Showing papers on "Tartrate-resistant acid phosphatase published in 2006"
Journal Article•
TL;DR: This review summarizes the scientific knowledge on the role of TRACP in osteoclastic bone resorption, the mechanism ofTRACP 5b generation in osteoblasts and its secretion into the blood circulation, the methodology of measuring TRACP 5a, diagnostic evidence for the use of TRACP 5b as a resorptive marker, and characteristics of TR ACP 5a compared to other commonly used bone turnover markers.
Abstract: Tartrate-resistant acid phosphatase (TRACP) is an enzyme that is expressed in high amounts by bone resorbing osteoclasts, inflammatory macrophages and dendritic cells. Two forms of TRACP circulate in human blood, TRACP 5a derived from macrophages and dendritic cells, and TRACP 5b derived from osteoclasts. Recent data have demonstrated the utility of TRACP 5b as a marker of osteoclast number and bone resorption, and serum TRACP 5a as a marker of inflammatory conditions. This review summarizes the scientific knowledge on the role of TRACP in osteoclastic bone resorption, the mechanism of TRACP 5b generation in osteoclasts and its secretion into the blood circulation, the methodology of measuring TRACP 5b, diagnostic evidence for the use of TRACP 5b as a resorption marker, and characteristics of TRACP 5b compared to other commonly used bone turnover markers.
225 citations
TL;DR: It is proposed that the MCP-1-induced TRAP+/CTR+ multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.
151 citations
TL;DR: Release of TRAP by osteoclasts represents not only a productive approach to the analysis of the mechanisms that modulate the rate of resorptive activity, but also a system whereby the mechanism through which bone substrates induce Resorptive behavior can be identified.
Abstract: There have been dramatic advances recently in our understanding of the regulation of osteoclastic differentiation. However, much less is known of the mechanisms responsible for the induction and modulation of resorptive behavior. We have developed a strategy whereby osteoclasts can be generated in vitro and released into suspension in a fully-functional state. We now exploit this approach to show that tartrate-resistant acid phosphatase (TRAP) is released by osteoclasts during bone resorption. TRAP release was inhibited by the secretion-inhibitor Brefeldin A, and was not accompanied by LDH release. This suggests that TRAP release is due to secretion, rather than cell death. Consistent with this, TRAP secretion was stimulated by resorbogenic cytokines, was inhibited by the resorption-inhibitor calcitonin, and correlated with excavation of the bone surface. We found that, in contrast to incubation on bone, incubation on plastic, glass, or vitronectin-coated plastic substrates did not induce secretion of TRAP. This suggests that the induction of resorptive behavior in osteoclasts depends upon stimulation by bone matrix of a putative osteoclastic "mineral receptor." Release of TRAP by osteoclasts thus represents not only a productive approach to the analysis of the mechanisms that modulate the rate of resorptive activity, but also a system whereby the mechanism through which bone substrates induce resorptive behavior can be identified.
134 citations
TL;DR: It is demonstrated for the first time that CLA inhibits osteoclastogenesis by modulating RANKL signaling, which may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption by excessive osteoporosis.
91 citations
TL;DR: The results suggest that tooth‐associated fibroblasts may trigger the formation of osteoclast‐like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formationof active osteoclasts.
Abstract: Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.
84 citations
TL;DR: It is shown here that MIP1α is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RankL.
Abstract: Chemokines MCP-1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP-1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1alpha is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1alpha was able to overcome the potent inhibition of GM-CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6- and 49-fold, P = 4.0 x 10(-7) and 4.0 x 10(-8), respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP-1, RANTES and MIP1alpha. Unexpectedly, treatment with MCP-1 in the absence of RANKL resulted in 458-fold induction of CCR4 (P = 1.0 x 10(-10)), while RANTES treatment resulted in twofold repression (P = 1.0 x 10(-4)). Since CCR2b and CCR4 are MCP-1 receptors, these data support the existence of an MCP-1 autocrine loop in human osteoclasts differentiated using RANKL.
80 citations
TL;DR: Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB ligand (RANKL), and these osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclastic differentiation and activation.
Abstract: Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-kappaB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed beta3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-kappaB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.
76 citations
TL;DR: The data suggest that neither cathepsin K nor L is essential in activating TRAP, and it is proposed that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in cal varia, which may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.
Abstract: Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.
60 citations
TL;DR: Observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans.
