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PubMed1975

The origin and fate of osteoclasts.

Despite intensive and ingenious investigation, the origins and ultimate fate of the osteoclast remain shrouded in mystery. This brief review evaluates some of the recent experimental approaches used in the study of the osteoclast, especially whether they form from intra- or extra-skeletal progenitor cells, whether from the same osteoprogenitor cell as the osteoblast, and whether, once formed, they may modulate to osteoblasts. That osteoprogenitor cells can, and do, become osteoclasts is well founded, as is the conclusion that such progenitor cells originate as blood-borne, extra-skeletal cells. Evidence that sessile, intra-skeletal, progenitor cells can form osteoclasts is less direct. There is good evidence that osteoclasts both shed and take-up nuclei, but no direct evidence that nuclear shedding is accompanied by death of the osteoclast, and no direct evidence for the fate of the shed nuclei. Whether the same osteoprogenitor cell can produce either an osteoblast or an osteoclast also remains an open question.

Animals↗

PubMed1986

Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells.

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.

Animals↗

PubMed2005

Synovial fibroblasts promote osteoclast formation by RANKL in a novel model of spontaneous erosive arthritis.

OBJECTIVE: Erosion of cartilage and bone is a hallmark of rheumatoid arthritis (RA). This study was undertaken to explore the roles of hyperproliferating synovial fibroblasts and macrophages in abnormal osteoclast formation, using the recently described BXD2 mouse model of RA. METHODS: Cell distribution in the joints was analyzed by immunohistochemistry, using tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclasts. To identify the defective cells in BXD2 mice, mouse synovial fibroblasts (MSFs) were cultured with bone marrow-derived macrophages. Osteoclast formation was assayed by TRAP staining and bone resorption pit assay, and the cytokine profiles of the MSFs and macrophages were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: In BXD2 mice, TRAP-positive osteoclasts were found at sites of active bone erosion, in close proximity to hyperproliferating synovial fibroblasts. On coculture, MSFs from BXD2 mice, but not C57BL/6 mice, produced high levels of RANKL messenger RNA, induced macrophages to form osteoclasts, and actively eroded bone slices, through a mechanism(s) that could be blocked by pretreatment with osteoprotegerin. Although macrophages from BXD2 mice expressed higher basal levels of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 than those from C57BL/6 mice, abnormal osteoclast formation was not due to enhanced sensitivity of the BXD2 mouse macrophages to RANKL. TNFalpha, produced by both BXD2 MSFs and BXD2 mouse macrophages, had a strong stimulatory effect on RANKL expression. CONCLUSION: BXD2 MSFs produce RANKL and induce the development of osteoclasts from macrophages. The enhanced production of RANKL is possibly due to autocrine stimulation, together with paracrine stimulation by factors produced by macrophages.

Animals↗

PubMed2003

The effect of sintered dicalcium pyrophosphate on osteoclast metabolism: an ultrastructural study.

Sintered dicalcium pyrophosphate (SDCP), a synthetic compound, has proved to be both bioabsorbable and biocompatible in vivo. Recent work in our institute also has demonstrated that the ingestion of SDCP can increase bone mass in the ovariectomized rat. In this study, we used an in vitro cell culture model to investigate the ultrastructural changes and fate of osteoclasts induced by SDCP. Quantitative evaluation of osteoblasts and osteoclasts after administration of SDCP was performed. We studied immunohistochemical and ultrastructural features of osteoclasts undergoing apoptosis. The results showed that at 10(-4) M SDCP, the osteoblast cell count increased significantly, whereas the osteoclast population decreased significantly. Apoptosis of the osteoclast population was well demonstrated by immunohistochemical study. Ultrastructural study showed that the Golgi apparatus was degraded or dispersed in the cytoplasm. Later, osteoclasts revealed pyknotic nuclei showing condensation and margination of heterochromatins and DNA fragmentation, which are typical features of apoptosis. In addition, disruption of nuclear envelopes leading to leakage of nuclear contents into the cytoplasm was observed in the late stage of apoptosis. In conclusion, SDCP-induced apoptosis of osteoclasts was characterized by ultrastructural changes of the nucleus accompanied by degradation of cellular organelles.

Animals↗

PubMed1987

Biological activity of chicken calcitonin: effects on neonatal rat and embryonic chick osteoclasts.

