The role of osteoclast differentiation in aseptic loosening.

The major cause of orthopaedic implant loosening is thought to be accelerated osteoclastic bone resorption due to the action of cytokines produced in response to phagocytosis of implant-derived wear particles. This accelerated osteoclastic bone resorption could be due to increases in any of the following processes: recruitment of osteoclast precursors to the local microenvironment, differentiation of precursors into mature multinucleated osteoclasts. activation of mature osteoclasts, and/or survival of osteoclasts. Our studies have focused on differentiation and survival to complement work by others who have focused on recruitment of precursors and activation. Taken together, our studies and those of other investigators provide strong evidence that increased recruitment of osteoclast precursors and their subsequent differentiation play major roles in wear particle-induced osteolysis. In contrast, increased osteoclast activation and survival appear to play minor roles. These studies suggest that development of therapeutic interventions that reduce either recruitment or differentiation of osteoclast precursors would improve the performance of orthopaedic implants.     

Stimulatory effect of insulin-like growth factor binding protein-5 on mouse osteoclast formation and osteoclastic bone-resorbing activity.

Insulin-like growth factor binding protein-5 (IGFBP-5) stimulates osteoblast proliferation directly or indirectly through IGF-I action, but its effects on osteoclast formation and osteoclastic activity are unknown. We tested the effects of IGFBP-5 on osteoclastic activity and osteoclast formation. IGFBP-5 significantly stimulated pit formation by pre-existent osteoclasts in mouse bone cell cultures and its stimulatory effect was completely blocked by IGF-I antibody (Ab). However, IGFBP-5 did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When IGFBP-5 was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, IGFBP-5 (77 pM-7.7 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of IGF-I Ab. Moreover, osteoclast-like cells newly formed by IGFBP-5 from unfractionated bone cells possessed the ability to form pits on dentine slices. We next examined the direct effect of IGFBP-5 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. IGFBP-5 dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors, irrespective of the presence of IGF-I Ab. Growth hormone (GH) as well as IGF-I significantly stimulated bone resorption by pre-existent osteoclasts in mouse bone cell cultures and these stimulatory effects were completely blocked by IGF-I Ab. GH as well as IGF-I stimulated osteoclast-like cell formation from unfractionated bone cells and this stimulatory effect of GH was significantly but partially blocked by IGF-I Ab. The direct stimulatory effect of GH on osteoclast-like cell formation from hemopoietic blast cells was not affected by IGF-I Ab. The present data indicate that IGFBP-5 stimulates bone resorption both by stimulation of osteoclast formation in an IGF-I-independent fashion and by IGF-I-dependent activation of mature osteoclasts, possibly via osteoblasts, in vitro.     

Chemically modified tetracyclines act through multiple mechanisms directly on osteoclast precursors

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption primarily through their ability to inhibit matrix metalloproteinases (MMPs). We have previously demonstrated that some tetracycline compounds (TCs) induce apoptosis in mature rabbit osteoclasts and inhibit osteoclastic resorption in mouse osteoblast/marrow co-cultures in vitro. In this report, we now show that non-antibiotic analogues of doxycycline (CMT-3) and minocycline (CMT-8) are potent inhibitors of osteoclastogenesis in vitro from human peripheral blood mononuclear cells (PBMC) stimulated with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kappaB ligand (RANKL), through an action that is independent of osteoblast-osteoclast interactions. Osteoclast formation over 20 days was completely abrogated when CMT-3 or CMT-8 were included in PBMC cultures at a concentration of 250 ng/ml, although doxycycline at this concentration reduced osteoclast formation to ca. 50% of control. CMT-3 and CMT-8 also significantly induced apoptosis over 24 h in mature osteoclasts generated over 20 days when added to cultures at 5 microg/ml or more. In a time-course experiment, apoptosis was evident after a delay of 1-2 h following treatment of mature osteoclasts with CMT-3 at 20 microg/ml. The broad-spectrum MMP inhibitor BB94 (Batimastat) did not recapitulate the apoptosis induced by CMT-3, even at a concentration where MMP-13 activity was completely inhibited. There was no evidence for an anabolic effect of any of the TCs on osteoblast lineage cells in a calcifying fibroblastic colony (CFU-f) formation assay, where CMT-3 partially inhibited CFU-f formation at 5 microg/ml. Our data indicate that inhibition of osteoclast formation and induction of osteoclast apoptosis are pharmacologically significant actions of CMTs in inhibiting bone resorption, and that osteoclast apoptosis cannot be attributed to the ability of CMTs to inhibit MMPs or to actions mediated by osteoblastic lineage cells.     

