The generation of highly enriched osteoclast-lineage cell populations.

Osteoclasts form when hematopoietic cells are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNFalpha). Osteoclast precursors derive from M-CSF-dependent proliferating hematopoietic cells but cannot yet be purified from mixed populations. M-CSF stimulation of bone marrow cells results in large numbers of nonadherent, proliferating macrophage precursors. These rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA-strain mouse bone marrow, when cocultured with ST2 cells (which express RANKL and M-CSF), formed numerous mononuclear osteoclasts, which resorbed bone and expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors (CTR). Addition of approximately 10 BMM precursors to ST2 cultures resulted in over 80% of these cocultures forming functional osteoclasts, suggesting that they are a highly enriched source of osteoclast progenitors. Supporting this, recombinant RANKL/M-CSF-stimulated BMM precursors formed populations in which all cells expressed TRAP. While only a small proportion of these cells (8.6%) expressed CTR, with transforming growth factor-beta (TGFbeta) present RANKL/M-CSF-stimulated BMM precursors formed almost pure (98.4%) CTR-positive osteoclasts after 7 days. This suggests that TGFbeta stimulated the maturation rate of these cells. Passaged or viably frozen BMM precursors gave rise to BMM that also all formed osteoclasts lineage cells after RANKL/M-CSF stimulation. These data suggest that BMM precursors derived from CBA mice are an expanded pool of osteoclast progenitors. These can be employed to generate osteoclast populations of high purity and in large numbers when stimulated by TGFbeta, which greatly augments the osteoclastogenic effects of RANKL.     

Generation of bone-resorbing osteoclasts from B220+ cells: its role in accelerated osteoclastogenesis due to estrogen deficiency.

Estrogen deficiency stimulates both osteoclastic bone resorption and pre-B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220+ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220+ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220+ and B220- cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross-contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate-resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220+ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220+ cells are capable of generating osteoclasts and that the increase in the number of B220+ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.     

Phosphodiesterase 4 inhibitor rolipram potentiates the inhibitory effect of calcitonin on osteoclastogenesis

To assess the combination effect of calcitonin and the phosphodiesterase 4 inhibitor rolipram on osteoclastogenesis, adherent cell-depleted bone marrow cells from mouse tibia and femur (ACD-BMCs), which were cultured in the presence of 25 ng/ml colony-stimulating factor 1 (CSF-1) and 100 ng/ml soluble receptor activator of NF-κB ligand (sRANKL), were utilized. Calcitonin inhibited formation of tartrate-resistant acid phosphatase-positive multinucleated cells, as mature osteoclasts, by 70% even at 20 pM, whereas rolipram (10 μM) scarcely affected osteoclast formation; in contrast, the combination of both agents led to significant inhibition of multinucleation and pit formation ability of osteoclasts. The combined administration of calcitonin and rolipram attenuated calcitonin receptor mRNA expression in comparison to treatment with either agent alone, whereas expression of RANK and CSF-1 receptor mRNAs was unchanged. Alone, these agents scarcely elevated intracellular cyclic AMP (cAMP) concentration; however, combination treatment with both agents significantly increased cAMP concentration in osteoclast progenitors and osteoclasts. The combination effect was abolished by H-89, an inhibitor of protein kinase A. It appears that rolipram inhibited hydrolysis of cAMP formed by calcitonin in cells and potentiated the inhibitory effect of calcitonin on osteoclastogenesis. The escape phenomenon following calcitonin treatment may also be prevented by concomitant treatment with the phosphodiesterase 4 inhibitor.     

Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development

Amylin and calcitonin gene-related peptide (CGRP) are homologous 37 amino acid peptides that are found in the circulation. Both peptides belong to the calcitonin family. Similar to calcitonin, amylin and CGRP inhibit osteoclast activity, although they are much less potent than calcitonin. Calcitonin is known to act on the latter stages of osteoclast development, inhibiting the fusion of committed preosteoclasts to form mature multinucleated cells; however, whether or not calcitonin acts earlier in the formation of the precursor osteoclasts is controversial. The question of osteoclast development has never been examined with respect to amylin and CGRP. These issues are addressed in the present study. We studied the effects of calcitonin (salmon and rat), amylin (human and rat), and CGRP (human and rat) in mouse bone marrow cultures stimulated to generate osteoclasts using 1alpha,25-dihydroxyvitamin D3. Calcitonin dose-dependently decreased the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as TRAP-positive mono-/binucleated cells at concentrations >10(-13) mol/L. Amylin and CGRP showed similar effects at concentrations >10(-9) mol/L. In addition, calcitonin substantially reduced the ratio of TRAP-positive multinucleated to mono-binucleated cells, indicating an effect on fusion of osteoclast precursors. The present data establish that this family of peptides not only acts on mature osteoclasts but also inhibits their development in bone marrow cultures. This activity is shared by amylin and CGRP. The much greater potency of calcitonin than amylin and CGRP is consistent with the action of these peptides being mediated by calcitonin receptors.     

Osteoclast precursors circulate in avian blood

Summary The osteoclast is known to be derived from a marrow-residing precursor that is a member of the mononuclear phagocyte family, but the means by which this cell moves from marrow to bone is unknown. We herein demonstrate that mononuclear progenitors capable of differentiating, in vitro, into cells exhibiting the osteoclast phenotype circulate in chickens. The mononuclear fraction was isolated on a density gradient from blood drawn from calcium-deprived laying hens and the plastic-adherent population was obtained. These cells are members of the mononuclear phagocyte family, as demonstrated by nonspecific esterase and tartrate-resistant acid phosphatase (TRAP) activities, expression of the macrophage-specific mannose receptor, and their ability to phagocytose latex particles. When cultured in the presence of devitalized bone, these cells undergo progressive multinucleation and ultimately become essentially indistinguishable from isolated osteoclasts and those generated from bone marrow precursors. Specifically, the blood-derived polykaryons are TRAP-positive, exhibit characteristic ruffled membranes, and express the osteoclast antigens 121F and 23C6. When placed on bone slices, these cells from typical resorptive pits. Moreover, when cultured with ³H-proline-labeled bone, the blood monocytegenerated osteoclasts mobilize matrix as effectively as those derived from marrow. Thus, osteoclast precursors circulate in the blood of laying hens and can be induced to differentiate in vitro.     

Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts.

Osteoclasts are thought to belong to a macrophage lineage. However, the nature of common precursors of osteoclasts and macrophages remains to be investigated. We have characterized the differentiation potential of mouse bone marrow macrophages into mature osteoclasts. Monocyte macrophage-colony-stimulating factor (M-CSF) stimulated the proliferation of bone marrow macrophages in a dose-dependent manner and these M-CSF-dependent bone marrow macrophage (MDBM) cells efficiently differentiated into the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the presence of soluble RANKL (sRANKL) and M-CSF in the in vitro culture. The macrophage-like cell line TMC16 was established from tsA58 (temperature-sensitive SV40 large T-antigen) transgenic mice in the same manner to the preparation of MDBM cells and also differentiated into mature osteoclasts. During this differentiation in vitro, the morphology of the cells changed from spindle to round and smaller (termed pOC) on day 2 and to multinuclear (termed multinucleated cells [MNCs]) on day 4. The surface expression of macrophage marker CD14 was down-regulated and that of CD43 was up-regulated on pOC, analyzed by flow cytometry. RNA analysis revealed that osteoclast marker genes such as calcitonin receptor (CTR), carbonic anhydrase II (CAII), cathepsin K (cath K), MMP9, and TRAP were strongly expressed in MNCs and weakly in pOC whereas MDBM cells did not express these genes. However, the osteopontin (OPN) gene was strongly expressed in MDBM cells and this expression became weakened after differentiation into pOC. The TMC16 cell line weakly expressed cath K, TRAP, and OPN, suggesting that the TMC16 cell line is immortalized at a stage slightly differentiated from MDBM cells. Furthermore, cell sorting analysis revealed that osteoclast early progenitors in bone marrow cells are preferentially present in the Mac-1- F4/80dull population, which differentiated into MDBM cells (the osteoclast progenitor) expressing Mac-1+ F4/80int, suggesting that M-CSF plays roles of a differentiation factor as well as a growth factor for osteoclast early progenitors. These results showed the transition of morphology, surface markers, and gene expression from the early to mature stage in osteoclast differentiation. We propose three differentiation stages in the osteoclast lineage: the pro-osteoclast (spindle-shaped macrophage cells), the pre-osteoclast (small round mononucleated TRAP-positive cells), and the mature osteoclast (multinucleated TRAP-positive cells) stage.     