Chicken calcitonin (cCT) has recently been synthesized according to nucleotide sequence data. We have investigated the in vitro effects of this hormone on the activity of disaggregated, neonatal rat and embryonic chick osteoclasts. While synthetic cCT inhibited bone resorption by neonatal rat osteoclasts at concentrations as low as 0.1 pg/ml, it failed to produce a significant reduction in bone resorption by embryonic chick osteoclasts, even at concentrations as high as 1 microgram/ml. Similarly, cCT at 1 pg/ml reproducibly produced the rapid and dramatic inhibition of rat osteoclast motility characteristic of other calcitonins, as judged by time-lapse video recording, but did not impair the motile behavior of chick osteoclasts at concentrations up to a million-fold higher. Previous studies showing that embryonic chick osteoclasts were unresponsive to synthetic salmon calcitonin left open the possibility that the native hormone was required to produce an inhibitory effect. We conclude that the osteoclast is not a target cell for calcitonin in the embryonic chick; further studies will be required to define a role, if any, for this potent but enigmatic hormone in birds.

Animals↗

PubMed1988

The distribution of podosomes in osteoclasts cultured on bone laminae: effect of retinol.

Osteoclasts, isolated and purified from the medullary bone of calcium-deficient egg-laying hens, adhere to glass coverslips in vitro by means of specialized protrusions of the ventral membrane, denoted podosomes. These structures represent dotlike close-contact adhesion sites in which most cytoskeletal proteins involved in linking the plasma membrane to microfilaments are organized according to a specific and previously described pattern also shared by many oncogene-transformed cells. We show now that podosomes are not only a feature of osteoclasts adhering to artificial glass surfaces but are also present in the ventral membrane of osteoclasts adhering to bone laminae. Moreover, the quantity and the topography of podosomes may be modulated by retinol, which increases bone-resorbing activity of osteoclasts both in vivo and in vitro. A comparative transmission electron microscopy study of osteoclasts adhering on bone laminae in vitro or in vivo indicates that podosomes with identical features are present in the clear zone of the osteoclasts in either condition. Since podosomes are the sealing structures of the clear zone, podosome formation may represent one of the modifications involved in the reorganization process of the osteoclast that precedes bone resorption.

Acid Phosphatase↗

PubMed1989

Organization of osteoclast microfilaments during the attachment to bone surface in vitro.

Rat and chicken osteoclasts were cultured on bone slices, where they showed active resorption with resorption lacunae, which cold be seen by toluidine blue staining or with a scanning electron microscope. Osteoclast microfilaments, F-actin, vinculin, and talin were studied by immunofluorescence. In attached osteoclasts, vinculin appeared as a double circle in the periphery of the cell, and the most intense F-actin staining was located between these vinculin zones. Some chicken osteoclasts showed also intense F-actin staining throughout the center of the cell. Talin appeared in a similar double circle to vinculin. This kind of distribution of microfilaments was always associated with resorption lacunae, and F-actin, vinculin, and talin zones correspond roughly to the edge of lacunae. Osteoclasts showing a diffuse staining pattern were not associated with a resorption pit. The results suggest that this specific microfilament arrangement is located at the attachment zone of the osteoclast and is obligatory for the attachment and resorption. However, this arrangement of microfilaments is quite different from the one that has been previously described in osteoclasts cultured on glass.

Acid Phosphatase↗

PubMed1990

Lysozyme synthesis in osteoclasts.

Osteoclasts may or may not be directly related to monocytes and macrophages, but it is well established that these cell types share a number of features in common. In the present study we sought to extend this comparison by assessing lysozyme synthesis in osteoclasts, an enzyme known to be produced and secreted in large amounts by monocytes and macrophages. Our data show that freshly isolated chicken osteoclasts and osteoclasts in situ contain an abundant amount of lysozyme and correspondingly high steady-state levels of the enzyme's messenger RNA. Marrow macrophages, at various stages of in vitro maturation, also possess lysozyme mRNA but in amounts approximately two to four times lower than osteoclasts. These observations reaffirm the monocyte-macrophage nature of the osteoclast but raise questions about the function of the lysozyme in this cell. At present, the role of the lysozyme in osteoclast activity remains unexplained.

Animals↗

PubMed1992

Effects of heparin on osteoclast activity.