Dissociation of angiogenesis and osteoclastogenesis during endochondral bone formation in neonatal mice.

Invasion of the mineralized matrix by endothelial cells and osteoclasts is a key event in endochondral bone formation. To examine the putative role of osteoclast activity in the angiogenic process, we used two in vivo models of suppressed bone resorption: mice treated with the bisphosphonate clodronate and in osteoclast-deficient, osteopetrotic mice. Angiogenesis was assessed in caudal vertebrae of these neonatal mice. This model enables us to study the interaction between osteoclasts and endothelial cells during endochondral bone formation. In control conditions, sinusoid-like structures were detected in the vicinity of tartrate resistance acid phosphatase positive (TRAcP+) osteoclasts. Treatment with clodronate completely abolished osteoclastic bone resorption, whereas angiogenesis remained unaffected. In line with these observations, in the osteopetrotic mouse mutants c-fos knockout mice and op/op mice, capillaries invaded the calcified cartilage in the absence of osteoclasts. In conclusion, our data strongly suggest that during endochondral bone formation, vascular invasion can occur in the absence of osteo(chondro)clastic resorption. In addition, bisphosphonates show no apparent effect on angiogenesis in this in vivo model. These findings may have important clinical implications in the management of skeletal disorders such as metastatic bone disease, in which both osteoclastic bone resorption and angiogenesis contribute to tumor growth. On the other hand, our results confirm that bisphosphonates can be used safely in the treatment of disorders that affect the growing skeleton, such as in juvenile osteoporosis.     

Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate

Osteoclast-devoid bone explants were cultured together with embryonic liver as a source of osteoclast precursors, but separated from each other by a filter. Cells migrated through the filter toward the calcified matrix and acquired the characteristics of mature, tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts upon contact with the bone explant. Migration and attachment could be visualized separately. Progressive reduction of filter pore size resulted in progressive reduction of resorption because the use of smaller pores made it increasingly difficult for cells to pass. Indeed, the use of 0.22-micron filters, through which no cells can pass, but which still allow full passage of medium, completely blocked the resorption. When migrating cells from fetal liver were arrested for 10 days by using a combination of filters with different pore sizes, the arrested cells showed a tendency to fuse just opposite the mineralized matrix. Furthermore, a great number of the arrested cells expressed the macrophage-specific cell-surface antigen F4/80 and showed acid phosphatase activity, but none of these cells were tartrate resistant. The acquisition of tartrate-resistant acid phosphatase activity upon contact with the bone explant and subsequent resorption of this explant could be prevented by exposure of the system to the bisphosphonate dimethyl-APD (Me2-APD), whereas migration of cells through the filter was not affected. We suggest that the bisphosphonate interferes with a matrix factor that is essential for the attachment and subsequent transformation of the osteoclast precursor into the mature phenotype.     

Microcracks and Osteoclast Resorption Activity In Vitro

During bone remodeling osteoclasts resorb bone, thus removing material, e.g., damaged by microcracks, which arises as a result of physiological loading and could reduce bone strength. Such a process needs targeted bone resorption exactly at damaged sites. Osteocytic signaling plays a key role in this process, but it is not excluded that osteoclasts per se may possess toposensitivity to recognize and resorb damaged bone since it has been shown that resorption spaces are associated with microcracks. To address this question, we used an in vitro setup of a pure osteoclast culture and mineralized substrates with artificially introduced microcracks and microscratches. Histomorphometric analyses and statistical evaluation clearly showed that these defects had no effect on osteoclast resorption behavior. Osteoclasts did not resorb along microcracks, even when resorption started right beside these damages. Furthermore, quantification of resorption on three different mineralized substrates, cortical bone, bleached bone (bone after partial removal of the organic matrix), and dentin, revealed lowest resorption on bone, significantly higher resorption on bleached bone, and highest resorption on dentin. The difference between native and bleached bone may be interpreted as an inhibitory impact of the organic matrix. However, the collagen-based matrix could not be the responsible part as resorption was highest on dentin, which contains collagen. It seems that osteocytic proteins, stored in bone but not present in dentin, affect osteoclastic action. This demonstrates that osteoclasts per se do not possess a toposensitivity to remove microcracks but may be influenced by components of the organic bone matrix.     

Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that LPS (lipopolisaccharide)-induced osteolysis occurred more rapidly and extensively in bgn deficient mice compared to wild type (WT) mice. To further understand the mechanism of action, we determined the effects of Bgn on 1, 25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate-resistant acid phosphatase-positive multinuclear cells appeared earlier and more extensively in the co-cultures containing calvarial cells from bgn deficient mice than WT mice, regardless of the genotype of osteoclast precursors. The osteoblast abnormality that stimulated osteoclast formation appeared to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte protease inhibitor (slpi) in bgn deficient osteoblasts treated with 1,25-(OH)₂D₃. These findings point to a novel molecular factor made by osteoblasts that could potentially be involved in LPS-induced osteolysis.     

PPAR agonists modulate human osteoclast formation and activity in vitro

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.     

Prostate-specific antigen induces apoptosis of osteoclast precursors: potential role in osteoblastic bone metastases of prostate cancer

BACKGROUND: Prostate cancer is frequently associated with bone metastases with marked osteoblastic changes and low osteoclastic activity but its mechanism is not well understood. We previously reported that prostate-specific antigen (PSA) stimulated the proliferation and the activation of osteoblasts. In this study, we investigated the effect of PSA on osteoclastogenesis. METHODS: Two human prostate cancer cell lines and PSA were directly injected into human adult bone (HAB) implanted into NOD/SCID mice, followed by morphological analysis. RAW 264.7 cells, murine osteoclast precursor, were treated with PSA. RESULTS: PSA-producing LNCaP and PSA caused a significant decrease of osteoclast precursors and osteoclasts in HAB accompanied by osteoblast proliferation and new bone formation, while PSA-nonproducing PC3 showed increasing osteoclasts with osteolysis. PSA induced apoptosis of RAW 264.7 cells in vitro. PSA-induced apoptosis was dependent of enzymatic activity of PSA and was specific to immature tartrate-resistant acid phosphatase-negative mononuclear RAW 264.7 cells. CONCLUSIONS: PSA plays a crucial role for osteoblastic bone metastasis by promoting both osteoblasts proliferation and apoptosis of osteoclast precursors.     

Macrophage colony stimulating factor increases bone resorption in dispersed osteoclast cultures by increasing osteoclast size.

Several reports indicate that macrophage colony stimulating factor (MCSF) is one of the major factors required for osteoclast proliferation and differentiation. Paradoxically, it has also been reported that MCSF inhibits osteoclastic activity. We therefore decided to investigate in detail the effects of MCSF on resorption and osteoclast formation to try and clarify this issue. Osteoclast-containing cultures were obtained from rabbit long bones and cultured on plastic culture dishes or devitalized bovine bone slices. MCSF (4-400 ng/ml) stimulated osteoclastic bone resorption in a time-dependent manner and at all doses examined. After 48 h of culture in the presence of MCSF, we observed a 2-fold increase in the total area of bone resorbed, as well as a significant increase in the area of bone resorbed per osteoclast and the number of resorption pits per osteoclast. This effect was paralleled by an increase in the number of larger osteoclasts (as determined by the number of nuclei per cell) and an increase in the size and depth of the resorption pits. Since the total number of osteoclasts remained the same, the MCSF-induced increase in resorptive activity appeared to be related to an increase in the average size of the osteoclasts. When resorption was expressed as the amount of bone resorbed per osteoclast nucleus, larger osteoclasts resorbed more per nucleus, suggesting that large osteoclasts, as a population, are more effective resorbers than small osteoclasts. Interestingly, when osteoclasts were plated at one-fifth the standard density, the amount of bone resorbed per osteoclast decreased considerably, indicating that resorptive activity is also affected by cell density of osteoclasts and/or of other cells present. However, at this lower density MCSF still increased osteoclast size and resorption by the same fold increase over control, suggesting that the effect of MCSF was independent of factors related to cell density.     

Regulation of osteoclast differentiation and function by phosphate: potential role of osteoclasts in the skeletal abnormalities in hypophosphatemic conditions.

