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PubMed1998

Adhesive and bone resorptive activities of isolated osteoclasts from hen medullary bone.

In the present study, osteoclasts were isolated from hen medullary bones at the formative and resorptive phases. The cells were cultured on glass culture dishes and bone slices. After culturing, the adhesion activity of the isolated osteoclasts with the substrates was estimated with a light microscope, and the surfaces of the bone slices were observed with a scanning electron microscope. The results showed that the adhesion activity of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts is higher at the bone resorptive phase than at the bone formative phase, and this tendency in isolated osteoclasts was observed more frequently on the bone slices than on the glass culture dishes. Furthermore, scanning electron microscopy showed that the isolated osteoclasts in the bone resorptive phase adhered to the bone surface with developed-cytoplasmic projections and formed broad pits where collagen fibrils were exposed. On the other hand, isolated osteoclasts in the bone formative phase adhered to the bone slice with board-shaped cytoplasmic projections and did not form any pits. These results suggest that isolated osteoclasts in the bone resorptive phase have a high level of adhesion activity and actively resorb the bone, whereas isolated osteoclasts in the bone formative phase have a low level of adhesion activity and cease bone resorption. The procedure reported here is useful for studying the bone-resorptive mechanism of authentic osteoclasts.

Acid Phosphatase↗
PubMed1991

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: light and electron microscopic study.

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

Adult↗
PubMed1993

The majority of osteoclasts require mRNA and protein synthesis for bone resorption in vitro.

Mithramycin is an mRNA synthesis inhibitor that has been used to decrease bone resorption in patients with humoral hypercalcemia and Paget's disease. During studies on the mechanism of action of mithramycin it became clear that the compound has a direct inhibitory effect on osteoclastic bone resorption in the in vitro bone slice assay. At concentrations of 0.1-100 nM mithramycin directly inhibited osteoclastic bone resorption dose-dependently up to 66 +/- 5% at 100 nM (mean +/- SEM, 3 expts.). Another mRNA synthesis inhibitor, actinomycin D (0.1-100 nM) and the protein synthesis inhibitor, cycloheximide (0.1-10 microM), also dose-dependently inhibited osteoclastic bone resorption by 78 +/- 7% at 100 nM and 76 +/- 7% at 10 microM, respectively. Mithramycin and actinomycin D at 100 nM did not affect osteoclast survival on bone slices and were therefore not cytotoxic at the concentrations used. Mithramycin (100 nM) and cycloheximide (10 microM) both slightly decreased osteoclast cytoplasmic spreading. Addition of 100 nM mithramycin 6 hr after osteoclast adhesion to bone slices still inhibited subsequent resorption by 50%, indicating a continued but lesser requirement for mRNA synthesis during bone resorption. These results show that approximately 75% of osteoclasts obtained from neonatal rat long bones are activated by adhesion to mineralized bone surfaces and require mRNA and protein synthesis in order to resorb bone in vitro.

Animals↗
PubMed2003

[Culture of human osteoclasts].

OBJECTIVE: To establish a stable, useful culture system for human osteoclasts and to investigate the effect of osteoblasts on the differentiation, proliferation and activation of osteoclasts so as to provide a base for the studies on prevention and treatment of osteolysis and osteoporosis. METHODS: In the presence of 1,25-(OH)2D3, monocytes abstracted from human bone marrow were cultured in three groups: co-culture of monocytes and osteoblasts, monocytes alone, monocytes with conditional media (CM) of osteoblasts. Differentiation process of the cultured cells was observed under biological microscope. HE staining and tartrate-resistant acid phosphatase (Trap) staining were employed to assay the cultured cells. The resorption pits on bone slices, on which cells were cultured, were observed under scanning electronic microscope (SEM). RESULTS: In the group of co-culture of monocytes and osteoblasts, monocytes gradually fused to form multinucleated cells (MNCs), and the MNCs were also indicated in HE staining and Trap staining. The SEM showed a number of resorption pits on bone slices. In the other two groups, Trap-positive MNCs were not obtained, and resorption pits were not observed on bone slices. CONCLUSION: In this culture, monocytes obtained from human marrow fused to form multinucleated cells (MNCs) that express the main characteristics of the osteoclast phenotype, such as Trap-positive and the ability to form resorption lacunae when cultured on bone slices. Cell-to-cell contact with osteoblasts was necessary for the differentiation, proliferation and activation of osteoclasts.