The effect of heparin on osteoclastic bone resorption was studied in vitro using the disaggregated osteoclast resorption assay. Bone resorption was assessed by counting the resorption lacunae on bone slices by light microscopy. Low concentrations of heparin (5 micrograms/ml) increased bone resorption by isolated chick and rat osteoclasts. Among other glycosaminoglycans tested at 5 micrograms/ml, only dextran sulfate showed a small but significant stimulation of resorption. Chondroitin sulfates A, B, and C were without effect at 25 and 100 micrograms/ml, whereas resorption was increased by 100 micrograms/ml of heparan sulfate. With chick osteoclasts, which could be maintained in serum-free conditions, a stimulatory effect of heparin was found both in the presence of 5% fetal calf serum and in serum-free media containing insulin, transferrin, and selenium. The magnitude of the heparin-induced increase in resorption was similar in the presence or absence of serum. The stimulation of resorption was associated with an increase in the number of osteoclasts on bone slices. Pretreatment of the bone slices with heparin also enhanced resorption. In time course experiments, 5 micrograms/ml of heparin caused a doubling of chick osteoclast activity index (number of resorption pits per number of osteoclasts) at 12 and 24 h. In 24 h cultures, treatment with 10 micrograms/ml of the arginine-rich basic protein, protamine, 1 microgram/ml of the immunosuppressant, cyclosporine A, or 5 micrograms/ml of the cysteine-proteinase inhibitor, leupeptin, negated the heparin effect on bone resorption. Leupeptin also inhibited basal resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Leukemia inhibitory factor inhibits osteoclastic resorption, growth, mineralization, and alkaline phosphatase activity in fetal mouse metacarpal bones in culture.

Leukemia inhibitory factor (LIF) has been reported to affect bone metabolism, but results are variable. We examined the effect of mouse recombinant LIF on osteoclastic resorption in fetal bone explants representing different stages of osteoclast development. In cultures of 17-day-old fetal mouse metacarpals in which only osteoclast progenitors and precursors are present, resorption (measured as 45Ca release) was significantly inhibited to 29.2% and to 96.6% in the presence of LIF 100 and 1000 U/ml, respectively. Histologic examination of the explants treated with 1000 U/ml of LIF confirmed the biochemical findings and showed that osteoclast progenitors and precursors remained in the periosteum and did not invade the mineralized matrix. In metacarpals of older fetuses (18- and 19-day-old) in which the mineralized cartilage has been invaded by mature osteoclasts, the inhibition of resorption by LIF (1000 U/ml) was 87.9 and 74.7%, respectively, the latter being significantly less than the inhibition observed in 17-day-old metacarpal cultures. The inhibitory effect of LIF was absent during concurrent administration of PTH or 1,25-(OH)2D3 and could be reversed by PTH. In addition, LIF was found to inhibit growth, mineralization, and alkaline phosphatase activity in metacarpals independently of osteoclastic resorption. These results suggest that LIF affects the development rather than the activity of osteoclasts, probably through an effect on the osteogenic cells. LIF may be an important endogenous regulator of bone metabolism.

Acid Phosphatase↗

PubMed1993

Modulation of vitronectin receptor-mediated osteoclast adhesion by Arg-Gly-Asp peptide analogs: a structure-function analysis.

This study details the investigation of induction of retractile shape change in the osteoclast through inhibition of adhesion between osteoclasts and matrix with (1) peptide analogs bearing an Arg-Gly-Asp (RGD) sequence, (2) antibodies to the integrin alpha V beta 3 vitronectin receptor, and (3) the RGD-containing snake venom peptide echistatin. Osteoclast retraction on dentin has been demonstrated for GRGDSP peptide, in contrast to the inactivity of the analog containing the conservative RGE sequence modification. An osteoclast adhesion assay employing rat or chick bone cells and serum-coated glass coverslips as substrate was developed for routine evaluation of inhibition of adhesion. Antibodies F4 and F11 to the beta 3 chain of rat vitronectin receptor were effective at submicromolar concentrations in rat osteoclasts (IC50 0.29 and 0.05 microM, respectively), whereas MAb 23C6 to human/chick vitronectin receptor was somewhat less effective against chick osteoclasts (IC50 1.6 microM). A rank order of RGD analog activity (mean IC50, microM) in the serum-coated glass adhesion assay was derived for the linear peptides GRGDSP (201 microM), GRGDTP (180 microM), Ac-RGDS-NH2 (84 microM), Ac-RGDV-NH2 (68 microM), RGDV (43 microM), GRGDS (38 microM), and RGDS (26 microM). The two most potent short peptides were the cyclic analog SK&F 106760 Ac-S,S-cyclo-(Cys-(N alpha Me)Arg-Gly-Asp-Pen)-NH2 (IC50 7.0 microM), and the Telios peptide H-Gly-S,S-cyclo-(Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys)-Ala-OH (IC50 6.6 microM). The snake venom peptide echistatin was the most potent substance evaluated in the serum-coated glass assay (IC50 0.78 nM) employing either rat or chick osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Effects of medium acidification by alteration of carbon dioxide or bicarbonate concentrations on the resorptive activity of rat osteoclasts.

