大鼠去卵巢后骨髓源性破骨细胞形成的动态变化及其与骨髓IL-6、IL-6受体表达的关系

目的 观察大鼠去卵巢后骨髓源性破骨细胞形成的动态变化及其与骨髓细胞IL-6、IL-6受体以及gpl30基因表达水平改变的关系。方法 健康3月龄雌性SD大鼠60只,30只作为假手术对照组,30只经腹手术去卵巢。分别于去卵巢后2,4,6,8,12周取去卵巢组和对照组各6只大鼠骨髓细胞作细胞培养和提取RNA。培养的第6天分别作瑞氏-吉姆萨染色和抗酒石酸酸性磷酸酶(TRAP)染色,以胞核3个或3个以上以及TRAP(+)为破骨细胞标志,计数细胞数。骨髓细胞提取总RNA进行逆转录PCR。结果 术后2周去卵巢组破骨细胞形成数即多于对照组(P<0.05);第4-6周,去卵巢组破骨细胞形成达高峰,明显多于2周(P<0.01);去卵巢后的8周,较峰值下降,直至12周去卵巢组破骨细胞形成数仍高于对照组(P<0.05)。与破骨细胞的变化相对应,术后2周,骨髓细胞IL-6及IL-6RmRNA表达均明显增高(分别为P<0.01,P<0.05);第4周时,IL-6表达水平达到高峰,保持至第6周,IL-6R表达则在第6周达最高;第8周,IL-6、IL-6R基因表达水平较峰值下降,但直至第12周仍高于对照组。去卵巢后全程未见gp130基因表达水平有明显变化。结论 大鼠去卵巢后骨髓干细胞分化形成破骨细胞明显增加,呈时间相关动态过程,第6周达高峰,这一过程与骨髓细胞IL-6、IL-6R基因表达的动态变化一致,提示去卵巢后骨髓细     

Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow.

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1alpha,25-dihydroxyvitamin D3 [1a,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1alpha,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D-treated mice as well as estrogen-treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the management of osteoporosis.     

Role of prostaglandins in interleukin-1-induced bone resorption in mice in vitro

The mechanism of bone resorption induced by interleukin 1 (IL-1) was examined in mice using three different in vitro assay systems: a fetal long bone organ culture system, a bone marrow culture system, and a coculture system of primary osteoblastic cell populations and spleen cells. In the organ culture system, recombinant human IL-1 alpha (rhIL-1 alpha) increased both bone resorption and osteoclast number. Both were partially suppressed in the presence of indomethacin. In the marrow culture, both rhIL-1 alpha and rhIL-1 beta stimulated osteoclastlike cell formation, which was completely inhibited by adding indomethacin concurrently. Furthermore, there was a good correlation between the number of osteoclastlike cells formed and the amount of prostaglandin E2 (PGE2) released into the culture media. This indicates that PGE2 is involved in the mechanism of IL-1-mediated osteoclastlike cell formation. In the coculture of primary osteoblastic cell populations and spleen cells, rhIL-1 again stimulated osteoclastlike cell formation, which was inhibited by adding indomethacin. In the cocultures in which direct interaction between osteoblastic cells and spleen cells was inhibited, PGE2 synthesis was similarly increased but no osteoclastlike cells were formed. These results indicate that IL-1 induces osteoclast formation by a mechanism involving PG (most likely PGE2). Furthermore, direct interaction between osteoclast progenitors and osteoblastic cells is required in the osteoclast recruitment induced by IL-1.     

