. [source] Osteoblast-Derived TGF-,1 Stimulates IL-8 Release Through AP-1 and NF-,B in Human Cancer Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008Yi-Chin Fong Abstract Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)-8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone-derived growth factors on the IL-8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast-derived TGF-,1 is associated with osteolytic bone diseases. Materials and Methods: IL-8 mRNA levels were measured using RT-PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF-,1, BMP-2, and IGF-1. DNA affinity protein-binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c- fos, c- jun, p65, and p50 to the IL-8 promoter. A transient transfection protocol was used to examine IL-8, NF-,B, and activator protein (AP)-1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL-8, AP-1, and NF-,B promoter in human cancer cells. Osteoblasts were transfected with TGF-,1, BMP-2, or IGF-1 small interfering RNA, and the medium was collected after 48 h. TGF-,1 but not BMP-2 or IGF-1 siRNA inhibited OBCM-induced IL-8 release in human cancer cells. In addition, TGF-,1 also directly induced IL-8 release in human cancer cells. Activation of AP-1 and NF-,B DNA-protein binding and MAPKs after TGF-,1 treatment was shown, and TGF-,1,induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF-,1, BMP-2, and IGF-1. TGF-,1 is the major contributor to the activation of extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to the activation of AP-1 and NF-,B on the IL-8 promoter and initiation of IL-8 mRNA and protein release, thereby promoting osteoclastogenesis. [source] Retention, Distribution, and Effects of Intraosseously Administered Ibandronate in the Infarcted Femoral Head,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007James Aya-ay Abstract The local distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted femoral heads were studied. Intraosseous administration effectively delivered and distributed ibandronate in the infarcted femoral heads and decreased the femoral head deformity in a large animal model of Legg-Calve-Perthes disease. Introduction: Bisphosphonate therapy has gained significant attention for the treatment of ischemic osteonecrosis of the femoral head (IOFH) because of its ability to inhibit osteoclastic bone resorption, which has been shown to contribute to the pathogenesis of femoral head deformity. Because IOFH is a localized condition, there is a need to explore the therapeutic potential of local, intraosseous administration of bisphosphonate to prevent the femoral head deformity. The purpose of this study was to investigate the distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted head. Materials and Methods: IOFH was surgically induced in the right femoral head of 27 piglets. One week later, a second operation was performed to inject 14C-labeled or unlabeled ibandronate directly into the infarcted head. 14C-ibandronate injected heads were assessed after 48 h, 3 weeks, or 7 weeks later to determine the distribution and retention of the drug using autoradiography and liquid scintillation analysis. Femoral heads injected with unlabeled ibandronate were assessed at 7 weeks to determine the degree of deformity using radiography and histomorphometry. Results: Autoradiography showed that 14C-Ibandronate was widely distributed in three of the four heads examined at 48 h after the injection. Liquid scintillation analysis showed that most of the drug was retained in the injected head, and almost negligible amount of radioactivity was present in the bone and organs elsewhere at 48 h. At 3 and 7 weeks, 50% and 30% of the 14C-drug were found to be retained in the infarcted heads, respectively. Radiographic and histomorphometric assessments showed significantly better preservation of the infarcted heads treated with intraosseous administration of ibandronate compared with saline (p < 0.001). Conclusions: This study provides for the first time the evidence that local intraosseous administration is an effective route to deliver and distribute ibandronate in the infarcted femoral head to preserve the femoral head structure after ischemic osteonecrosis. In a localized ischemic condition such as IOFH, local administration of bisphosphonate may be preferable to oral or systemic administration because it minimizes the distribution of the drug to the rest of the skeleton and bypasses the need for having a restored blood flow to the infarcted head for the delivery of the drug. [source] Expression of Mouse Osteoclast K-Cl Co-Transporter-1 and Its Role During Bone Resorption,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2006Hiroshi Kajiya PhD Abstract To assess the role of Cl, transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl, co-transporters (KCCs). KCC1 and chloride channel-7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)-butylindazone (DIOA), KCC1 antisense oligo-nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl, extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl, extrusion mechanism accompanying the H+ extrusion during bone resorption. Introduction: Mice with deficient chloride (Cl,) channels, ClC7, show severe osteopetrosis, resulting from impairment of Cl, extrusion during osteoclastic bone resorption. However, the expression and functional role of Cl, transporters other than ClC7 in mammalian osteoclasts is unknown. The aim of this study was to determine expression of K+/Cl, co-transporters (KCCs) and their functional role for bone resorption in mouse osteoclasts. Materials and Methods: Mouse osteoclasts were derived from cultured bone marrow cells with macrophage-colony stimulating factor (M-CSF) and RANKL or from co-culture of bone marrow cells and primary osteoblasts. We examined the expression of Cl, transporters using RT-PCR, immunochemical, and Western blot methods. The effects of Cl, transport inhibitors on H+ and Cl, extrusion were assessed by measuring intracellular H+ ([H+]i) and Cl, ([Cl,]i). The effects of inhibitors, antisense oligo-nucleotides, and siRNA for Cl, transporters on bone resorption activities were evaluated using a pit formation assay. Results and Conclusions: Mouse osteoclasts express not only ClC7 but also K+/Cl, co-transporter mRNA. The existence of KCC1 in the cell membrane of mouse osteoclasts was confirmed by immunochemical staining and Western blot analysis. KCC inhibitors and Cl, channels blockers increased [Cl,]i and [H+]i in resorbing osteoclasts, suggesting that the suppression of Cl, extrusion through KCC and Cl, channels leads to reduced H+ extrusion activity. The combination of both inhibitors greatly suppressed these extrusion activities. KCC inhibitors and Cl, channel blockers also decreased osteoclastic bone resorption in our pit area essay. Furthermore, KCC1 antisense oligo-nucleotides and siRNA suppressed osteoclastic pit formation as well as treatment of ClC7 inhibitors. These results indicate that K+/Cl, co-transporter-1 expressed in mouse osteoclasts acts as a Cl, extruder and plays an important role for H+ extrusion during bone resorption. [source] Disease Status in Autosomal Dominant Osteopetrosis Type 2 Is Determined by Osteoclastic Properties,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2006Kang Chu Abstract Asymptomatic gene carriers and clinically affected ADO2 subjects have the same ClCN7 mutation. We examined osteoclastic bone resorption in vitro as well as osteoclast formation, several markers, acid secretion, and cytoskeletal structure. We found that ADO2 expression results from osteoclast specific properties. Introduction: Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder that results from heterozygous mutations in the ClCN7 gene. However, of those individuals with a ClCN7 mutation, one third are asymptomatic gene carriers who have no clinical, biochemical, or radiological manifestations. Disease severity in the remaining two thirds is highly variable. Materials and Methods: Human peripheral blood mononuclear cells were isolated and differentiated into osteoclasts by stimulation with hRANKL and human macrophage-colony stimulating factor (hM-CSF). Study subjects were clinically affected subjects, unaffected gene carriers, and normal controls (n = 6 in each group). Pit formation, TRACP staining, RANKL dose response, osteoclast markers, acid secretion, F-actin ring, and integrin ,v,3 expression and co-localization were studied. Results: Osteoclasts from clinically affected subjects had severely attenuated bone resorption compared with those from normal controls. However, osteoclasts from unaffected gene carriers displayed similar bone resorption to those from normal controls. In addition, the resorption lacunae from both unaffected gene carriers and normal controls appeared much earlier and spread much more rapidly than those from clinically affected subjects. As time progressed, the distinction between clinically affected subjects and the other two groups increased. No significant difference was found in acidic secretion or osteoclast formation between the three groups. Osteoclast cytoskeletal organization showed no difference between the three groups but there was low cellular motility in clinically affected subjects. Conclusions: Osteoclasts from the unaffected gene carriers, in contrast to those from the clinically affected subjects, functioned normally in cell culture. This finding supports the hypothesis that intrinsic osteoclast factors determine disease expression in ADO2. Further understanding of this mechanism is likely to lead to the development of new approaches to the treatment of clinically affected patients. [source] Metabolic Acidosis Stimulates RANKL RNA Expression in Bone Through a Cyclo-oxygenase-Dependent Mechanism,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2003Kevin K Frick Abstract Metabolic acidosis inhibits osteoblastic bone formation and stimulates osteoclastic resorption. To determine whether acidosis alters expression of RNA for the osteoclastic differentiation factor RANKL, mouse calvariae were incubated in neutral or physiologically acidic media. Acidosis resulted in a significant cyclo-oxygenase-dependent