Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in osteoclast formation. However, its actions on mature cells have not been fully characterized. We now report that M-CSF dramatically stimulates osteoclastic motility and spreading; osteoclasts responded to a gradient of M-CSF with orientation, and random cell polarization occurred after isotropic exposure. M-CSF also supported the survival of osteoclasts by preventing apoptosis. Paradoxically, M-CSF inhibits bone resorption by isolated osteoclasts. We found that this was effected predominantly by reduction in the number of excavations. Thus, M-CSF showed a propensity to suppress resorption through a reduction in the proportion of cells that were resorbing bone. Our data suggest that apart from the established role of M-CSF in the provision of precursors for osteoclastic induction, a major role for M-CSF in bone resorption is to enhance osteoclastic survival, migration, and chemotaxis. It seems appropriate that during these processes resorptive functions should be suppressed. We suggest that M-CSF continues to modulate osteoclastic activity once osteoclasts are on resorptive sites, through regulation of the balance between resorption and migration, such that not only the quantity, but the spatial pattern of resorption can be controlled by adjacent M-CSF- secreting cells of osteoblastic lineage.     

Multiple immunoreactive forms of osteocalcin in uremic serum.

Circulating osteocalcin, which normally reflects the rate of bone formation, is elevated in uremia. In 18 patients receiving maintenance hemodialysis, serum osteocalcin levels were directly related to the bone formation rate (r = 0.88, P less than 0.001), osteoblastic osteoid surface density (r = 0.65, P less than 0.01), and osteoclastic resorptive surface density (r = 0.75, P less than 0.001). Multiple regression analysis showed that osteocalcin levels remained positively correlated with osteoclastic resorption when the bone formation rate was held constant (P less than 0.01). The intimation that the coupling of bone formation and resorption could not explain the relationship between osteocalcin and resorption led us to determine whether fragments of this abundant matrix protein are released by bone resorption and retained in uremia. Sera from dialysis patients with renal osteodystrophy were fractionated by sequential gel filtration and HPLC, and assayed for immunoreactive osteocalcin. When normal serum was analyzed, a single sharp peak was found. In pooled sera from patients with high osteoclastic resorptive surfaces identified by histomorphometry, we found five additional immunoreactive peaks, while three additional peaks were detected in sera from patients with lower osteoclastic surfaces. Bio-Gel P-10 chromatography showed that these multiple peaks were of lower molecular weight than intact osteocalcin. We suggest that the liberation of bone matrix by osteoclasts contributes to the circulating osteocalcin immunoreactivity in uremia.     

Nanoparticulate mineralized collagen glycosaminoglycan materials directly and indirectly inhibit osteoclastogenesis and osteoclast activation

The ability of the extracellular matrix (ECM) to direct cell fate has generated the potential for developing a materials‐only strategy for tissue regeneration. Previously, we described a nanoparticulate mineralized collagen glycosaminoglycan (MC‐GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion. In this work, we evaluated the interactions between MC‐GAG and primary human osteoclasts (hOCs). In the absence of hMSCs, mineralized Col‐GAG materials directly inhibited hOC viability, proliferation, and resorption in contrast to nonmineralized Col‐GAG, which demonstrated a modest inhibition of resorptive activity only. Cocultures containing differentiating hMSCs with hOCs demonstrated increased hOC‐mediated resorption only on Col‐GAG while MC‐GAG cocultures continued to inhibit resorption. Unlike Col‐GAG, hMSCs on MC‐GAG expressed increased amounts of osteoprotegerin (OPG) protein, the major endogenous osteoclast inhibitor. Interestingly, OPG expression was found to be antagonized by small mothers against decapentaplegic1/5 (Smad1/5) phosphorylation, an obligate pathway for osteogenic differentiation of hMSCs on MC‐GAG, and potentiated by extracellular signal‐regulated kinase (ERK1/2) phosphorylation. Collectively, these results suggested that the MC‐GAG material both directly inhibited the osteoclast viability, proliferation, and resorptive activity as well as induced hMSCs to secrete osteoprotegerin, an antiosteoclastogenic factor, via a signalling pathway distinct from osteogenic differentiation.     