Cell Differentiation↗
PubMedDate not supplied

Measurement of mandibles with microfocus x-ray computerized tomography and compact computerized tomography for dental use.

PURPOSE: The trabecular bone patterns in jaws and tooth structure were analyzed using microfocus x-ray computerized tomography (micro-CT), and the usefulness of this method was reported. This study was performed to clarify the accuracy of micro-CT and to determine whether micro-CT could replace bone slice segments as a means for assessment of the accuracy of medical radiography units. In addition, the accuracy of compact CT for dental use (compact CT) was evaluated using the same method. MATERIALS AND METHODS: Three dried hemimandibles were used in this study. Images of mandibular interdental alveolar bone in the premolar and molar regions were obtained using micro-CT and compact CT. Measurement of the mandibular shape at 6 sites using micro-CT images and at 4 sites using compact CT images was performed, and then the values of the micro-CT and compact CT were compared with those of bone slice segments. RESULTS: The accuracy of mandibular measurement and the ratios of agreement for micro-CT were significantly higher than those for compact CT. Moreover, the coefficient of correlation of the rate of trabecular bone between compact CT images and bone slice segment images was 0.916. DISCUSSION: The significant difference in mandibular measurement between micro-CT and compact CT was considered to be related to pixel size. CONCLUSION: Micro-CT can replace bone slice segments for assessment of the accuracy of medical radiography units. In addition, compact CT can be used for imaging diagnosis in dental implant treatment. It is suggested that the ratio of trabecular bone could be used to evaluate the bone density.

Alveolar Process↗
PubMed1991

Inhibition of bone resorption by bisphosphonates: interactions between bisphosphonates, osteoclasts, and bone.

Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl2MBP) inhibits bone resorption through injury to the cells that resorb Cl2MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorption in vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl2MBP. The greater sensitivity of resorption to AHPrBP in vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormones in vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl2MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generation in vivo in AHPrBP-treated rats. These results suggest that the comparisons of potency in vitro do not include all the factors responsible for determining bisphosphonate potency in vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10(-3) M AHPrBP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation.

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1alpha,25-dihydroxyvitamin D3[1alpha,25-(OH)2D3] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7 kDa and 6.8 kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4-7) or to early-stage (days 0-3) cultures of calvarial cells pretreated with 1alpha,25-(OH)2D3, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1+ c-Fms+ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

Evidence that c-src is involved in the process of osteoclastic bone resorption.

Transgenic mice lacking a functional c-src gene have osteopetrosis, a bone disorder characterized by defective osteoclast function. We have investigated the effects of selective protein tyrosine kinase inhibitors that are known to inhibit c-src, on osteoclast activity in the bone slice assay. Geldanamycin, herbimycin A and monorden (0.001-10 microM) all dose-dependently inhibited bone resorption with IC50 values of 8, 70 and 86 nM, respectively. At concentrations of 0.001-1 microM, the compounds were not cytotoxic as judged by osteoclast morphology and survival on bone slices. In order to determine whether c-src plays a role in signal transduction associated with osteoclast activation prior to bone resorption commencing, or in the resorptive process itself, we performed kinetic experiments using human calcitonin as a positive control. Calcitonin inhibited all bone resorption subsequent to its addition at t = 0, 3 or 6 hr (100%, approximately 90% and approximately 50% inhibition, respectively), after the start of the 24 hr bone slice assay. Similar results were obtained with herbimycin A and geldanamycin (1 microM) added at t = 0, 3 or 6 hr, and with monorden (1 microM) added at t = 0 and 6 hr. These results indicate that c-src plays a crucial and continuous role in the process of osteoclastic bone resorption, most likely related to the translocation and/or fusion of exocytic vesicles to the ruffled border membrane.