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

1alpha-Hydroxyvitamin D3 suppresses trabecular bone resorption by inhibiting osteoclastogenic potential in bone marrow cells after ovariectomy in mice

To test the hypothesis that 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) suppresses bone resorption after ovariectomy (ovx) by inhibiting osteoclastogenic potential in bone marrow cells, the bilateral tibiae of ddY mice, 8 weeks of age, subjected to ovx were obtained. 1alpha(OH)D3, at doses of 0, 0.2 (low dose), or 0.4 microg/kg body weight (high dose), was administered orally by canula three times a week for 2 or 6 weeks. Histomorphometric analysis of the proximal tibiae revealed that 1alpha(OH)D3 administration had no significant effect on trabecular bone volume of ovx limbs, which was reduced after ovx. The bone formation rate, increased by ovx, was significantly decreased by the administration of high-dose 1alpha(OH)D3. The ovx-induced increases in osteoclast number and surface at 2 weeks postsurgery were suppressed by the administration of high-dose 1alpha(OH)D3. With regard to bone marrow cells, the number of nonadherent cells per tibia obtained from ovx limbs increased, and this increase was suppressed by the administration of low- and high-dose 1alpha(OH)D3. The formation of mineralized nodules in marrow cultures obtained from ovx limbs was increased after surgery and unaltered by 1alpha(OH)D3 administration. The number of osteoclast-like multinucleated cells obtained from ovx limbs was reduced by low- and high-dose 1alpha(OH)D3 administration alike. The number of colony forming units-fibroblast and the number of colony forming units for granulocytes and macrophages was unaltered by ovx or the administration of 1alpha(OH)D3. The present study clearly demonstrates that high-dose 1alpha(OH)D3 suppresses osteoclast numbers and surface after ovx. The inhibitory effects of low- and high-dose 1alpha(OH)D3 on bone marrow cells after ovx were marked in the differentiation from osteoclast precursors to mature osteoclasts. Administration of 1alpha(OH)D3 suppressed ovx-promoted trabecular bone resorption by inhibiting osteoclastogenic potential in bone marrow cells.     

Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclastlike cells in vitro

Interleukin-1 (IL-1) alpha and beta and tumor necrosis factor (TNF) alpha and beta are potent stimulators of bone resorption in vitro and in vivo. However, the mechanisms underlying this increased bone resorption have not been clearly defined. Increased bone resorption can result from increased activity of individual osteoclasts, increased numbers of osteoclasts, or both. Therefore, we have used a long-term human marrow culture system that forms multinucleated cells (MNC) with the characteristics of osteoclasts to examine the effects of IL-1 and TNF on osteoclast formation. Human recombinant IL-1 alpha and IL-1 beta, and human recombinant TNF-alpha and TNF-beta stimulated MNC formation from 4- to 60-fold. IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta significantly increased MNC formation at very low concentrations: 2.5 x 10(-13) M for IL-1 alpha and IL-1 beta, 10(-11) M for TNF-alpha, and 10(-10) M for TNF-beta In addition, these cytokines enhanced MNC formation in the presence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], a potent osteotropic factor that stimulates MNC formation by stimulating fusion of mononuclear precursor cells. Simultaneous addition of IL-1 and TNF to the cultures resulted in a synergistic stimulation of MNC formation. These results suggest that: (1) IL-1 and TNF stimulate bone resorption in part by increasing osteoclast formation and (2) an extremely low concentration of these factors can synergistically increase osteoclast formation in the absence of other factors, such as 1,25-(OH)2D3. These data suggest that synergistic interactions among cytokines play an important role in maintaining bone cell activity in normal and pathologic states.     

Direct and indirect actions of fibroblast growth factor 2 on osteoclastic bone resorption in cultures.

Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10(-9) M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10(-8) M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (> or = 10(-12) M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10(-11) M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10(-9) M - 10(-8) M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (> or =10(-12) M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (> or = 10(-9) M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently.     