Mice fed with a low Pi diet exhibited decreased osteoclast number. Hyp mice also showed decreased osteoclasts, and high Pi reversed it. Low Pi reduced osteoclast formation and bone resorption in vitro. Hypophosphatemia may suppress osteoclast differentiation/function, leading to skeletal abnormalities. INTRODUCTION: Skeletal abnormalities seen in hypophosphatemic disorders indicate a critical role of phosphate (Pi) in skeletogenesis. However, the role of osteoclasts in the pathogenesis of the disturbed skeletogenesis is unclear. MATERIALS AND METHODS: Mice fed with a low-Pi diet and Hyp mice that are characterized by hypophosphatemia and impaired osteogenesis were studied. Effects of Pi on osteoclast formation and bone resorption were also examined in vitro. RESULTS: Histomorphometric examination showed that mice on a low-Pi diet exhibited decreased osteoclast number. Furthermore, osteoclast number in Hyp mice was also decreased compared with wildtype (WT) mice. Of note, feeding of Hyp mice with high-Pi diet significantly reversed hypophosphatemia, improved disturbed osteogenesis, and increased osteoclast number. Osteoclast-like cell (OLC) formation and bone resorption in Hyp bone marrow cells was not different from WT bone marrow cells. On the other hand, OLC formation and bone resorption were decreased in conjunction with reduced mRNA expression of RANKL in WT bone marrow cells cultured in the medium containing low Pi (0.5 mM). Recombinant human matrix extracellular phosphoglycoprotein (MEPE), a candidate for phosphatonin, also decreased osteoclast formation, whereas fibroblast growth factor 23 (FGF23), another phosphatonin candidate, showed no effects. CONCLUSIONS: Our results suggest that Pi controls the differentiation and function of osteoclasts. These actions of Pi on osteoclasts may be associated with the pathogenesis of the skeletal abnormalities in hypophosphatemic disorders.     

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)     

Bergapten prevents lipopolysaccharide mediated osteoclast formation,bone resorption and osteoclast survival

Purpose

This study was designed to investigate the potential effect of bergapten on lipopolysaccharide (LPS)-mediated osteoclast formation, bone resorption and osteoclast survival in vitro.

Methods

After osteoclast precursor RAW264.7 cells were treated with bergapten (5, 20, 40 μmol/L) for 72 hours in the presence of LPS (100 ng/ml), osteoclastogenesis was identified by tartrate-resistant acid phosphatase (TRAP) staining, and the number of TRAP-positive multinucleated cells [TRAP(+)MNCs] per well were counted. To investigate the effect of bergapten on osteoclastic bone resorption, RAW264.7 cells were treated with bergapten for six days in the presence of LPS, and the area of bone resorption was analyzed with Image Pro-Plus. Next, we examined apoptosis of RAW264.7 cells after bergapten incubation for 48 hours by flow cytometer using annexin V/propidium iodide (PI) double labeling. Finally, osteoclast survival was observed by Hoechst 33342 labeling and Western blotting after bergapten treatment for 24 hours.

Results

Data showed that bergapten (5–40 μmol/L) dose-dependently inhibited LPS-induced osteoclast formation and bone resorption. Treatment with bergapten triggered apoptotic death of osteoclast precursor RAW264.7 cells in a dose-dependent manner. Furthermore, bergapten significantly reduced the survival of mature osteoclast, as demonstrated by emergence of apoptotic nuclei and activation of apoptotic protein caspase 3/9.

Conclusions

These findings suggest that bergapten effectively prevents LPS-induced osteoclastogenesis, bone resorption and survival via apoptotic response of osteoclasts and their precursors. The study identifies bergapten as an inhibitor of osteoclast formation and bone resorption and provides evidence that bergapten might be beneficial as an alternative for prevention and treatment of inflammatory bone loss.     

Retinoid-induced bone thinning is caused by subperiosteal osteoclast activity in adult rodents