55 citations
TL;DR: It is demonstrated that p-hydroxycinnamic acid (HCA) has stimulatory effects on bone formation and inhibitory effects onBone resorption in tissue culture in vitro.
Abstract: The effect of cinnamic acid or its related compounds, which is present in many plants, on bone metabolism has not been clarified yet. The effect of cinnamic acid, p-hydroxycinnamic acid (HCA), ferulic acid, caffeic acid, or 3,4-dimethoxycinnamic acid (DCA) on bone calcium content in vitro was investigated. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48,h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of HCA (10−5 or 10−4,M) caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. Such an effect was not observed in the presence of cinnamic acid or other compounds at the concentration of 10−5 or 10−4,M. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in the presence of HCA (10−5 or 10−4,M). The effect of HCA (10−4,M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal or metaphyseal tissues was completely prevented in the presence of cycloheximide (10−6,M), an inhibitor of protein synthesis. Thus HCA had anabolic effects on bone components. The presence of parathyroid hormone (PTH; 10−7,M), a bone-resorbing factor, caused a significant decrease in calcium content and a corresponding elevation in medium glucose consumption, lactic acid production or tartrate-resistant acid phosphatase (TRACP) activity in the diaphyseal or metaphyseal tissues. These alterations were completely prevented in the presence of HCA (10−5 or 10−4,M). This study demonstrates that p-hydroxycinnamic acid (HCA) has stimulatory effects on bone formation and inhibitory effects on bone resorption in tissue culture in vitro.
50 citations
TL;DR: The results suggest that tacrolimus or cyclosporine A acts directly on human osteoclast precursors in RA patients and exerts their immunosuppressive effects on human monocyte–osteoclast formation via targeting both the calcineurin-dependent NFAT pathway and activation pathway for c-Jun or MITF.
Abstract: In the present study, we aimed to determine whether tacrolimus (FK506) and cyclosporine A act directly on human osteoclast precursors obtained from patients with rheumatoid arthritis (RA) and influence monocyte–osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL) in vitro, the stage at which differentiation was affected and the manner in which tacrolimus or cyclosporine A affected the osteoclast signaling pathway. Peripheral blood mononuclear cells (PBMCs) were isolated from RA patients and cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF). Tacrolimus or cyclosporine A was added to these cultures to determine the effect on the osteoclast differentiation. Osteoclast formation was determined by assessing the number of tartrate resistant acid phosphatase (TRAP) staining cells and measuring the extent of lacunar resorption. The expression of osteoclast transcription factors, such as TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells c1 (NFATc1), c-Fos, c-Jun, microphthalmia transcription factor (MITF) and PU.1 in mononuclear cells (MNCs) was assayed by quantitative reverse transcription-polymerase chain reaction. Addition of tacrolimus or cyclosporine A resulted in a decrease in the number of TRAP-positive multinucleated cells (TRAP+ MNCs) and a decrease in the extent of lacunar resorption pit formation as compared to the control cultures; thus, human monocyte–osteoclast differentiation was more effectively inhibited at the late stage and addition of tacrolimus or cyclosporine A resulted in a decrease in the mRNA expression of NFATc1, c-Jun, and MITF at the late stage. Our results suggest that tacrolimus or cyclosporine A acts directly on human osteoclast precursors in RA patients and exerts their immunosuppressive effects on human monocyte–osteoclast formation via targeting both the calcineurin-dependent NFAT pathway and activation pathway for c-Jun or MITF.
TL;DR: The net results demonstrate that Canova medication is an effective stimulator of macrophage activity and stimulates an increase of the endosomal/lysosomal system.
TL;DR: Liver-derived IGF-I is permissive for ovx-induced trabecular bone loss and might exert this permissive action by modulation of the number of T-cells and the expression of IL-7, which in turn is of importance for the RANKL/OPG ratio and consequently osteoclastogenesis in the bone marrow.
TL;DR: It is found that ADO2 expression results from osteoclast specific properties, and several markers, acid secretion, and cytoskeletal structure are examined.
Abstract: Asymptomatic gene carriers and clinically affected ADO2 subjects have the same ClCN7 mutation. We examined osteoclastic bone resorption in vitro as well as osteoclast formation, several markers, acid secretion, and cytoskeletal structure. We found that ADO2 expression results from osteoclast specific properties.
Introduction: Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder that results from heterozygous mutations in the ClCN7 gene. However, of those individuals with a ClCN7 mutation, one third are asymptomatic gene carriers who have no clinical, biochemical, or radiological manifestations. Disease severity in the remaining two thirds is highly variable.