Little is known about the extracellular conditions or factors that stimulate mature osteoclasts to resorb mineralized tissues. Isolated mammalian osteoclasts are strongly stimulated by protons in HEPES-buffered culture media in the absence of CO2 and HCO3-, but it has been reported that cell-mediated Ca2+ release from bone organ cultures is increased only when media are acidified by reduction of HCO3- concentrations, and not by increasing PCO2 (considered models of metabolic and respiratory acidosis, respectively). We investigated this question using disaggregated rat osteoclasts cultured on dentin slices for 24 h. The number of pits resorbed per osteoclast was stimulated in media acidified by manipulation of either HCO3- or CO2 concentrations. In experiments in which incubator CO2 was varied, resorption was almost abolished in the presence of 2.5% CO2 at pH 7.61 but increased in a stepwise manner up to 1.3 pits per osteoclast when dentin slices were cultured with 10% CO2 at pH 6.97. The depths and widths of pits, measured using a confocal laser reflection microscope, also tended to increase with increasing CO2 and decreasing pH. However, in experiments where pH was lowered by reducing medium HCO3-, pit size decreased, partially offsetting the increased number of pits resorbed per osteoclast. These findings suggest that rat osteoclasts may be more sensitive to stimulation by CO2 acidosis than by HCO3- acidosis, at least in the short term, and may possibly reflect local regulatory processes in bone.

Acid Phosphatase↗

PubMed1994

Platelet-activating factor induces pseudopod formation in calcitonin-treated rabbit osteoclasts.

We demonstrated previously that platelet-activating factor (PAF), a potent inflammatory mediator, acts on osteoclasts to elevate cytosolic [Ca2+] and stimulate resorption. However, it is not clear whether the effects of PAF on resorptive activity are direct or indirect. In the present study, we investigated the effects of PAF on osteoclast motility. Osteoclasts were isolated from the long bones of neonatal rabbits, and cell motility and morphology were monitored using time-lapse video microscopy. Calcitonin, a hormone known to induce retraction of pseudopods and inhibit resorptive activity, was used to render osteoclasts quiescent. Within 10 minutes of calcitonin treatment (100 ng/ml, final), pronounced retraction of pseudopods was observed in 68 of 112 cells tested. When PAF (200 nM, final) was added 10 minutes after calcitonin treatment, pseudopods were evident 1 h later in 15 of 37 calcitonin-responsive cells tested. In contrast, pseudopods were evident in only 4 of 31 calcitonin-responsive cells treated with control solutions (PAF-vehicle or S-PAF, the biologically inactive stereoisomer of PAF). Pseudopod formation was quantified by measuring the planar area of pseudopods with a computer-based video analysis system. When assessed 60 minutes following PAF treatment, the pseudopod area was significantly greater in PAF-treated cells than in control cells. In some calcitonin-treated osteoclasts, PAF induced pseudopod formation when applied focally using an extracellular micropipette, consistent with a direct action of PAF. We conclude that PAF directly induces pseudopod formation in calcitonin-inhibited osteoclasts, a morphologic response indicative of osteoclast activation.

Animals↗

PubMed1996

The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase.

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.

Alendronate↗

PubMed2003

Co-detection of PTH/PTHrP receptor and tartrate resistant acid phosphatase in osteoclasts.

Serial sections of rat metaphyses were prepared from paraffin embedded tissue blocks and analyzed in sets of three. The central section was stained for tartrate resistant acid phosphatase (TRAP) in order to identify osteoclasts, one adjacent section was immunostained with an affinity purified antibody to a 15 amino acid sequence unique to rat PTH/PTHrP receptor, and the other adjacent section in the set served as an immunostaining control. This allowed each of the 110 osteoclasts examined to be identified by TRAP and to be tested for the presence or absence of PTH/PTHrP receptor. All antibody solutions and rinses contained 1% donkey serum and 0.5% Tween 20 to ensure antibody integrity and good rinsing procedure. Confocal microscopy was used to evaluate fluorescence intensity of the immunostained osteoclasts. Pixel intensities of 58 osteoclasts from young (4 month) rats and 52 osteoclasts from old (15 month) rats were obtained. Pixel intensities were similar (P = 0.89) for both young and old animals. However, the number of PTH/PTHrP receptor deficient osteoclasts was greater for the older animals (14.29% vs. 7.24%). This provides direct evidence of PTH/PTHrP receptors in osteoclasts.