The surface antigen CD45R identifies a population of estrogen-regulated murine marrow cells that contain osteoclast precursors

We examined the osteoclastogenic potential of murine bone marrow cells that were fractionated according to their expression of the surface antigen CD45R. Osteoclast-like cells (OCL) with many authentic osteoclast characteristics readily formed in purified CD45R⁺ murine bone marrow cell cultures after treatment with receptor activator of nuclear factor κB ligand (RANKL) and M-CSF. Ovariectomy (Ovx) caused a 1.5- to 2-fold increase in OCL number in unfractionated and CD45R⁺ murine bone marrow cell cultures without affecting OCL formation in CD45R⁻ marrow cells. Limiting dilution assays confirmed that Ovx caused an increase in osteoclast precursor cell number in CD45R⁺ but not CD45R⁻ cells. Mice deficient in the type 1 IL-1 receptor (IL-1R1 KO) do not lose bone mass after Ovx. We found that unfractionated, CD45R⁺, and CD45R⁻ bone marrow cells from IL-1R1 KO mice showed no increase in OCL formation in vitro after Ovx. In both the wild-type (WT) and the IL-1R1 KO mice Ovx was associated with a 2-fold increase in pre-B-lymphocytes. About 1.3–3.5% of murine marrow cells expressed surface RANK (the receptor for RANKL) while about 11.9–15% of murine bone marrow cells expressed c-Fms (the receptor for M-CSF). There was little effect of Ovx on cells expressing either RANK or c-Fms. These results demonstrate that CD45R expression identifies a subset of murine bone marrow cells whose ability to form OCL in vivo is regulated by estrogen in WT but not IL-1R1 KO cells. The effects of estrogen on bone mass may be related to these responses.     

RANKL-stimulated osteoclast-like cell formation in vitro is partially dependent on endogenous interleukin-1 production

Receptor activator of NF-kappaB ligand (RANKL) and interleukin-1 (IL-1) individually plays a critical role in the differentiation and activation of osteoclasts in bone. In addition, both RANKL and IL-1 activate similar signal transduction pathways including p38 MAP kinase and c-Jun NH(2) terminal kinase (JNK). We examined if endogenously produced IL-1 influenced osteoclast-like cell (OCL) formation in murine bone marrow and bone marrow monocyte (BMM) cultures that were stimulated with M-CSF and RANKL. RANKL stimulated OCL formation in a dose-dependent manner in bone marrow cultures, and this response was significantly inhibited by IL-1 RA (100 ng/ml), a specific IL-1 antagonist. Interleukin-1 further increased OCL formation in BMM cultures that were treated with M-CSF (30 ng/ml) and RANKL (1, 3, 10 and 30 ng/ml). In addition, BMM cultures from IL-1 type I receptor-deficient mice, which do not respond to IL-1, demonstrated significantly less OCL formation compared to wild-type BMM cultures. We examined the time course and dose response of IL-1alpha protein expression by ELISA in BMM cultures that were treated with or without M-CSF and RANKL. RANKL dose dependently stimulated IL-1alpha protein significantly (up to 46%) in 6-day cultures. The interaction of RANKL and IL-1 on osteoclastogenesis did not appear significantly dependent on prostaglandin synthesis since PGE(2) expression in the conditioned medium of BMM cultures was nearly undetectable and the PGHS-2 specific inhibitor, NS-398, was without effect. We also investigated the effect of IL-1 on p38 MAP kinase and JNK in BMM cultures. The combination of RANKL and IL-1 had additive effects on JNK but not p38 MAP kinase compared to results in cultures treated with RANKL or IL-1 alone. In addition, SP600125, a specific JNK inhibitor, markedly reduced OCL formation in BMM cultures that were treated with RANKL or the combination of RANKL and IL-1. These findings demonstrate that endogenously produced IL-1 augments the response of bone marrow cells to RANKL, and this effect appears mediated by mechanisms that are associated with enhancement of JNK activity.     

A murine model of inflammatory bone disease

We have recently reported the identification of a new recessive mutation on murine chromosome 18 that results in tail kinks and deformity in the lower extremities of mice. Preliminary examination of the bones of these mice showed that there are abnormalities present that resembled chronic recurrent multifocal osteomyelitis. Accordingly, this new mutation was named "CMO." In this report, we describe the histology of bones in CMO mice, as well as the capacity of the bone marrow cells from these animals to form osteoclasts (OCLs). In addition, we tested conditioned media from non-adherent marrow cells and total marrow cells from CMO mice for their capacity to induce OCL formation in normal murine marrow cultures. These studies demonstrated that the bone disease in these animals is inflammatory in nature, and a soluble factor(s) that is not IL-1alpha, IL-6 or TNF-alpha is released by marrow cells from CMO animals and enhances OCL formation in normal murine marrow cultures.     