NF-kB signaling blockade abolishes implant particle-induced osteoclastogenesis.

In this study we investigated the effect of NF-kB signaling blockade on polymethylmethacrylate (PMMA) particle-induced osteoclastogenesis in vitro. We first established effective blockade of NF-kB activity as tested by electrophoretic mobility shift assays (EMSA). Particle-induced NF-kB activation in murine osteoclast precursor cells (CSF-1-dependent bone marrow macrophages) was markedly reduced by co-treatment of the cells with the NF-kB inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Calpain Inhibitor I (CPI). This inhibition of NF-kB activity was associated with blockade of p50 NF-kB subunit nuclear translocation. We then established a direct NF-kB inhibition approach by utilizing a TAT-bound, mutant IkB (TAT:IkB(46-317)), and demonstrated an inhibitory effect evidenced by decreased NF-kB DNA binding activity. Having established that these strategies (TPCK, CPI, TAT: IkB(46-317)) effectively block NF-kB activation, we next investigated the effect of these agents on particle-stimulated osteoclast formation. PMMA particle stimulation of mature osteoclast formation from RANKL-primed osteoclast precursor cells was blocked by all three inhibitors. To further test the efficacy of NF-kB blockade, experiments were performed with the TAT:IkB(46-317) mutant peptide in whole bone marrow cultures that contain supporting stromal cells. Again, this inhibitor efficiently blocked particle-induced osteoclastogenesis. Thus, we have shown that pharmaceutical and molecular blockade of NF-kB activation inhibits PMMA particle-directed osteoclastogenesis in vitro.     

Ipriflavone inhibits murine osteoclast formationIn Vitro

Ipriflavone, one of the iprflavone derivatives, is a therapeutic drug for osteoporosis. The mechanism is thought to be the inhibition of bone resorption. In the present paper, we report that ipriflavone inhibited formation of osteoclasts from murine spleen cells co-cultured with stromal cells cloned from murine bone marrow. In this system, ipriflavone inhibited osteoclast generation in a dose-dependent manner (10^–7-10^–5 M). Ipriflavone also inhibited prostaglandin E₂ production in MC3T3-E1 cells, which are widely employed as osteoblasts. Moreover, ipriflavone inhibited the proliferation of stromal cells (10^–6-10^–5 M), but not osteoblastic cells. These results suggest that one mechanism for the inhibitory effects of ipriflavone on bone resorption is the inhibition of osteoclast formation through inhibiting prostaglandin E₂ production in osteoblasts and thereby suppressing proliferation of stromal cells.     

Oscillatory fluid flow-induced shear stress decreases osteoclastogenesis through RANKL and OPG signaling

Physical activity creates deformation in bone that leads to localized pressure gradients that drive interstitial fluid flow. Due to the cyclic nature of the applied load, this flow is oscillatory by nature. Oscillatory fluid flow (OFF) may lead to positive bone remodeling through effects on both osteoblasts and osteoclasts but its effect on osteoclastogenesis is poorly understood. In this study, the effects of OFF on expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), two important regulators of osteoclast differentiation, were investigated. In addition, its effect on osteoclast formation was quantified. ST-2 murine bone marrow stromal cells were plated on glass slides and cultured with 1,25-dihydroxyvitamin D(3) to express RANKL. Cells were exposed to various durations of OFF resulting in a peak shear stress of 1 Pa. Time course and dose-response studies were performed and real-time RT-PCR was used to quantify levels of RANKL, OPG mRNA. ST-2 cells exposed to OFF were also co-cultured with RAW 264.7 monocytes and osteoclast number quantified. Decrease in RANKL/OPG was maximal immediately after end of flow and there existed a significant increase in OPG and decrease in RANKL with increasing load duration of up to 2 h. OFF resulted in a decrease in osteoclast formation by ST-2 cells co-cultured with RAW 264.7 cells compared to co-culture of control (non-loaded) ST-2 cells with RAW 264.7 cells. These results suggest that indeed OFF is a potent regulator of bone remodeling, and that shift towards positive bone remodeling mediated by loading-induced fluid flow may occur via suppression of the formation of osteoclasts.     