Graves' disease and bone metabolism

Thyroid hormone stimulates osteoclastic bone resorption, through increased expression of receptor activator of nuclear factor kappa B ligand (RANKL) in osteoblasts as well as via non-RANKL-mediated pathway. Therefore, in hyperthyroid patients with Graves' disease, bone resorption (urinary excretion of calcium, phosphate, deoxypyridinoline, N-terminal telopeptide of collagen type I) is increased. Due to accelerated bone remodeling, bone formation is also increased. However, the amount of bone formation is less than that of bone resorption, leading to a gradual decrease in bone mineral density (BMD). In young patients, the decreased BMD is reversible, but not in post-menopausal women. Therefore, in these patients with rapid bone looser, bisphosphonates may be beneficial treatment for prevention of osteoporosis and will prevent bone fractures in senile period.     

Glucocorticoids modulate macrophage surface oligosaccharides and their bone binding activity

The circumstantial evidence that indicates that glucocorticoids (GC) may stimulate osteoclastic resorption in vivo has recently found support in observations that demonstrate that these compounds effectively increase the activity of isolated resorptive cells (osteoclasts, macrophage polykaryons, and elicited macrophages [MO] ) in vitro. Data are presented here that indicate that this stimulation by GC is due to an enhancement of the initial stage of the resorption process, the attachment of cells to bone, and that this is caused by alterations of cell surface oligosaccharides. Specifically, dexamethasone and cortisol enhance by 80% the attachment of MO to bone surfaces in a dose dependent manner but do not alter or reduce the binding of these cells to other surfaces (plastic, collagen, and hydroxyapatite crystals). The effect of GC on cell-bone attachment is blocked by the glycosylation inhibitor, tunicamycin, and the glycosylation modifier, swainsonine; this demonstrates that asparagine-linked oligosaccharides are involved in the stimulatory process. Flow cytometric analysis of GC-treated cells using a panel of fluoresceinated lectins confirms this by indicating a selective, enhanced exposure of plasma membrane-associated N-acetylglucosamine and N-acetylgalactosamine residues, sugars we have previously shown to be pivotal in MO-bone binding. Finally, progesterone, a known GC antagonist, blocks GC-stimulated resorption, macrophage-bone binding, and membrane oligosaccharide modification, presumably by competing for the GC receptor. Progesterone alone alters none of these processes. Thus, GC stimulates the resorptive activity of macrophages by enhancing the initial events in the degradative process (cell-bone binding) and does so, apparently, via receptor-mediator alteration of cell surface glycoproteins.     

The role of osteoclasts in bone tissue engineering

The success of scaffold‐based bone regeneration approaches strongly depends on the performance of the biomaterial utilized. Within the efforts of regenerative medicine towards a restitutio ad integrum (i.e. complete reconstruction of a diseased tissue), scaffolds should be completely degraded within an adequate period of time. The degradation of synthetic bone substitute materials involves both chemical dissolution (physicochemical degradation) and resorption (cellular degradation by osteoclasts). Responsible for bone resorption are osteoclasts, cells of haematopoietic origin. Osteoclasts play also a crucial role in bone remodelling, which is essential for the regeneration of bone defects. There is, however, surprisingly limited knowledge about the detailed effects of osteoclasts on biomaterials degradation behaviour. This review covers the relevant fundamental knowledge and progress made in the field of osteoclast activity related to biomaterials used for bone regeneration. In vitro studies with osteoclastic precursor cells on synthetic bone substitute materials show that there are specific parameters that inhibit or enhance resorption. Moreover, analyses of the bone–material interface reveal that biomaterials composition has a significant influence on their degradation in contact with osteoclasts. Crystallinity, grain size, surface bioactivity and density of the surface seem to have a less significant effect on osteoclastic activity. In addition, the topography of the scaffold surface can be tailored to affect the development and spreading of osteoclast cells. The present review also highlights possible areas on which future research is needed and which are relevant to enhance our understanding of the complex role of osteoclasts in bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Increased levels of biochemical markers of bone turnover in relation to persistent immune activation in common variable immunodeficiency

BACKGROUND: Based on the involvement of cytokines and growth factors in bone homeostasis, we hypothesised that patients with common variable immunodeficiency (CVI), characterised by persistent immune activation in vivo, may have disturbed bone metabolism as evaluated by biochemical markers of bone turnover. MATERIALS AND METHODS: Serum levels of tumour necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), bone-specific alkaline phosphatase (B-ALP), osteocalcin, carboxyterminal crosslinking telopeptide of type I collagen (CTX-I), insulin-like growth factor (IGF)-I and IGF binding protein-3 (IGFBP-3) were measured in 25 patients with CVI and compared to 25 age- and sex-matched healthy controls. RESULTS: Patients with CVI had significantly higher serum levels of CTX-I and B-ALP, and significantly lower serum levels of IGF-I and IGFBP-3 compared to controls as shown in cross-sectional, and as for B-ALP and CTX-I, also during longitudinal testing. No differences were observed for osteocalcin between the two groups. The elevated B-ALP and decreased IGF-I and IGFBP-3 levels were most pronounced in a subgroup of CVI patients characterised by persistent activation of proinflammatory cytokines in vivo. Raised B-ALP and decreased IGF-I and IGFBP-3 were also significantly correlated with enhanced IL-6 and TNF-alpha levels in these patients. CONCLUSIONS: The present study suggests that persistent immune activation in vivo, with raised levels of proinflammatory cytokines, may be related to disturbed bone homeostasis in CVI patients, further supporting an interaction between immune related mediators and bone metabolism in humans.     