Eosinophil chemotactic factor-L (ECF-L): a novel osteoclast stimulating factor.

Screening a cDNA library enriched for genes expressed in OCLs identified ECF-L. ECF-L enhanced OCL formation without increasing RANKL levels. Anti-ECF-L inhibited RANKL-induced OCL formation. These results support a potent role of ECF-L in osteoclastogenesis. INTRODUCTION: To investigate the molecular mechanisms that control osteoclastogenesis, we developed an immortalized osteoclast (OCL) precursor cell line that forms mature OCLs in the absence of stromal cells and used it to form pure populations of OCLs. MATERIALS AND METHODS: Polymerase chain reaction (PCR) selective cDNA subtraction was used to identify genes that are highly expressed in mature OCLs compared with OCL precursors employing OCL and OCL precursors derived from this cell line. RESULTS: Eosinophil chemotactic factor-L (ECF-L), a previously described chemotactic factor for eosinophils, was one of the genes identified. Conditioned media from 293 cells transfected with mECF-L cDNA, or purified ECF-L Fc protein, increased OCL formation in a dose-dependent manner in mouse bone marrow cultures treated with 10(-10) M 1,25(OH)2D3. OCLs derived from marrow cultures treated with ECF-L conditioned media formed increased pit numbers and resorption area per dentin slice compared with OCLs induced by 1,25(OH)2D3 (p < 0.01). Addition of an antisense S-oligonucleotide to mECF-L inhibited OCL formation in murine bone marrow cultures treated only with 10(-9) M 1,25(OH)2D3 compared with the sense S-oligonucleotide control. Time course studies demonstrated that ECF-L acted at the later stages of OCL formation, and chemotactic assays showed that mECF-L increased migration of OCL precursors. mECF-L mRNA was detectable in mononuclear and multinucleated cells by in situ hybridization. Interestingly, a neutralizing antibody to ECF-L blocked RANKL or 10(-9) M 1,25(OH)2D3-induced OCL formation in mouse bone marrow cultures, although ECF-L did not induce RANKL expression. CONCLUSIONS: These data show ECF-L is a previously unknown factor that is a potent mediator of OCL formation, which acts at the later stages of OCL formation and enhances the effects of RANKL.     

A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures. In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor beta (TGF-beta). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.     

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.     

Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts

The proliferation inhibitor of the macrolide class, everolimus, is a drug shown to be effective in the prevention of organ transplant rejection and to have a potential in the treatment of rheumatoid arthritis and certain cancers. As these diseases or their current treatments are associated with bone loss, we examined the effect of everolimus on mouse and human bone cells in vitro and on bone in an ovariectomized (OVX) rat model. Everolimus potently inhibited primary mouse and human osteoclast activity in the pit assay (IC50 values of 0.6-4.0 nM), as well as osteoclast formation, measured as the number of tartrate-resistant acid phosphatase (TRAP) multinucleated cells (IC50 values of 7.7-10.5 nM). Inhibition of osteoblastic differentiation was also observed (IC50 value of 13.5 nM). As expected, everolimus inhibited proliferation of osteoclast precursors and stimulated apoptosis, albeit with insufficient potency and efficacy to explain inhibition of osteoclast activity. Thus, everolimus appeared to directly inhibit bone resorption, which is in accord with the detected inhibition of mRNA and protein expression of cathepsin K; the main collagen-degrading protease in osteoclasts. Despite the in vitro antiproliferative activity of everolimus and the observed inhibition of osteoblast differentiation, no detrimental effects were detected at different skeletal sites in mature OVX rats at doses up to 3 mg/kg/day. This everolimus dose also prevented the OVX-induced loss of cancellous bone by 60%, an effect predominantly associated with decreased osteoclast-mediated bone resorption, resulting in a partial preservation of the cancellous bone network. Everolimus inhibited S6 kinase 1 activity in rat blood cells, skin, and bone, at doses equivalent to those used for efficacy experiments in the OVX rat model, which demonstrated in vivo targeting of the expected molecular pathway. In conclusion, everolimus directly inhibits bone resorption by osteoclasts and thus could at least be neutral or protective for bone in vivo, which would favor its use in disease indications associated with bone loss.     