Jaw and Long Bone Marrows Have a Different Osteoclastogenic Potential

Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP⁺ multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow.     

Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function

Transforming growth factor (TGF) beta 1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-beta 1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-beta 1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-beta 1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-beta 1 in osteoclast generation.     

Osteoclasts and a small population of peripheral blood cells share common surface antigens

Summary Several methods have been tried to identify mononuclear osteoclast precursors. We used a panel of 13 osteoclast-recognizing monoclonal antibodies (mabs) for the identification of osteoclast precursor cells from the bone, bone marrow, and peripheral blood of egg laying hens. Almost all mabs stained some mononuclear cells in the bone. Seven mabs recognized few mononuclear cells in the bone marrow and five mabs gave the positive immunofluorescence reaction in the white blood cell fraction. Possible immediate osteoclast precursor cells differing from osteoclasts in their densities were identified in the bone. Three mabs (K38, K52, and K70) stained the same amount of mononuclear cells (2.6–3.4%) enriched in Percoll density centrifugation. Of the monoclonal antibodies that recognized few cells in blood, K41 stained only osteoclasts. K47 and K52 also recognized some mononuclear cells in the bone marrow. Other monoclonal antibodies K51 and K70 were more unspecific, since they stained cells derived from other tissues. Blood cells detected with these different monoclonal antibodies were negative for tartrate-resistant acid phosphatase (TRAP). On the basis of our results, we suggest that there is in the blood a specific TRAP-negative cell population, which is a good candidate for osteoclast precursor.     

Breast cancer increases osteoclastogenesis by secreting M-CSF and upregulating RANKL in stromal cells

BACKGROUND: Breast cancer metastasis to bone causes resorption of the mineralized matrix by osteoclasts.Macrophage colony stimulating factor (M-CSF)and receptor activator of the NF-kappaB ligand (RANKL) are produced by stromal cells and are essential for osteoclast formation. The human breast cancer cell line, MDA-MB-231, reliably forms bone metastases in a murine model and stimulates osteoclast formation in culture. We hypothesized that MDA-MB-231 stimulates osteoclast formation through secretion of M-CSF and/or RANKL. MATERIALS AND METHODS: We cocultured MDA-MB-231 and a bone marrow derived cell line, UAMS-33, and evaluated the expression of M-CSF and RANKL mRNA. Osteoclast formation was assessed using these cells added to hematopoietic cell cultures. RESULTS: MDA-MB-231 exhibited constitutive expression of M-CSF mRNA. As expected, addition of recombinant M-CSF (30 ng/ml) and RANKL (30 ng/ml) to hematopoietic osteoclast precursors supported osteoclast formation, while the addition of soluble RANKL alone or MDA-231 without added RANKL did not. Notably, coculture of MDA-231 with hematopoietic cells and added soluble RANKL stimulated significant osteoclast formation, indicating that MDA-231 served as an effective source for M-CSF. MDA-231 did not express RANKL. However, when cocultured with the murine bone marrow stromal cell line UAMS-33, RANKL expression was significantly increased in the latter cells. MDA-231 also stimulated osteoclast formation in coculture with UAMS-33 and hematopoietic cells. CONCLUSIONS: We conclude that MDA-MB-231 increases osteoclast formation by secreting adequate amounts of M-CSF protein and enhancing the expression of RANKL by stromal support cells. The ability to stimulate osteoclasts may explain the ability to metastasize to bone.     