1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption.

Although 1,25-dihydroxyvitamin D3 stimulates osteoclastic bone resorption in vivo and in organ culture, the mechanism by which it effects this stimulation is unknown. We have recently found that the agent does not stimulate resorption by osteoclasts mechanically disaggregated from bone and incubated on slices of cortical bone. This suggests that the osteoclasts were removed by disaggregation from the influence of some cell type, present in intact bone, that mediates hormone responsiveness. We therefore tested the ability of osteoblastic cells derived from neonatal rat calvariae and of cloned, hormone-responsive osteosarcoma cells (UMR106) to restore hormone responsiveness to unresponsive populations of osteoclasts. We found that osteoblastic cells from both sources induced a two- to fourfold stimulation of osteoclastic bone resorption in the presence of 1,25-dihydroxyvitamin D3. Stimulation was observed at concentrations of 10(-10) M and above. Actinomycin D and cycloheximide did not affect bone resorption by osteoclasts incubated alone, but abolished the capacity of osteoblastic cells to stimulate osteoclastic resorption in the presence of 1,25-dihydroxyvitamin D3. When calvarial cells or osteoblastlike UMR cells were incubated with the hormone, they produced a factor in cell-free supernatants that stimulated bone resorption by disaggregated osteoclasts. These experiments suggest that 1,25-dihydroxyvitamin D3 stimulates bone resorption through a primary action on osteoblastic cells, that are induced by the hormone to produce a factor that stimulates osteoclastic bone resorption.     

Serum beta2-microglobulin reflects increased bone resorption in immobilized stroke patients

OBJECTIVE: The so-called bone-derived growth factor, beta2-microglobulin, has a regulatory function in bone metabolism by stimulating osteoclast activity. We undertook this study because osteoclast activity is known to be enhanced in patients with immobilized stroke, suggesting that their beta2-microglobulin concentrations may be increased. DESIGN: We studied 79 patients with acute stroke hemiplegia, including 36 men and 43 postmenopausal women ranging in age between 51 and 70 yr. RESULTS: The mean Barthel Index was 43 and 42 for men and women, respectively. The serum beta2-microglobulin concentration was increased in male and female patients, compared with the findings of 44 age-matched control subjects, and the serum concentration of pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen was also increased in male and female patients, compared with the findings of the control subjects. Serum concentrations of pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen correlated negatively with Barthel Index scores in both genders, indicating increased bone resorption caused by immobilization in these patients. Linear regression analysis revealed a positive correlation between beta2-microglobulin and pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen in both genders. CONCLUSIONS: These findings suggest that beta2-microglobulin reflects osteoclastic activity in response to stroke-induced immobilization in both genders. Beta2-microglobulin is a useful indicator of bone resorption in patients with immobilized stroke.     

Bone turnover 18 months after a single intravenous dose of zoledronic acid

Zoledronic acid inhibits bone resorption for up to 12 months. It is not known whether the duration of this antiresorptive effect extends beyond this period of time. The aim of this study was to evaluate the changes in bone turnover at 12 months (T12) and 18 months (T18) after a single injection of 4 mg of zoledronic acid. It is a prospective, longitudinal study, with a follow-up for 18 months. We studied male and female patients (60.5 +/- 11.0 years old), with low bone mineral density (BMD) coming from the outpatient clinic in a metabolic bone unit of a tertiary care hospital. All patients received a single intravenous dose of 4 mg of zoledronic acid, bone turnover markers [serum carboxyterminal telopeptide of type I collagen (CTX-I), bone-specific alkaline phosphatase (BSAP)] and BMD [lumbar spine (LS) and total hip (TH)] were measured at baseline, and after 12 months (T12) and 18 months (T18). Median serum CTX-I and BSAP levels were suppressed at T12 in comparison with baseline values: 0.183 to 0.039 ng/ml for CTX-I (p = 0.0002) and 16.95 to 13.96 U/l for BSAP (p = 0.005). At T18, both CTX-I and BSAP continued to be suppressed below baseline at 0.108 ng/ml and 12.23 U/l (p = 0.009 and p = 0.02, vs. T0). Significant increases in BMD at T18 as compared with T12 were observed in patients (median increase 6.1% for LS and 2.0% for TH). Zoledronic acid inhibits bone turnover effectively for 12 months, with evidence for continued suppression and gains in BMD even after 18 months.     