Acid Phosphatase↗

PubMed2004

Expression of Nox4 in osteoclasts.

A new superoxide-generating enzyme, NADPH oxidase 4 (Nox4), contributes to osteoclastic superoxide production. In this study, we demonstrated that Nox4 is expressed at a higher level in osteoclasts than that in precursor cells. This result suggested that Nox4 is upregulated during the differentiation and development of osteoclasts. Cotransfection of Nox4/P22 DNA resulted in enhanced superoxide production in osteoclasts, indicating that P22 may be a necessary factor for the Nox4 activity. In addition, expression of both cathepsin K and TRAP is increased significantly in osteoclasts cotransfected with Nox4/P22. Further study revealed that JNK was activated and that NF-kappa B was inhibited in Nox4/P22 cotransfected osteoclasts. These findings suggest that superoxide and/or superoxide derived molecules may modulate the signal transduction pathways necessary for osteoclasts to function.

Animals↗

PubMed2006

Calmodulin is a critical regulator of osteoclastic differentiation, function, and survival.

Increased osteoclastic resorption and subsequent bone loss are common features of many debilitating diseases including osteoporosis, bone metastases, Paget's disease, and rheumatoid arthritis. While rapid progress has been made in elucidating the signaling pathways directing osteoclast differentiation and function, a comprehensive picture is far from complete. Here, we explore the role of the Ca(2+)-activated regulator calmodulin in osteoclastic differentiation, functional bone resorption, and apoptosis. During active bone resorption, calmodulin expression is increased, and calmodulin concentrates at the ruffled border, the organelle utilized for acid transport and bone dissolution. Pharmacologic inhibitors of calmodulin, several of which are already used clinically as anti-cancer and anti-psychotic agents, inhibit osteoclastic acid transport, suggesting their potential as bone-sparing drugs. Recent studies also implicate calmodulin in osteoclast apoptosis through a mechanism involving its direct interaction with the death receptor Fas. During osteoclastogenesis, RANKL-induction stimulates a rise in intracellular Ca2+, which in turn activates calmodulin and its downstream effectors. In particular, the Ca(2+)/calmodulin-dependent phosphatase calcineurin and its targets, the NFAT family of transcription factors, have been posited as the master regulators of osteoclastogenesis. However, recent in vivo and in vitro studies demonstrate that another Ca(2+)/calmodulin-regulated effector protein, CaMKII, is also involved. CaMKII(+/-) mutant mice have reduced osteoclast numbers, and CaMKII antagonists inhibit osteoclastogenesis in vitro. Furthermore, CaMKII is known to activate AP-1 transcription factors, which are also required for RANKL-induced osteoclast gene transcription, and recent findings suggest that CaMKII can down-regulate gp130, a cytokine receptor involved in bone remodeling and implicated in numerous osteo-articular diseases.

Animals↗

PubMed1992

Reversible inhibition of osteoclastic activity by bone-bound gallium (III).

Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7-12 microM Ga(III) reduces calcium release from bone, but surprisingly, 10-100 microM Ga3+ added to isolated avian osteoclasts did not reduce their degradation of L-(5-3H)-proline bone. 3H-proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) greater than 100 microM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured 67Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 microM gallium from 500 microliters of tissue culture medium, with steady state at greater than 24 h. Osteoclasts on bone inhibited gallium binding capacity approximately 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum-containing medium than in phosphate-buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre-bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 micrograms of bone pre-incubated with 1 ml of 1 microM Ga3+, with 10 pmoles Ga3+/micrograms bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/micrograms bone, resorption was essentially zero. In contrast, pre-treatment of bone with [Ga3+] as high as 15 microM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below approximately 150 pg/micrograms bone acts for a limited time and does not permanently damage the cells. We conclude that bone-bound Ga(III) from medium concentrations less than 15 microM inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] greater than 50 microM.

Animals↗