Excess of Vitamin A (retinol) and related compounds (retinoids) induces bone fragility and is associated with increased hip fracture incidence in humans. Yet, their impact on the adult skeleton has been studied in relatively little detail. It is assumed that they induce generalized bone loss and decrease long-bone thickness due to reduction of radial bone growth. Here we characterized early skeletal responses of adult rodents to retinoid treatment, revealing novel aspects of retinoid action on the mature skeleton. The retinoid Ro 13-6298, given subcutaneously for 4 days, induced bone loss in the hind limbs of 12- and 56-week-old rats and of 15-week-old mice. In vivo monitoring of bone mass and geometry changes by peripheral quantitative computed tomography demonstrated that bone mass decline was due to subperiosteal cortical bone loss, which induced a shrinkage of bone diameter, whilst cancellous bone mass was preserved. We observed that the native retinoic acid isomer all-trans RA induced an identical pattern of bone loss. Histomorphometric evaluation revealed that increased subperiosteal osteoclastic bone resorption caused the cortical bone destruction. Interestingly, bone resorption was suppressed in cancellous bone, which was in agreement with reduced in vitro formation of osteoclasts from bone marrow cells that were derived from the proximity of cancellous bone. The retinoid-induced increase in subperiosteal bone resorption could be blocked by bisphosphonate as direct potent inhibitor of osteoclast action, but not by estradiol. Retinoid treatment induced a reduction of bone-forming surfaces at the subperiosteal site, but not in cancellous bone. In vitro osteoblast performance was also reduced or unchanged, depending on the cellular system used and assay type/duration. In conclusion, our studies revealed that the impact of retinoids on bone is highly bone-compartment-specific at early treatment phases. Furthermore, we showed that bone diameter shrinks in the adult skeleton after retinoid treatment due to subperiosteal osteoclastic bone resorption. Thus, retinoid-induced bone thinning is not only due to reduced radial bone growth as previously assumed. Our findings might explain why high intake of retinol is associated with increased hip fracture risk in the elderly and suggest a therapy to prevent such potential negative effects.     

Fusion disability of embryonic osteoclast precursor cells and macrophages in the microphthalmic osteopetrotic mouse

Osteoclast formation in the microphthalmic osteopetrotic (mi) mouse was studied from very early embryonic to newborn stages. Embryonic and fetal milmi osteoclasts, generated during the period before bone marrow is formed in the long bones, were predominantly mononuclear and lacked ruffled borders. These cells did, however, show many osteoclastic morphologic and functional properties, such as an abundance of mitochondria, positive succinic dehydrogenase and acid phosphatase reactions, and close contact with and resorption of the calcified cartilage matrix (though diminished). These osteoclastic mononuclear cells appeared in vivo as well as in organ cultures of fetal metatarsal bones with their intact periostea. They also were observed in cocultures of periosteum-free fetal metatarsal bones, with several extraneous sources of osteoclast precursors: yolk sacs and abdominal regions of 9- and 11-day-old embryos, fetal livers, and precultured mononuclear phagocytes isolated from the fetal liver. In contrast, +/+ osteoclasts were always multinuclear, functioned normally in resorbing the calcified cartilage matrix, and had ruffled borders in vivo as well as when derived from the above-mentioned sources. Fetal liver-derived milmi macrophages also failed to form multinuclear foreign body giant cells as opposed to +/+ macrophages in granulomas on implanted pieces of Melinex. The fusion failure of cells derived from embryonic and fetal extramedullary milmi monocyte/macrophage sources contrasted with the occurrence of multinuclear osteoclasts and foreign body giant cells derived from precursors from the bone marrow in young milmi mice. We conclude that the fusion defect of milmi osteoclast precursor cells is already present in their ancestry in blood cell-forming organs of very young embryos and that these cells differentiate into mononuclear osteoclasts that function inefficiently in prenatal bone. We presume that in fully developed bone marrow, local factors are favorable for abolishing the fusion defect.     

Avian osteoblast conditioned media stimulate bone resorption by targeting multinucleating osteoclast precursors

Summary Osteoblasts are thought to secrete factors that regulate the rate of osteoclastic bone resorption. We studied the effect of osteoblast conditioned medium on bone degradation by multinucleated osteoclast-like cells generated in vitro from mononuclear precursors and found that the medium stimulates bone degradation primarily through interactions with osteoclast precursors. The conditioned medium also stimulates expression of the osteoclast-specific antigen 121F. The increased bone degradation, but not increased 121F expression, is due to the conditioned medium maintaining activity of the osteoclast precursors. Although the osteoclast precursors exhibit the DNA fragmentation characteristic of apoptosis, the osteoblast conditioned medium does not prevent such fragmentation. Chicken macrophage growth factor neither mimics nor augments the ability of the conditioned medium to stimulate bone degradation. Studies of osteoclast generation or function should carefully consider whether the effects are dependent on the viability of the resorbing cells.A preliminary account of these results was presented at the 12th Annual Meeting of the American Society for Bone and Mineral Research, August 28–31, 1990     