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.     

Acid pH Increases Carbonic Anhydrase II and Calcitonin Receptor mRNA Expression in Mature Osteoclasts

Numerous resorptive stimuli have been shown to enhance osteoclast differentiation, increasing osteoclast numbers and accelerating bone resorption. Currently, there is much less understanding of regulation of mature osteoclast activity. Indeed, there is presently only minimal evidence of changes in gene expression as a mechanism for altering bone resorption. We investigate here, in the mature osteoclast, regulation of 2 genes—carbonic anhydrase II (CAII) and calcitonin receptor (CTR) in response to acidosis, which is known to increase bone resorption. We studied the effect of acid pH on CAII and CTR mRNA expression in mature osteoclasts raised in coculture of ST-2 and primary marrow cells. On day 6 of culture, stromal cells were removed with collagenase, the remaining osteoclasts were incubated overnight, and then exposed to varying pH. RT-PCR was performed on total RNA using primers for CAII, CTR, or glyceraldehyde dehydrogenase phosphate (GAP). Expression of CTR mRNA was increased 2.14 ± 0.41 and 2.56 ± 0.45 (P < 0.05)-fold by a 4-hour exposure to pH 6.75 and 6.5, respectively. CAII mRNA was similarly increased 2.18 ± 0.42 and 2.63 ± 0.48 (P < 0.05)-fold by pH 6.75 and 6.5, respectively. Increased expression of CAII and CTR mRNA was seen by 2 hours and maximally by 4 hours. Increased expression of CTR and CAII mRNA was not explained by increases in osteoclast numbers: pH 7.4–100 ± 3.7, 6.75–133 ± 8.3, 6.5–124 ± 7.8. These results demonstrate upregulation of two osteoclast genes in response to acidosis, illustrating the ability of the mature osteoclast to respond to resorptive signals with increased functional gene expression. Received: 28 June 1999 / Accepted: 4 February 2000     

Ultrastructural and cytobiological studies on possible interactions between PTHrP-secreting tumor cells,stromal cells,and bone cells

Parathyroid hormone-related peptide (PTHrP) induces pathological bone resorption in an endocrine manner, resulting in hypercalcemia of malignancy. However, the histopathological aspect of the action of PTHrP secreted by tumor cells on bone resorption has not well been documented. Therefore, we studied cell–cell interactions between bone cells, stromal cells, and PTHrP-secreting tumor cells (EC-GI-10) morphologically. Tumor cells injected subcutaneously into the parietal region formed a tumor mass, invading the bone marrow. The tumor mass was surrounded by a membrane structure consisting of stromal cells. These stromal cells were positive for alkaline phosphatase (ALPase). Tartrate-resistant acid phosphatase (TRAPase)-positive osteoclasts were localized close to the ALPase-positive cells, and numerous osteoclasts were observed on the neighboring bone surfaces. PTHrP, vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-9 were detected in the tumor cells. Using RT-PCR, expression of interleukin (IL)-1, IL-1, and PTHrP, which are strong bone resorption factors, was detected in the tumor cells. Some ALPase-positive cells localizing on the neighboring bone surfaces and endothelial cells revealed PTH/PTHrP receptor immunoreactivity. Ultrastructurally, numerous blood vessels were observed between the tumor nests and the stromal cells. The nests were surrounded by a basement membrane, but it was discontinuous, therefore permitting direct contact between the tumor cells and the stromal cells. These results indicate that PTHrP secreted by tumor cells appears to stimulate osteoclast differentiation and bone resorption in a paracrine manner through PTH/PTHrP receptor-immunopositive cells. IL-1, IL-1, VEGF, and MMP-9 may also be involved in facilitating osteoclast formation and the subsequent bone resorption.     

Production of IL-7 is increased in ovariectomized mice,but not RANKL mRNA expression by osteoblasts/stromal cells in bone,and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220⁺ cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.     

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.     

Bovine dentine organic matrix down-regulates osteoclast activity

Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)₂-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this.