increase in RANKL RNA levels, which would be expected to induce the associated increase in bone resorption. Introduction: Metabolic acidosis increases net calcium efflux from bone, initially through physicochemical mechanisms and later through predominantly cell-mediated mechanisms. Acidosis decreases osteoblastic bone formation and increases osteoclastic resorption. The growth and maturation of osteoclasts, derived from hematopoietic precursors in the monocyte/macrophage lineage, are dependent on the interplay of a number of factors. Commitment of pre-osteoclasts to osteoclasts is induced by the interaction of the osteoclastic cell-surface receptor RANK with a ligand expressed by osteoblasts, RANKL. The RANK/RANKL interaction not only initiates a differentiation cascade that culminates in mature bone-resorbing osteoclasts but also increases osteoclastic resorptive capacity and survival. Methods: To test the hypothesis that metabolic acidosis increases expression of RANKL, we cultured neonatal mouse calvariae in acidic (initial medium pH ,7.1 and [HCO3,] ,11 mM) or neutral (initial medium pH ,7.5 and [HCO3,] ,25 mM) medium for 24 and 48 h. We determined the relative expression of RANKL RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitated the expression by Northern analysis. Results: In this model of metabolic acidosis, there was significantly increased expression of RANKL RNA at both 24 (2-fold) and 48 h (5-fold) compared with respective controls. Net calcium efflux from bone was also increased in acidic medium compared with control medium. At 48 h, net calcium efflux correlated directly with RANKL expression (r = 0.77, n = 15, p < 0.001). Inhibition of prostaglandin synthesis with indomethacin blocked the acid-induced increase in RANKL RNA as well as the increased calcium efflux. Conclusions: Metabolic acidosis induces osteoblastic prostaglandin synthesis, followed by autocrine or paracrine induction of RANKL. This increase in RANKL would be expected to augment osteoclastic bone resorption and help explain the increase in cell-mediated net calcium efflux. [source] Effects of Genistein and Hormone-Replacement Therapy on Bone Loss in Early Postmenopausal Women: A Randomized Double-Blind Placebo-Controlled Study,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2002Nunziata Morabito Abstract The natural isoflavone phytoestrogen genistein has been shown to stimulate osteoblastic bone formation, inhibit osteoclastic bone resorption, and prevent bone loss in ovariectomized rats. However, no controlled clinical trial has been performed so far to evaluate the effects of the phytoestrogen on bone loss in postmenopausal women. We performed a randomized double-blind placebo-controlled study to evaluate and compare with hormone-replacement therapy (HRT) the effect of the phytoestrogen genistein on bone metabolism and bone mineral density (BMD) in postmenopausal women. Participants were 90 healthy ambulatory women who were 47,57 years of age, with a BMD at the femoral neck of <0.795 g/cm2. After a 4-week stabilization on a standard fat-reduced diet, participants of the study were randomly assigned to receive continuous HRT for 1 year (n = 30; 1 mg of 17,-estradiol [E2] combined with 0.5 mg of norethisterone acetate), the phytoestrogen genistein (n = 30; 54 mg/day), or placebo (n = 30). Urinary excretion of pyridinoline (PYR) and deoxypyridinoline (DPYR) was not significantly modified by placebo administration either at 6 months or at 12 months. Genistein treatment significantly reduced the excretion of pyridinium cross-links at 6 months (PYR = ,54 ± 10%; DPYR = ,55 ± 13%; p < 0.001) and 12 months (PYR = ,42 ± 12%; DPYR = ,44 ± 16%; p < 0.001). A similar and not statistically different decrease in excretion of pyridinium cross-links was also observed in the postmenopausal women randomized to receive HRT. Placebo administration did not change the serum levels of the bone-specific ALP (B-ALP) and osteocalcin (bone Gla protein [BGP]). In contrast, administration of genistein markedly increased serum B-ALP and BGP either at 6 months (B-ALP = 23 ± 4%; BGP = 29 ± 11%; p < 0.005) or at 12 months (B-ALP = 25 ± 7%; BGP = 37 ± 16%; p < 0.05). Postmenopausal women treated with HRT had, in contrast, decreased serum B-ALP and BGP levels either at 6 months (B-ALP = ,17 ± 6%; BGP = ,20 ± 9%; p < 0.001) or 12 months (B-ALP = ,20 ± 5%; BGP = ,22 ± 10%; p < 0.001). Furthermore, at the end of the experimental period, genistein and HRT significantly increased BMD in the femur (femoral neck: genistein = 3.6 ± 3%, HRT = 2.4 ± 2%, placebo = ,0.65 ± 0.1%, and p < 0.001) and lumbar spine (genistein = 3 ± 2%, HRT = 3.8 ± 2.7%, placebo = ,1.6 ± 0.3%, and p < 0.001). This study confirms the genistein-positive effects on bone loss already observed in the experimental models of osteoporosis and indicates that the phytoestrogen reduces bone resorption and increases bone formation in postmenopausal women. [source] High-Turnover Periprosthetic Bone Remodeling and Immature Bone Formation Around Loose Cemented Total Hip JointsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2001Michiaki Takagi Abstract Aseptic loosening and periprosthetic osteolysis are the major problems awaiting solution in total hip surgery. The clinical investigation focused on the analysis of periprosthetic bone remodeling to clarify one important key event in the cascade of periprosthetic connective tissue weakening and osteolysis around loose artificial hip joints. Twelve acetabular bone samples adjacent to granulomatous synovial-like membrane of loose hip prosthesis were retrieved at revision surgery and processed for Villanueva bone staining for morphological observation and bone histomorphometric analysis. Eight well-fixed bony samples were used as control. Although osteoclastic surface and eroded surface by osteoclasts were evident in the periprosthetic bone from loose hip joints (p = 0.003 and p = 0.027), increased osteoid/low-mineralized bone matrix (p < 0.001) and osteoid width (p < 0.001) also were significant findings in structural analysis. In addition, not only elevated mineral apposition rate (MAR; p = 0.044) but also increased mineralizing surface (p = 0.044) and bone formation rate (BFR; p = 0.002) in loose periprosthetic bones were shown in dynamic data analysis. These results were confirmed by precise morphological observation by confocal laser scanning microscopy. Active coupling of bone formation and resorption and increased osteocytes with abundant bone canalicular projections were found in combined with the presence of immature bone matrices (osteoid and low-mineralized bone areas) in periprosthetic bones from loose hip joints. These results indicated that active osteoclastic bone resorption and/or defective bone formation are coupled with monocyte/macrophage-mediated foreign body-type granuloma in the synovial-like interface membrane of loose hip joints. Thus, this unique high-turnover periprosthetic bone remodeling with bad bone quality probably is caused by the result of cellular host response combined with inappropriate cyclic mechanical loading. The fragile periprosthetic bone may contribute to hip prosthesis loosening. [source] Prostaglandin E2 Induces Expression of Receptor Activator of Nuclear Factor,,B Ligand/Osteoprotegrin Ligand on Pre-B Cells: Implications for Accelerated Osteoclastogenesis in Estrogen DeficiencyJOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2000Masahiro Kanematsu Abstract Estrogen deficiency causes bone loss as a result of accelerated osteoclastic bone resorption. It also has been reported that estrogen deficiency is associated with an increase in the number of pre-B cells in mouse bone marrow. The present study was undertaken to clarify the role of altered B lymphopoiesis and of the receptor activator of nuclear factor-,B ligand (RANKL), a key molecule in osteoclastogenesis, in the bone loss associated with estrogen deficiency. In the presence of prostaglandin E2 (PGE2), the activity to form tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells was significantly greater in bone marrow cells derived from ovariectomized (OVX) mice than in those from sham-operated mice. Northern blot analysis revealed that PGE2 increased the amount of RANKL messenger RNA (mRNA) in bone marrow cells, not only adherent stromal cells but nonadherent hematopoietic cells; among the latter, RANKL mRNA was more abundant in OVX mice than in sham-operated mice and was localized predominantly in B220+ cells. Flow cytometry revealed that most B220+ cells in bone marrow were RANKL positive and that the percentage of RANKL-positive, B220low cells was higher in bone marrow from OVX mice than in that from sham-operated mice. The increase in the expression of RANKL and the percentage of these cells in OVX mice was abolished by the administration of indomethacin in vivo. PGE2 also markedly increased both the level of RANKL mRNA and cell surface expression of RANKL protein in the mouse pre-B cell line 70Z/3. Finally, osteoclastogenic response to PGE2 was reduced markedly by prior depletion of B220+ cells, and it was restored by adding back B220+ cells. Taken together with stimulated cyclo-oxygenase (COX)-2 activity by tumor necrosis factor , (TNF-,) and interleukin-1 (IL-1) in estrogen deficiency, these results suggest that an increase in the number of B220+ cells in bone marrow may play an important role in accelerated bone resorption in estrogen deficiency because B220+ cells exhibit RANKL on the cell surface in the presence of PGE2, thereby leading to accelerated osteoclastogenesis. [source] Direct Measurement of Hormone-Induced Acidification in Intact BoneJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2000Glenn S. Belinsky Abstract Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6 -2,-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively. [source] Study of the Nonresorptive Phenotype of Osteoclast-like Cells from Patients with Malignant Osteopetrosis: A New Approach to Investigating PathogenesisJOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2000Adrienne M. Flanagan Abstract Osteopetrosis manifests as failure of osteoclastic bone