Materials and Methods: Human peripheral blood mononuclear cells were isolated and differentiated into osteoclasts by stimulation with hRANKL and human macrophage-colony stimulating factor (hM-CSF). Study subjects were clinically affected subjects, unaffected gene carriers, and normal controls (n = 6 in each group). Pit formation, TRACP staining, RANKL dose response, osteoclast markers, acid secretion, F-actin ring, and integrin αvβ3 expression and co-localization were studied.
Results: Osteoclasts from clinically affected subjects had severely attenuated bone resorption compared with those from normal controls. However, osteoclasts from unaffected gene carriers displayed similar bone resorption to those from normal controls. In addition, the resorption lacunae from both unaffected gene carriers and normal controls appeared much earlier and spread much more rapidly than those from clinically affected subjects. As time progressed, the distinction between clinically affected subjects and the other two groups increased. No significant difference was found in acidic secretion or osteoclast formation between the three groups. Osteoclast cytoskeletal organization showed no difference between the three groups but there was low cellular motility in clinically affected subjects.
Conclusions: Osteoclasts from the unaffected gene carriers, in contrast to those from the clinically affected subjects, functioned normally in cell culture. This finding supports the hypothesis that intrinsic osteoclast factors determine disease expression in ADO2. Further understanding of this mechanism is likely to lead to the development of new approaches to the treatment of clinically affected patients.
TL;DR: The results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.
Abstract: Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.
TL;DR: Bone turnover markers are hardly useful to diagnose bone metastases in patients with renal cell carcinoma, however, osteoprotegerin together with clinicopathological characteristics may be helpful as prognosticator of cancer specific death.
TL;DR: High concentrations of TRAP were observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease, and it might be useful as a marker of progression of malignant disease.
Abstract: Tartrate-resistant acid phosphatase (TRAP) is a metalloprotein enzyme that belongs to the acid phosphatases and is known to be expressed by osteoclasts. It has already been investigated as a marker of bone metastases in cancer patients. In this study, which examined the value of serum TRAP concentrations as a marker of bone disease in breast cancer patients, we observed high concentrations of TRAP even in patients without bone metastases. To elucidate this phenomenon, we examined the expression of TRAP in breast cancer cells and the cells of several other malignancies. TRAP concentrations in the serum of tumor patients were determined by ELISA. The expression of TRAP in breast, ovarian, and cervical cancer and malignant melanoma was analyzed by immunohistochemistry. RT-PCR and immunocytology were used to evaluate TRAP expression in cultured tumor cells. A marked increase in serum TRAP concentrations was observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease. TRAP expression was found in breast and ovarian cancers and malignant melanoma, while cervical cancer showed only minimal expression of TRAP. Expression of TRAP was absent in benign tissue or was much less marked than in the corresponding malignant tissue. TRAP expression was also demonstrated in cultured primary cancer cells and in commercially available cell lines. Overexpression of TRAP was detected in the cells of various different tumors. TRAP might be useful as a marker of progression of malignant disease. It could also be a potential target for future cancer therapies.
TL;DR: In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFκB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry.
Abstract: The transcription factor, nuclear factor-kappa B (NFkappaB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkappaB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkappaB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkappaB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkappaB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkappaB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkappaB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkappaB might be potential therapy in various bone metabolic diseases.
TL;DR: β-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals, and this brief article is presented as a caution to those testing genetic models of skeletal gene expression using β-gal as a marker gene.
Abstract: Escherichia coli beta-galactosidase (beta-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by beta-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined beta-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). beta-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using beta-gal as a marker gene.
TL;DR: It can be concluded that SA possesses strong anti-arthritic property by regulating bone turnover by significantly reverted the alterations in the bone turnover observed in arthritic animals by modulating the levels of calcium, phosphorus and the activities of the enzymes names tartrate resistant acid phosphatase, acidosphatase and alkaline phosphatases.
TL;DR: At concentrations found in tissues of individuals exposed to geochemical AsO(2), osteoclasts underwent an H(2)O( 2)-dependent differentiation, and chronic exposure to low-level amounts of AsO (2) could result in increased bone resorption and contribute to bone related pathologies.
TL;DR: Light microscopy and scanning electron micrographs demonstrated that when applied on an intact bioactive glass surface, osteoclasts were unable to dissolve the glass material within this culture period, which indicates that they contribute to this process.
