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Human Osteoclasts (human + osteoclast)
Selected AbstractsGlucocorticoids maintain human osteoclasts in the active mode of their resorption cycleJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2010Kent Søe Abstract Osteoclasts are known to exert their resorptive activity through a so-called resorption cycle consisting of alternating resorption and migration episodes and resulting typically in the formation of increasing numbers of discrete round excavations on bone slices. This study shows that glucocorticoids deeply modify this resorptive behavior. First, glucocorticoids gradually induce excavations with a trenchlike morphology while reducing the time-dependent increase in excavation numbers. This indicates that glucocorticoids make osteoclasts elongate the excavations they initiated rather than migrating to a new resorption site, as in control conditions. Second, the round excavations in control conditions contain undegraded demineralized collagen as repeatedly reported earlier, whereas the excavations with a trenchlike morphology generated under glucocorticoid exposure appear devoid of leftovers of demineralized collagen. This indicates that collagenolysis proceeds generally at a lower rate than demineralization under control conditions, whereas collagenolysis rates are increased up to the level of demineralization rates in the presence of glucocorticoids. Taking these observations together leads to a model where glucocorticoid-induced increased collagenolysis allows continued contact of osteoclasts with mineral, thereby maintaining resorption uninterrupted by migration episodes and generating resorption trenches. In contrast, accumulation of demineralized collagen, as prevails in controls, acts as a negative-feedback loop, switching resorptive activity off and promoting migration to a new resorption site, thereby generating an additional resorption pit. We conclude that glucocorticoids change the osteoclastic resorption mode from intermittent to continuous and speculate that this change may contribute to the early bone fragilization of glucocorticoid-treated patients. © 2010 American Society for Bone and Mineral Research. [source] Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman PrimateJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2001George B. Stroup Abstract Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis. [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] Gap junctional communication in human osteoclasts in vitro and in vivoJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6a 2008A. F. Schilling Abstract Bone-forming cells are known to be coupled by gap junctions, formed primarily by connexin43 (Cx43). The role of Cx43 in osteoclasts has so far only been studied in rodents, where Cx43 is important for fusion of mononuclear precursors to osteoclasts. Given the potential importance for human diseases with pathologically altered osteoclasts, we asked whether a similar influence of Cx43 can also be observed in osteoclasts of human origin. For this purpose, Cx43 mRNA expression was studied in a time course experiment of human osteoclast differentiation by RT-PCR. Localization of Cx43 in these cells was determined by immunohistochemistry and confocal microscopy. For the assessment of the effect of gap junction inhibition on cell fusion, gap junctions were blocked with heptanol during differentiation of the cells and the cells were then evaluated for multinuclearity. Paraffin sections of healthy bone and bone from patients with Paget's disease and giant cell tumour of the bone were used to study Cx43 expression in vivo. We found mRNA and protein expression of Cx43 in fully differentiated osteoclasts as well as in precursor cells. This expression decreased in the course of differentiation. Consistently, we found a lower expression of Cx43 in osteoclasts than in bone marrow precursor cells in the histology of healthy human bone. Blockade of gap junctional communication by heptanol led to a dose-dependent decrease in multinuclearity, suggesting that gap junctional communication precedes cell fusion of human osteoclasts. Indeed, we found a particularly strong expression of Cx43 in the giant osteoclasts of patients with Paget's disease and giant cell tumour of the bone. These results show that gap junctional communication is important for fusion of human mononuclear precursor cells to osteoclasts and that gap junctional Cx43 might play a role in the regulation of size and multinuclearity of human osteoclasts in vivo. [source] Optimized transfection of diced siRNA into mature primary human osteoclasts: Inhibition of cathepsin K mediated bone resorption by siRNAJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2005Christina I. Selinger Abstract Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by ,60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P,=,0.018) and resorbed area (P,=,0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects. © 2005 Wiley-Liss, Inc. [source] NFAT expression in human osteoclastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Christopher J. Day Abstract Nuclear factor of activated T-cells cytoplasmic (NFATc) is a family of transcription factors originally identified in T-cells. The gene family is currently known to have four members (NFATc1 through NFATc4) which have roles both within and outside the immune system. We show that NFATc1 is the major induced NFAT in human osteoclasts, with expression greatly exceeding that of NFATc2 through NFATc4. In macrophage-like cells in culture, NFATc1 through NFATc4 are expressed at similar low levels. NFATc1 is comprised of five mRNA transcript variants known to encode three different protein isoforms. The mRNA encoding isoform C (mRNA variant 3) was the most expressed with 38 copies per nanogram followed by isoform B (mRNA variant 5) with 17 copies per nanogram of total RNA. Isoform A (mRNA variant 1) and mRNA variants 2 and 4 made up less than 1% of the total NFATc1 expressed. NFATc1 is activated by calcineurin after calcium-calmodulin signalling. The induction of NFATc1 in osteoclasts was not altered in the presence of cyclosporin A, an inhibitor of calcineurin, suggesting that NFATc1 does not participate in autoregulatory activation of its own promoter. The NFATc1 variants expressed by human osteoclasts are not those normally expressed by effector T-cells but are similar to those seen in naïve T-cells. © 2005 Wiley-Liss, Inc. [source] CXCL12 chemokine up-regulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid arthritis patientsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Francesco Grassi Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases. J. Cell. Physiol. 199: 244,251, 2004© 2003 Wiley-Liss, Inc. [source] Human osteoclast formation and activity on an equine spongy bone substituteCLINICAL ORAL IMPLANTS RESEARCH, Issue 1 2009Vittoria Perrotti Abstract Objectives: The aim of the present study was to evaluate the in vitro formation and activity of human osteoclasts (OCLs) generated on a new type of xenograft for bone substitution, an equine spongy bone. Material and methods: Peripheral blood mononuclear cells from healthy volunteers were used to generate OCLs in vitro in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-,B ligand (RANKL) on bovine bone slices (positive control) and equine spongy bone. Morphological and biochemical methods were used to assess OCLs formation and activity. Results: Cells generated after 21 days of culture on equine spongy bone showed similar morphology to those on the positive control and displayed typical OCL markers and features, indicating that this material supported OCL formation. Moreover, these cells were functionally active on equine spongy bone with statistically significant differences compared with the control in the release of tartrate-resistant acid phosphatase (TRAcP5b) at days 14 and 21 of culture. With regard to the resorption, on equine bone, OCLs formed smaller discontinuous island-like lacunae rather than the typical lobulated, tracking resorption lacunae observed on the control. Conclusions: This study enables clinicians to tailor the usage of equine spongy bone and presents a model, which can be applied to the preclinical assessment of bone substitute material's resorbability and resorption rates. [source] | |||||||||||||