Two distinct cellular mechanisms of osteoclast formation and bone resorption in periprosthetic osteolysis.

PURPOSE: TNFalpha and IL-1alpha are proinflammatory cytokines that are abundant in periprosthetic tissues. These cytokines stimulate bone resorption and have recently been shown to directly induce osteoclast formation in mouse marrow cultures. We examined whether TNFalpha and IL-1alpha can directly induce osteoclast formation from human arthroplasty-derived (CD14(+)) macrophages by a mechanism independent of RANKL-induced osteoclastogenesis. METHODS: TNFalpha and M-CSF (+/-IL-1alpha) were added to cultures of magnetically sorted (CD14(+)) and unsorted (CD14(+)/CD14(-)) cells isolated from the pseudomembrane of loosened hip arthroplasties. Osteoprotegerin (OPG), RANK:Fc and antibodies to TNF receptors (p55 and p75) were added to these cultures to distinguish the pathway of osteoclastogenesis. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and lacunar resorption. RESULTS: The addition of TNFalpha (+/-IL-1alpha) resulted in differentiation of CD14(+) macrophages into TRAP(+) and VNR(+) multinucleated cells capable of extensive lacunar resorption. Both OPG and RANK:Fc (which inhibit RANKL-induced osteoclastogenesis) did not block osteoclastogenesis. The addition of antibodies directed against the p55 receptor subunit of TNF resulted in significant inhibition of osteoclast formation and lacunar resorption. CONCLUSIONS: Our results indicate that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from arthroplasty macrophages and that TNFalpha acts synergistically with IL-1alpha to stimulate lacunar resorption. This process is distinct from the RANK/RANKL signalling pathway and is likely to operate in periprosthetic tissues when there is heavy wear particle deposition and cytokine production.     

Inhibition of RANKL-induced osteoclast formation in mouse bone marrow cells by IL-12: involvement of IFN-gamma possibly induced from non-T cell population

IL-12 was shown to have the potential to inhibit osteoclast formation in mouse bone marrow cells treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). When bone marrow macrophages (BMM) were used as osteoclast precursors, IL-12 failed to inhibit M-CSF/RANKL-induced osteoclast formation from BMM. In coculture experiments using transwells, IL-12 did inhibit osteoclast formation from BMM cocultured with whole bone marrow cells. These results indicated that IL-12 indirectly affected M-CSF/RANKL-induced osteoclastogenesis in bone marrow cells and that the inhibition of IL-12 on osteoclast formation was caused by a humoral factor from bone marrow cells treated with IL-12. Experiments with anti-interferon (IFN)-gamma antibody and bone marrow cells from IFN-gamma receptor knockout mice revealed that IFN-gamma might be involved in the inhibition of osteoclast formation in this system. The expression of osteoprotegerin mRNA in bone marrow cells was not affected by treatment with IL-12. The inhibitory effect of IL-12 on osteoclast formation was also seen in the T cell-depleted bone marrow cells of normal mice and the whole bone marrow cells of athymic nude mice, while the inhibitory effect of IL-12 was partially suppressed in the B cell-depleted bone marrow cells. The inhibitory effect of IL-12 on M-CSF/RANKL-induced osteoclastogenesis was not accompanied with cell death, in contrast with our previous finding that the inhibitory effect of IL-12 on M-CSF/TNF-alpha-induced osteoclastogenesis is attributable to Fas and FasL-mediated apoptosis.     