resorption. The cause of the disease lies either in the hematopoietic lineage or in the bone marrow stromal microenvironment. It has not been possible to define the cell type involved in the various forms of the human disease because of the inability to form human osteoclasts in vitro. Using the recently described method for generating human osteoclasts from peripheral blood in coculture with rat osteoblastic UMR 106 cells, we demonstrate that a defect lies in the mature osteoclast-like cells in four cases of this disease. Control and osteopetrotic cocultures generated large numbers of osteoclast-like cells (calcitonin and vitronectin receptor positive, and F-actin ring,positive cells) with similar morphology. Bone resorption did not occur in three of the four osteopetrotic cultures. In case 1, in which bone resorption was identified, the area of resorption was negligible compared with the number of osteoclast-like cells in the culture and was detected only by scanning electron microscopy. In contrast, up to 20% of the bone surface in controls was resorbed. The normal and osteopetrotic osteoclast-like cells had a similar phenotype except that two of the osteopetrotic cases did not express CD44 and two expressed CD44 weakly, whereas CD44 was strongly expressed in the controls. This study shows that it is possible to reproduce in vitro the pathological features of human osteopetrosis, and the assay provides a means of acquiring a greater understanding of the pathogenesis of human osteopetrosis. (J Bone Miner Res 2000;15:352,360) [source] Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2002J. Jeffrey Goater Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone-implant interface, which is caused by wear debris-induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the efficacy of stably transfected fibroblast-like synoviocytes (FLS) expressing OPG in preventing wear debris-induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/106 cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly, implantation of 107 FLS,OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS-LacZ control cells, which persisted for 3 days as determined by X-gal staining, together with the Ti particles, had a 6-fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS-OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] The role of osteoclast differentiation in aseptic loosening,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2002Edward M. Greenfields The major cause of orthopaedic implant loosening is thought to be accelerated osteoclastic bone resorption due to the action of cytokines produced in response to phagocytosis of implant-derived wear particles. This accelerated osteoclastic bone resorption could be due to increases in any of the following processes: recruitment of osteoclast precursors to the local microenvironment, differentiation of precursors into mature multinucleated osteoclasts, activation of mature osteoclasts, and/or survival of osteoclasts. Our studies have focused on differentiation and survival to complement work by others who have focused on recruitment of precursors and activation. Taken together, our studies and those of other investigators provide strong evidence that increased recruitment of osteoclast precursors and their subsequent differentiation play major roles in wear particle-induced osteolysis. In contrast, increased osteoclast activation and survival appear to play minor roles. These studies suggest that development of therapeutic interventions that reduce either recruitment or differentiation of osteoclast precursors would improve the performance of orthopaedic implants. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] PTHrP-independent hypercalcemia with increased proinflammatory cytokines and bone resorption in two children with CD19-negative precursor B acute lymphoblastic leukemiaPEDIATRIC BLOOD & CANCER, Issue 7 2007Hidetaka Niizuma MD Abstract Hypercalcemia in childhood acute lymphoblastic leukemia (ALL) is rare and occasionally associated with parathyroid hormone-related protein (PTHrP). However, the pathogenesis of PTHrP-independent hypercalcemia remains unclear. We report two children with precursor B ALL who had marked hypercalcemia (15.8 and 16.6 mg/dl, respectively) and disseminated osteolysis. Serum tumor necrosis factor-, (TNF-,) and IL-6 were markedly elevated, whereas 1,25(OH)2 vitamin D3, intact PTH and PTHrP were decreased or undetected. Analysis of urinary deoxypyridinoline (DPY) or bone biopsy of the osteolytic lesion showed an increased bone resorption, and administration of bisphosphonate improved the hypercalcemia. Patients had ALL with immunophenotype positive for CD10, CD34, and HLA-DR but negative for CD19 and obtained remission with chemotherapy. These findings suggest that increased osteoclastic bone resorption via stimulation with TNF-, and IL-6 may be mechanism causing PTHrP-independent hypercalcemia in some patients with precursor B ALL lacking CD19 expression. Pediatr Blood Cancer 2007;49:990,993. © 2006 Wiley-Liss, Inc. [source] Adenosine A1 receptors regulate