Role of mineralizing cartilage in osteoclast and osteoblast recruitment

Periost-free, live and/or devitalized cartilaginous long bone rudiments of fetal mice were transplanted under the renal capsule of adult syngeneic mice to study the role of cells and intercellular matrix in the recruitment and formation of osteoclasts and osteoblasts, both identified by means of enzyme- and immunohistochemical methods. Live bone rudiments recruited host-derived osteoclasts within 5 days after transplantation. Osteoblasts developed as rapidly as osteoclasts and participated in the modeling of the rudiments into hemopoietic bone marrow containing ossicles. Devitalized bone rudiments, killed before osteoclastic invasion had occurred, did not recruit osteoclasts or osteoblasts, and were not resorbed up till 35 days after transplantation. Co-transplantation of live and devitalized bone rudiments however resulted in osteoclastic resorption of the killed rudiments, starting 9 days after transplantation. Again the live rudiments were modeled into ossicles. Devitalized bone rudiments which had been invaded by osteoclasts before killing and transplantation, did recruit host osteoclasts, but at a slower rate than live rudiments, and depending on the number of resorption sites at the time of transplantation. Osteoblasts were not formed. These data suggest that in developing long bones chondrocyte activity is involved in the recruitment of osteoclasts as well as osteoblasts. Matrix components diffusing from resorbing surfaces seem to be involved in osteoclast recruitment.     

重组人骨保护素与重组核因子κb活化因子受体蛋白对破骨前体细胞分化的影响

目的:对比重组人骨保护素(rhOPG-Fc)与重组核因子κb活化因子受体蛋白(rhRANK)对破骨前体细胞分化的影响.方法:采用成骨细胞与破骨前体细胞RAW264.7混合培养,在地塞米松、1,25 (OH) 2VitD3诱导下生成破骨细胞的方法.研究分3组:rhRANK组:10-5 g/L;rhOPG-Fc组:10-5 g/L;空白对照组.作用9d后观察破骨细胞数目和形态,抗酒石酸酸性磷酸酶(TRAP)染色阳性细胞个数,骨磨片吸收陷窝计数.结果:在空白对照组,小鼠成骨细胞与破骨前体细胞RAW264.7混合培养6d后,开始出现多核细胞,9d时可见大量成熟多核细胞,经TRAP染色证实为成熟破骨细胞,而rhRANK组及rhOPG-Fc组TRAP染色阳性多核细胞数较对照组均减少,特别是rhRANK组减少更明显.骨片吸收陷窝计数显示rhRANK组及rhOPG-Fc组较对照组也明显减少,而相对来说,rhRANK组较rhOPG-Fc组更少.结论:rhOPG-Fc与rhRANK均可以有效抑制破骨前体细胞分化成为成熟破骨细胞,且rhRANK较rhOPG-Fc抑制效果更明显.     

低分子量褐藻糖胶对破骨细胞凋亡的影响

目的探讨低分子量褐藻糖胶(LMWF)对小鼠单核细胞RAW264.7诱导成熟破骨细胞凋亡的影响。方法通过100ng/m L RANKL诱导RAW264.7细胞株分化为破骨细胞,经TRAP特异性染色和骨吸收陷窝对诱导后的细胞进行鉴定。鉴定成功后,用100 ng/m L RANKL诱导RAW264.7细胞株5 d后,使用含有LMWF的培养基继续培养3 d,通过对TRAP阳性细胞计数和分析骨吸收面积来观察低分子量褐藻糖胶对破骨细胞的抑制和骨吸收功能情况;采用流式细胞术检测LMWF对破骨细胞凋亡的影响,capsase-3活性测试试剂盒检测LMWF对capsase-3活性进行测定;RT-PCR检测LMWF对成熟破骨细胞BAX与BCL-2基因表达的影响。结果单纯采用100 ng/m L的RANKL可成功诱导成熟的、有功能的破骨细胞。LMWF可以明显抑制RANKL诱导成熟破骨细胞的形成以及成熟破骨细胞的骨吸收功能;流式细胞术显示LMWF可增加成熟破骨细胞的早期凋亡率;并且能升高capsase-3的活性;PCR显示LMWF可明显下调破骨细胞凋亡相关的BCL-2和上调BAX基因mRNA表达,降低BCL-2/BAX的比值。结论低分子量褐藻糖胶可抑制破骨细胞的活性与骨吸收能力,促进破骨细胞凋亡,其主要机制是通过下调BCL-2和上调BAX mRNA基因表达实现的。     

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.