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption.

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.     

咖啡酸对破骨细胞形成及组织蛋白酶K基因表达的抑制

背景：咖啡酸对破骨细胞形成分化方面影响的研究很少,对破骨细胞性骨吸收机制的研究更少。目的：观察咖啡酸对破骨细胞形成分化及组织蛋白酶K基因表达的影响,分析甘薯提取物咖啡酸对破骨细胞性骨吸收的抑制机制。方法：采用3种不同细胞培养系,观察咖啡酸对破骨细胞形成分化的影响,同时利用反转录-聚合酶链反应技术观察咖啡酸对组织蛋白酶K基因表达的抑制作用。结果与结论：咖啡酸在3种不同细胞培养系中均显示出直接抑制破骨细胞形成和分化的作用,根据半定量反转录-聚合酶链反应基因表达分析,咖啡酸对RANKL受体RANKmRNA的表达完全无影响,但显著抑制了组织蛋白酶K的表达。提示甘薯提取物有效成分咖啡酸不仅能直接抑制破骨细胞的形成及分化,而且可显著抑制组织蛋白酶K的表达,其可能是甘薯提取物抑制破骨细胞性骨吸收的作用机制。     

Variability in urinary excretion of bone resorption markers: limitations of a single determination in clinical practice.

In this study we assessed the within and between-subject variability of the concentrations of two urinary markers, free deoxypyridinoline (DPD) and C telopeptide (CTX-I), in healthy patients with the aim of setting reliable thresholds to enable physicians to take decisions about individual patients with confidence.Between-subject variability for the women was 25.4% for DPD and 38.2% for CTX-I, and for the men was 12.9% for DPD and 23.8% for CTX-I. The coefficients of variation were similar for daily, weekly and monthly determinations, giving means of 13.8 and 28.1% for DPD and CTX-I respectively. Critical difference (CD) was lower for DPD than for CTX-I (about 44 and 80% respectively). The number of samples required to determine the true mean with a CD at the 5% level was 29 for DPD and more than 113 for CTX-I.DPD was the least biologically variable. One determination was not sufficient to determine bone resorption status and a 44% decrease in DPD levels and an 80% decrease in CTX-I levels were required to demonstrate the efficacy of antiresorptive therapy in individual patients.     

Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts

A monocyte-derived factor with IL-1-like properties has recently been shown to cause resorption of bone in organ culture. We have investigated the action of IL-1 on disaggregated populations of osteoclasts, incubated alone or in the presence of osteoblastic cells, in an attempt to identify the target cell for IL-1 in bone, and to elucidate the mechanism by which IL-1 induces osteoclastic resorption. Osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Under these conditions, the majority of osteoclasts form distinctive excavations in the bone surface within 24 h, the volume of which can be quantified by computer-assisted morphometric and stereophotogrammetic techniques. IL-1 had no effect on bone resorption by osteoclasts alone, but when incubated in the presence of calvarial cells or cloned osteosarcoma cells, it induced a 3.8 (+/- 0.38)-fold increase in osteoclastic bone resorption, with significant enhancement at concentrations of greater than or equal to 30 pg/ml. The osteoblastic populations themselves did not resorb bone. The mechanism by which osteoblastic cells stimulate osteoclasts did not appear to depend upon PG synthesis; nor could we detect a diffusible substance in the medium of stimulated cocultures. These results indicate that IL-1 stimulates bone resorption through a primary action on osteoblasts, which are induced by IL-1 to transmit a short-range signal that stimulates osteoclastic bone resorption.     

DNA synthesis is not necessary for osteoclastic responses to parathyroid hormone in cultured fetal rat long bones.