Animals↗
PubMed1998

Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces.

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40-45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as alphaVbeta3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.

Alendronate↗
PubMed2003

Total coumarins from fruits of Cnidium monnieri inhibit formation and differentiation of multinucleated osteoclasts of rats.

AIM: To determine the effects of TCFC (total comarins from the fruits of Cnidium monnieri) on the activity of osteoclasts in vitro. METHODS: Osteoclasts isolated from rat marrow cells were co-cultured with osteoblasts under the 1,25-dihydroxyvitamine D3. The tartrate-resistant acid phosphatase (TRAP) stain was used to identify osteoclast morphology. The activity of TRAP was measured by p-nitrophenyl sodium phosphate assay. The resorption pit area on the bone slices formed by osteoclasts was measured by computer image processing. Calcium concentration in the medium of co-culture of bone slices and osteoclasts was determined by atomic absorption spectra. RESULTS: TCFC 2.5-25 mg/L inhibited osteoclast formation and differentiation. TCFC 0.25-25 mg/L inhibited TRAP activity of osteoclasts and TCFC 25 mg/L decreased the TRAP activity by 26.3 % and 24.1 % after 48 h and 72 h, respectively. TCFC 25 mg/L decreased the osteoclastic bone resorption pit area by 25.05 % and Ca2+ release from bone slices by 41.73 %. CONCLUSION: TCFC reduced the bone lose by decreasing the osteoclast formation, its TRAP activity, and osteoclastic bone resorption.

Acid Phosphatase↗
PubMed2000

Does high-resolution computed tomography image analysis of the distal radius provide information independent of bone mass?

This study aimed to investigate the usefulness of computed tomography (CT) image analysis of the distal radius for comparing two groups of postmenopausal women matched for age and bone mineral density at both the lumbar spine and femoral neck. The first one consisted of 16 women with at least one vertebral fracture and the second consisted also of 16 women without disease affecting bone mass or bone metabolism. Eight slices were selected in each patient: four consecutive coronal slices and four consecutive axial slices. Bone texture analysis was performed using structural methods leading to the measurement of 24 features. Most of the structural variables derived from histomorphometric parameters and were measured after segmentation from a binary or a skeletonized image. Nine variables were significantly different between the two groups on axial slices: valley number, valley surface area, apparent bone volume/tissue volume (BV/TV), apparent trabecular separation, apparent trabecular number, trabecular bone pattern factor, trabecular skeletal length, node count, and node-to-node strut count. Also four variables were significantly different between osteoporotic women and controls on coronal slices: apparent BV/TV, trabecular partition, node-to-node strut count, and terminus-to-terminus strut count. In conclusion this study suggests that bone texture analysis could yield additional data on bone mass for explaining bone strength and therefore could be used for improving the prediction of fracture risk.

Absorptiometry, Photon↗
PubMed2001

Alendronate disturbs vesicular trafficking in osteoclasts.

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.

Acid Phosphatase↗
PubMed1999

Predictive value of bone mineral density and Singh index for the in vitro mechanical properties of cancellous bone in the femoral head.