Abstract: Bioactive glass reacts with body fluids and is gradually dissolved in tissues and in cell cultures. We investigated whether osteoclasts contribute to this process, by culturing newborn rat bone-marrow cells containing osteoclasts on polished bioactive glass plates (glass S53P4). The cultures were inspected at days 1-5 and stained for alkaline phosphatase (ALP) to demonstrate osteoblasts and for tartrate resistant acid phosphatase (TRAP) to visualize osteoclasts. Nonosteoclastic cells proliferated several-fold both on bioactive glass and on plastic, whereas osteoclasts and their precursors matured into multicellular giant cells and degenerated. Most cells on bioactive glass became ALP-positive, whereas on plastic the majority of cells remained ALP-negative. Osteoclasts survived on bioactive glass for 4-5 days, whereas on plastic they degenerated and disappeared after 3 days. Condensed nuclei indicating apoptosis were detected both in degenerating osteoclasts and osteoblasts. The surface of the bioactive glass reacted rapidly forming rounded pits, erosions, and cracks within 24 h in areas occupied by osteoblasts. Light microscopy and scanning electron micrographs demonstrated, however, a smooth surface below the cytoplasm of osteoclasts. This indicates that when applied on an intact bioactive glass surface, osteoclasts were unable to dissolve the glass material within this culture period.
TL;DR: There was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoarthritis formation.
Abstract: Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP+ and VNR+ cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.
TL;DR: Osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracllular matrices.
Abstract: The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.
TL;DR: The results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.
TL;DR: The data strongly suggest that osteoblast-like cells recognize TRACP released by osteoclast precursors and that upon endocytosis inactivation of the enzyme occurs, thereby preventing degradation of matrix constituents.
Abstract: Tartrate-resistant acid phosphatase (TRACP) is generally used as a marker of osteoclasts. Yet, other bone-associated cells, such as osteoblasts and osteocytes, may also express activity of this enzyme. Osteoblasts containing TRACP activity are seen particularly in the vicinity of areas of bone resorption, suggesting that osteoclasts somehow induce TRACP activity in osteoblasts. In a recent study, we found that osteoblast-like cells appeared to have the capacity to endocytose TRACP released by osteoclast precursors. In the present study, we investigated the endocytosis of TRACP in more detail as well as the fate of the endocytosed enzyme. We found that incubation of osteoblast-like cells with TRACP-coated beads resulted in attachment of a high number of beads to the cells. After culturing osteoblast-like cells with medium conditioned by blood monocytes that contain TRACP, activity of the enzyme was found in the cells. Following replacement of the medium by normal medium that did not contain TRACP, a decrease in the level of TRACP activity in osteoblast-like cells occurred. Our data strongly suggest that osteoblast-like cells recognize TRACP released by osteoclast precursors and that upon endocytosis inactivation of the enzyme occurs. We propose that uptake of the enzyme is important for the control of enzyme activity, thereby preventing degradation of matrix constituents.
TL;DR: Elevated levels of serum IL-6 and bone remodeling markers, namely, bAP and TRACP5b which are common features of SHP, are effectively suppressed by calcitriol therapy, indicating that hyperparathyroidism not only accelerates bone remodelling but may also aggravate inflammation in patients on maintenance hemodialysis.
Abstract: Background/Aims: Secondary hyperparathyroidism (SHP) is characterized by high bone turnover and elevated serum bone remodeling markers. Elevation of serum interleukin-6 (IL-6) level
TL;DR: The results show that the size and multinuclearity of osteoclasts and the number of osteoclast formed are different in the distal and proximal areas of the tibia, and that alendronate and etidronate may suppress different types of osteoblasts as discriminated by the numberof nuclei.