Osteoclasts, the principal cells mediating bone resorption, are believed to increase their size, number, and resorbing activity in response to parathyroid hormone (PTH) through mechanisms dependent upon the fusion of specific mononuclear precursor cells into either new or existing multinucleated osteoclasts. To address the question of whether these actions of PTH are dependent on the replication of osteoclast precursor cells, we examined the ability of an inhibitor of DNA synthesis, hydroxyurea (HU), to alter bone resorption, osteoclast formation, and DNA synthesis in cultured fetal rat bones treated with PTH. We found that HU significantly reduced [3H]thymidine incorporation into the bones and labeling of osteoclast nuclei by greater than 90%, but did not prevent PTH from stimulating bone resorption, measured as the release of 45Ca, or from increasing the number of osteoclasts in the bones. In bones cultured without PTH, HU decreased the rate of bone resorption, but not the number of osteoclasts per bone. We conclude that in fetal rat bone cultures, PTH can increase osteoclast number and stimulate bone resorption by affecting existing osteoclasts and osteoclast precursors, and that replication of osteoclast precursor cells is not necessary for PTH to stimulate a resorptive response. In unstimulated cultures it appears that HU inhibits bone resorption by affecting mechanisms that are independent of changes in osteoclast number and that may be influenced by cell replication or other unknown factors.     

TRANCE Is Necessary and Sufficient for Osteoblast-mediated Activation of Bone Resorption in Osteoclasts

TRANCE (tumor necrosis factor–related activation-induced cytokine) is a recently described member of the tumor necrosis factor superfamily that stimulates dendritic cell survival and has also been found to induce osteoclastic differentiation from hemopoietic precursors. However, its effects on mature osteoclasts have not been defined. It has long been recognized that stimulation of osteoclasts by agents such as parathyroid hormone (PTH) occurs through a hormonal interaction with osteoblastic cells, which are thereby induced to activate osteoclasts. To determine whether TRANCE accounts for this activity, we tested its effects on mature osteoclasts. TRANCE rapidly induced a dramatic change in osteoclast motility and spreading and inhibited apoptosis. In populations of osteoclasts that were unresponsive to PTH, TRANCE caused activation of bone resorption equivalent to that induced by PTH in the presence of osteoblastic cells. Moreover, osteoblast-mediated stimulation of bone resorption was abrogated by soluble TRANCE receptor and by the soluble decoy receptor osteoprotegerin (OPG), and stimulation of isolated osteoclasts by TRANCE was neutralized by OPG. Thus, TRANCE expression by osteoblasts appears to be both necessary and sufficient for hormone-mediated activation of mature osteoclasts, and TRANCE-R is likely to be a receptor for signal transduction for activation of the osteoclast and its survival.     

Lack of a T-cell dependent subpopulation of macrophages in (dichloromethylene) diphosphonate-treated mice

In Cl2MDP-osteopetrotic mice, one subpopulation of thioglycollate-induced peritoneal exudate macrophages (M phi) is missing. This subpopulation is precisely the one whose differentiation is known to be dependent on T-lymphocytes, as it is also missing in the athymic nu/nu mice. Cl2MDP-induced osteopetrosis being partially attributable to deficient osteoclastic bone resorption, raises the possibility that this missing M phi subpopulation might represent the precursors of osteoclasts. It is suggested from this work that the interplays between T-cells and M phi, so well known in immunity and inflammation, may also be relevant to osteoclastic differentiation and therefore, to bone remodeling.     

Colony-stimulating factors regulate the development of multinucleated osteoclasts from recently replicated cells in vitro.

Osteoclasts mediate the process of bone resorption. However, little is known about the mechanisms that regulate the formation of either osteoclasts or osteoclast precursors. In contrast, colony-stimulating factors (CSFs) are well-known to regulate the formation of myeloid cells and their precursors. Because osteoclasts and myeloid cells may originate from a common stem cell, we examined the effects of two CSFs, granulocyte-macrophage CSF (GM-CSF) and interleukin 3 (IL-3), on bone resorption, osteoclast formation, and the incorporation of recently replicated nuclei into the osteoclasts of mouse bone cultures. CSFs had little effect on the formation rate of osteoclasts or their resorptive activity but significantly decreased the percentage of recently replicated osteoclast progenitor cell nuclei present in the osteoclasts of bones treated with parathyroid hormone. GM-CSF also increased the number of myeloid cells in the marrow space of the cultures and the percentage of these cells derived from recently replicated progenitors. These results demonstrate that GM-CSF and IL-3 can regulate the development of osteoclasts from recently replicated precursor cells in cultured fetal mouse long bones. However, the mechanisms by which CSFs influence osteoclast formation are difficult to determine from these studies because markers for the osteoclast progenitor and precursor do not exist. These data also provide evidence that the differentiation of osteoclast progenitors is regulated by different factors at different points in their ontogeny.