Stereophotogrammetric observations on bone resorption by isolated rabbit osteoclasts

Although the osteoclast has long been recognized as the major agent of bone resorption, it has previously only been possible to assess resorption by these cells in bone, where their response to environmental factors may be modified by, or even mediated by, the other cell types also present. We used a carborundum saw to prepare slices of human cortical bone which demonstrate a predictable surface appearance. Osteoclasts were disaggregated from neonatal rabbit long bone and settled onto these slices. After incubation for 24 hr we found that osteoclasts had formed distinctive excavations in the bone surface. This technique makes it possible, for the first time, to analyse the direct and indirect effects of hormones and cells on osteoclastic bone resorption.     

Gallium inhibits bone resorption by a direct effect on osteoclasts

Gallium nitrate has been used clinically to treat cancer-related hypercalcemia. It has been suggested that gallium may reduce calcium release from bone by inhibiting bone resorption, but the mechanism(s) involved remain to be elucidated. Therefore, we have examined the effect of gallium on bone resorption in vitro using osteoclasts isolated from neonatal rat long bones cultured on slices of cortical bone. Gallium nitrate (0.01–100 μg/ml) produced a concentration-dependent inhibition of bone resorption. Morphological studies showed that even (100μg/ml) gallium nitrate induced no light microscopical change in osteoclast morphology and did not affect their survival on bone slices. Pretreatment of bone slices with gallium nitrate (100μg/ml for 18 h), followed by extensive washing also inhibited subsequent osteoclastic bone resorption. These results suggest that gallium can be adsorbed onto the calcified surface of bone and inhibit osteoclastic bone resorption.     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.     

Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.     

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.     

破骨细胞骨吸收功能的检测方法

破骨细胞是一种多核的,具有骨吸收功能的骨组织细胞,在骨吸收过程中起着至关重要的作用.破骨细胞骨吸收功能的异常会引发一系列的临床病症,如骨质疏松症、关节置换术后假体松动、骨硬化症和牙周病变等.破骨细胞骨吸收功能的进一步研究对于各类骨疾病的防治具有重要的意义.然而破骨细胞骨吸收功能的检测方法一直以来是制约破骨细胞研究的瓶颈之一.为此,围绕破骨细胞骨吸收功能的检测方法做一综述.     

The in vitro effect of pH on osteoclasts and bone resorption in the cat: implications for the pathogenesis of FORL

Dental disease due to osteoclast over-activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio-pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (approximately 400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells-in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 +/- 0.003 mm(2), whereas an 8.6-fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 +/- 0.004 mm(2)). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5-fold increase in the area of bone slices covered by resorption lacunae ( approximately 70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3-fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL.     

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.     

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis

We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.     

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures--also stained by hematoxylin--were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images--round/ ovoid bodies with dense crescent-like chromatin--on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis.     

Uptake of peroxidase by calcitonin inhibited osteoclasts

Summary The present electron microscopic histochemical study demonstrates that osteoclasts from calcitonin treated bone are able to take up organic macromolecules even though the ruffled border has disappeared. The absorption occurs around the entire periphery of the osteoclast, but the amount of absorbed peroxidase seems to be reduced in comparison with that of untreated cells. It is concluded that the effect of calcitonin on osteoclasts is primarily a cessation of the exocytosis and the concomitant disappearance of the ruffled border.This research was supported by the Danish Medical Research Council, grant No. 512-7184     

Interaction of cyclosporine A and calcitonin on bone resorption in vitro

Cyclosporine A (CsA), which is a potent immunosuppressive agent, inhibits bone resorption in vitro. The inhibition of bone resorption by CsA is sustained, unlike the transient inhibition of bone resorption produced by calcitonin (CT). These different patterns of inhibition were studied by examining the interaction between CsA and CT on stimulated bone resorption in the neonatal mouse calvarial resorptive system. "Escape" from the CT inhibition of PTH stimulated bone resorption occurred after 24 hr of organ culture. Coincubation with CsA (1 micrograms/ml) delayed the "escape" response of CT + PTH treated bones, so that the full "escape" response did not occur until after 48 hr of organ culture. Likewise, a pretreatment of 24 hr with CsA (1 micrograms/ml) was sufficient to delay "escape" from CT inhibition of PTH stimulated bone resorption until after 48 hr of organ culture. A higher concentration of CsA (10 micrograms/ml) completely prevented the "escape" response. Our data could indicate an interaction between the CsA and CT inhibitory effects on resorption.     

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone.

Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.