Abstract: Patients with rheumatoid arthritis commonly suffer both systemic and periarticular osteoporosis. Bisphosphonates (BPs) are inhibitors of bone resorption, and several derivatives have been developed for treatment of enhanced bone resorption. We aimed to characterize osteoclast formation in two different sites, the proximal tibial and distal tibial areas, in rats with adjuvant arthritis, and to investigate the impact of amino or non-amino types of bisphosphonate. Adjuvant arthritis was initiated in rats while administering daily injections of either etidronate, a non-amino BP, or alendronate, an amino BP, for 3 weeks. On the day following the last injection, bone mineral density (BMD) was measured in the proximal tibia to assess systemic osteoporosis and in the distal tibia for periarticular osteoporosis using dual-energy X-ray absorptiometry. Subsequently, bone marrow cells from either end of the tibia were collected and incubated for 7 days before staining and counting tartrate-resistant acid phosphatase positive cells. In the rats with adjuvant arthritis, BMD of either end of the tibia was lower than in normal rats. Although etidronate prevented bone mineral loss at both ends, distal loss was significantly less than proximal. In contrast, alendronate significantly inhibited mineral loss primarily in the proximal area. Large osteoclasts, defined as having five or more nuclei, formed preferentially in the proximal tibia, while small osteoclasts with fewer than four nuclei were found mainly distally. The suppressive effect of alendronate was greater on the large osteoclasts, while etidronate had a greater effect on the small osteoclasts. These results show that the size and multinuclearity of osteoclasts and the number of osteoclasts formed are different in the distal and proximal areas of the tibia, and that alendronate and etidronate may suppress different types of osteoclasts as discriminated by the number of nuclei.
TL;DR: It is suggested that carbon ion irradiation acts primarily on osteoblastic cells, leading to a decreases in the RANKL/OPG mRNA ratio, which leads to a decrease in osteoclastogenesis and osteOClast activity, which results in an increase in bone volume.
Abstract: We investigated the effects of carbon ion and gamma-irradiation on osteoblastic MC3T3-E1 cells by comparing mRNA expression levels for RANKL and osteoprotegerin by RT-PCR. MC3T3-E1 cells were irradiated with 2, 4, or 6 Gy of carbon ions or gamma-rays, and total RNA was harvested 1, 2, 3, 5, or 7 days after irradiation. The RANKL mRNA/OPG mRNA ratio in carbon ion-irradiated MC3T3-E1 cells was lower, while in gamma-irradiated MC3T3-E1 cells this ratio was higher than in non-irradiated cells. To evaluate osteoclastogenesis of MC3T3-E1 cells, carbon ion- or gamma-irradiated cells were co-cultured with non-irradiated cells from murine bone marrow. Staining for tartrate-resistant acid phosphatase (TRAP) in co-cultures showed that carbon ion irradiation suppressed osteoclastogenesis. This result is consistent with the lower RANKL/OPG mRNA ratio for carbon ion-irradiated cells. These results suggest that carbon ion irradiation acts primarily on osteoblastic cells, leading to a decrease in the RANKL/OPG mRNA ratio. This effect, in turn, leads to a decrease in osteoclastogenesis and osteoclast activity, which results in an increase in bone volume.
Journal Article•
TL;DR: The dietary bor on supplement can increase the serum content of boron of osteoporotic rats to stimulate bone formation and inhibit bone resorption, producing therefore obvious therapeutical effect against osteoporeosis.
Abstract: Objective To observe the therapeutic efficacy of dietary boron supplement on retinoic acid-induced osteoporosis in rats, so as to provide experimental evidence for clinical management of osteoporosis with boron. Methods Thirty-two SD rats were randomized into normal control group (8 rats) and osteoporotic group (24 rats), and osteoporosis was induced in rats of the latter group by intragastric retinoic acid administration at the daily dose of 80 mg/kg for 15 consecutive days. The osteoporotic rats were subdivided into control group (8 rats) without treatment, boron treatment group (8 rats) and estradiol treatment group (8 rats). After 30 days of treatment, the serum contents of Ca, P, boron and the activities of alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (TRAP) in the rats were assayed, the bone mineral density (BMD) of the whole body, lumbar vertebrae and tibia were determined, and the morphological changes of the femurs were observed. Results The serum contents of Ca and P in the rats of the 4 groups differed scarcely, but the content of boron in boron treatment group was markedly higher than that in the other three groups. In the osteoporotic control group, the activities of serum AKP and TRAP, the masses of spongy bone and cortical bone of the femurs, and the quantity of the osteoclasts were increased, with the BMD of the lumbar vertebrae and tibia decreased, suggesting osteoporotic conditions. The mean trabecular plate density and thickness, trabecular bone volume and cortical bone volume of the femurs in the osteoporotic rats treated with boron or estradiol were significantly increased, but the active osteoclast quantity in the spongy bone and serum TRAP activities were obviously decreased, and the bone quality was comparable with that of the normal group. In addition, the serum AKP activity and the active osteoblast quantity in the spongy bone were obviously increased in boron treatment group. Conclusion The dietary boron supplement can increase the serum content of boron of osteoporotic rats to stimulate bone formation and inhibit bone resorption, producing therefore obvious therapeutical effect against osteoporosis.