Odanacatib reduces bone turnover and increases bone mass in the lumbar spine of skeletally mature ovariectomized rhesus monkeys

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once‐weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen‐deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle‐treated OVX monkeys. Treatment with ODN also led to dose‐dependent increases in serum 1‐CTP and maintained estrogen deficiency–elevated Trap‐5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L₁ to L₄) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose‐dependently increased L₁ to L₄ BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX‐vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX‐vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN‐treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long‐term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates. © 2012 American Society for Bone and Mineral Research     

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.     

Short-term effects of organic silicon on trabecular bone in mature ovariectomized rats

Summary Silicon is known to ensure an essential role in the formation of cross-links between collagen and proteoglycans during bone growth. In this study, we have evaluated the short-term effects of a preventive treatment with silanol, a soluble organic silicon (Si), on trabecular bone in mature ovariectomized rats. Three-month-old rats were shamoperated (sham) or were ovariectomized (OVX) and treated with 10 g/kg/day of 170 estradiol (E2), or with 0.1 mg Si/kg/day or 1.0 mg Si/kg/day of silanol for 1 month. Plasma alkaline phosphatase and osteocalcin levels were increased by 50% in OVX rats compared with sham rats and were corrected by E2 but not by silanol treatment. The trabecular bone volume measured at the tibial metaphysis was decreased by 48%, and histomorphometric indices of bone resorption and formation were increased in OVX rats compared with sham, and these parameters were corrected by E2 treatment. Treatment of OVX rats with silanol decreased the osteoclast surface by 31% and the number of osteoclasts by 20%. The mineral apposition rate, the bone formation rate, and the osteoblast surface at the tibia metaphyseal area were increased by 30% at the higher dose of silanol compared with OVX rats. In contrast, silanol treatment had no effect on the periosteal apposition rate. The reduction of the metaphyseal bone resorption and the increased bone formation induced by silanol resulted in a slight improvement of the trabecular bone volume (+ 14%) compared with controls. The results indicate that a short-term preventive treatment with the organic silicon silanol partially prevented the trabecular bone loss in mature OVX rats by reducing bone resorption and increasing bone formation, possibly through stimulatory effects on the formation and/or the stability of the organic bone matrix.     

Macrophage colony-stimulating factor and interleukin-6 release by periprosthetic cells stimulates osteoclast formation and bone resorption.

Periprosthetic bone loss is an important contributory factor for aseptic loosening of total joint replacements. It has recently been shown that osteoclast precursor cells are present in the wear particle-associated macrophage infiltrate found in the membrane surrounding loose implants and that these cells are capable of differentiating into osteoclastic bone-resorbing cells. Long-term co-culture of arthroplasty-derived macrophages and the rat osteoblast-like cell line, UMR-106, in the presence of 1,25(OH)2D3 results in the formation of numerous multinucleated cells that are positive for tartrate-resistant acid phosphatase and vitronectin receptor and capable of extensive lacunar bone resorption. The aim of this study was to determine the effect of cytokines/growth factors, known to be present in the arthroplasty membrane, on this process of osteoclast differentiation. During osteoclast formation, increased levels of macrophage colony-stimulating factor, interleukin-6, and to a lesser extent, interleukin-1beta, but not tumour necrosis factor alpha, were detected in the co-culture supernatants. Addition of neutralising antibodies to human interleukin-1beta or tumour necrosis factor alpha to the co-culture system did not inhibit osteoclast formation. In contrast, co-cultures to which neutralising antibodies to human macrophage colony-stimulating factor or interleukin-6 were added contained fewer cells positive for tartrate-resistant acid phosphatase and vitronectin receptor and formed significantly fewer resorption pits. Time-course studies showed that macrophage colony-stimulating factor and interleukin-6 increase osteoclast formation mainly in the early stages of osteoclast differentiation. These results indicate that the release of macrophage colony-stimulating factor and interleukin-6 by activated cells in the arthroplasty membrane is likely to contribute to pathological bone resorption associated with aseptic loosening by stimulating differentiation of mononuclear phagocyte osteoclast precursors into mature bone-resorbing cells.