bone resorption in mice: Adenosine A1 receptor blockade or deletion increases bone density and prevents ovariectomy-induced bone loss in adenosine A1 receptor,knockout miceARTHRITIS & RHEUMATISM, Issue 2 2010Firas M. Kara Objective Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A1 receptor blockade or deletion on bone density. Methods The bone mineral density (BMD) in adenosine A1 receptor,knockout (A1R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A1R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. Results Micro-CT analysis of bones from A1R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A1R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A1R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A1R-knockout mice. Conclusion These results suggest that the adenosine A1 receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening. [source] The ratio of circulating osteoprotegerin to RANKL in early rheumatoid arthritis predicts later joint destructionARTHRITIS & RHEUMATISM, Issue 6 2006P. P. Geusens Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease that may result in debilitating joint deformities with destruction of bone and cartilage. Inflammation is still considered the pivotal inducer of both components of joint damage. Results of recent animal studies suggested a prominent contribution of osteoclastic bone resorption that could be dissociated from inflammation. RANKL and its natural decoy receptor, osteoprotegerin (OPG), play key roles in osteoclast activation. In a group of patients with early RA not treated with disease-modifying drugs, we tested the hypothesis that osteoclast activation, reflected by the serum OPG:RANKL ratio at baseline, is negatively associated with progression of bone damage, independent of inflammation. Methods OPG and RANKL levels, together with a parameter of inflammation (first-year time-averaged erythrocyte sedimentation rate [tESR]), were measured in 92 patients with newly diagnosed early active RA who were participants in a randomized study. The tESR and the OPG:RANKL ratio were evaluated for the ability to predict 5-year radiographic progression of joint damage. Results The first-year tESR and the OPG:RANKL ratio, as measured at baseline, independently predicted 5-year radiographic progression of joint damage (both P , 0.001). Progression of radiographic damage was greatest in patients with a high tESR and a low OPG:RANKL ratio and was lowest in patients with a low tESR and a high OPG:RANKL ratio. Conclusion This study in patients with early untreated RA is the first to confirm the findings in animal models of arthritis, that radiographic progression of the bone component of joint destruction is dependent on both inflammation (tESR) and osteoclast activation (the OPG:RANKL ratio). [source] Calcium sensing and cell signaling processes in the local regulation of osteoclastic bone resorptionBIOLOGICAL REVIEWS, Issue 1 2004Mone Zaidi ABSTRACT The skeletal matrix in terrestrial vertebrates undergoes continual cycles of removal and replacement in the processes of bone growth, repair and remodeling. The osteoclast is uniquely important in bone resorption and thus is implicated in the pathogenesis of clinically important bone and joint diseases. Activated osteoclasts form a resorptive hemivacuole with the bone surface into which they release both acid and osteoclastic lysosomal hydrolases. This article reviews cell physiological studies of the local mechanisms that regulate the resorptive process. These used in vitro methods for the isolation, culture and direct study of the properties of neonatal rat osteoclasts. They demonstrated that both local microvascular agents and products of the bone resorptive process such as ambient Ca2+ could complement longer-range systemic regulatory mechanisms such as those that might be exerted through calcitonin (CT). Thus elevated extracellular [Ca2+], or applications of surrogate divalent cation agonists for Ca2+, inhibited bone resorptive activity and produced parallel increases in cytosolic [Ca2+], cell retraction and longer-term inhibition of enzyme release in isolated rat osteoclasts. These changes showed specificity, inactivation, and voltage-dependent properties that implicated a cell surface Ca2+ receptor (CaR) sensitive to millimolar extracellular [Ca2+]. Pharmacological, biophysical and immunochemical evidence implicated a ryanodine-receptor (RyR) type II isoform in this process and localized it to a unique, surface membrane site, with an outward-facing channel-forming domain. Such a surface RyR might function either directly or indirectly in the process of extracellular [Ca2+] sensing and in turn be modulated by cyclic adenosine diphosphate ribose (cADPr) produced by the ADP-ribosyl cyclase, CD38. The review finishes by speculating about possible detailed models for these transduction events and their possible interactions with other systemic mechanisms involved in Ca2+ homeostasis as well as the possible role of the RyR-based signaling mechanisms in longer-term cell regulatory processes. [source] | |||||||||||||