OBJECTIVE: The purpose of this study was to assess the validity of two methodically different radiological parameters, bone mineral density and Singh Index, for the prediction of mechanical properties in femoral cancellous bone. DESIGN: Coherence between in vitro evaluation of mechanical properties and bone mineral density on a femoral bone slice, combined with clinical determination of Singh Index on ordinary X-rays. BACKGROUND: It is accepted that bone densitometry yields excellent prediction of mechanical bone quality, but is considered to be an expensive and not widely available method for routine diagnostics in clinical practice. In contrast, determination of Singh Index is an inexpensive and simple technique, but its predictive value for bone mechanics is still controversially discussed. METHODS: We used cortically confined bone slices from 33 femoral heads. Bone mineral density was measured using quantitative computed tomography. Strength and elastic modulus were assessed by mechanical testing in up to 39 circular positions on each slice. Singh Index was evaluated on ordinary X-rays of the hip by two independent readers. RESULTS: Bone mineral density showed strong correlations with strength (r=0.86) and good correlations with elastic modulus (r=0.68). Singh Index correlated well with strength (r=0.70), but only moderately with elastic modulus (r=0.52). CONCLUSIONS: The strong validity of bone mineral density in predicting mechanical bone quality was confirmed. Singh Index assessment permits a rough estimation of mechanical strength in particular and can therefore be used for first estimations of mechanical bone quality, provided that readings were performed by experienced clinicians. RELEVANCE: Reduced mechanical bone quality induces an increase in fracture risk. Whenever performed, bone mineral density measurement allows an excellent evaluation of the mechanical properties of cancellous bone in the hip and can be recommended for screening evaluations. The assessment of Singh Index on ordinary X-rays of the hip is an inexpensive and simple method, and allows a rough estimation of the mechanical quality of the femur. However, due to its subjective character, its predictive value for the mechanical quality of bone in individual patients remains uncertain.

Aged↗
PubMed1995

Taxol inhibits osteoclastic bone resorption.

We have examined the effect of the anti-tumor compound taxol, on osteoclastic bone resorption. In the bone slice assay, taxol (0.1-0.001 microM) dose-dependently inhibited bone resorption with an IC50 of 0.08 microM. Osteoclast survival on bone slices was unaffected by 0.01-1 microM taxol, but 10 microM was cytotoxic. Taxol (1 microM) also inhibited osteoclast spreading (45%) on fibronectin-coated slides. The antiproliferative effects of taxol are due to its unique ability to stabilize microtubules. Primary osteoclasts are nonproliferating end cells, so taxol probably inhibits bone resorption by interfering with other microtubule-dependent functions such as cell polarization, motility or vesicle exocytosis. Since these inhibitory effects on osteoclasts in vitro are seen with therapeutically relevant concentrations, taxol therapy may have beneficial side-effects e.g. inhibition of hypercalcemia and bone metastases.

Animals↗
PubMed1989

Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts.

The reversible calcitonin (CT)-induced inhibition of osteoclastic activity has been studied to clarify the mechanisms responsible for the so-called "escape phenomenon." Osteoclasts disaggregated from neonatal rabbits were cultured on glass coverslips or thin bovine bone slices. Resorption activity was evaluated by using time-lapse recording and scanning electron microscopy. Addition of CT to the cultures caused most osteoclasts on glass surfaces to be immotile and contracted. From 1.5 h onward, in cultures with CT, osteoclasts started to escape from CT-induced quiescence independently of other cells. CT also prevented osteoclasts on bone slices from excavating bone while concomitant cell immobility occurred. Inhibited osteoclasts were able to regain apparent bone-resorbing potency only after resumption of cytoplasmic immobility. The resumption of bone resorption could begin as early as 9.7 h after CT addition. The observations indicate that CT-induced inhibition of osteoclastic bone resorption is associated with inhibition of cytoplasmic motility and that the "escape" phenomenon reflects resumption of activity of osteoclasts that were previously inhibited by CT action rather than the resportive activity of newly formed osteoclasts.

Animals↗
PubMed1991

Identification of osteoclast precursors in multilineage hemopoietic colonies.

The osteoclast is known to be derived from the hemopoietic stem cell, but its lineage and the mechanisms by which its differentiation is regulated are largely unknown. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hemopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semi-solid medium from mouse spleen cells in the presence of erythropoietin with either Wehi 3B-conditioned medium or interleukin 3 (IL3). After 7 days, individual colonies were picked. Half of each colony was phenotyped by the morphology of cells in cytospin preparations; the second half of each was incubated for 7 days with a bone marrow-derived cell line (ts8) that induces osteoclastic differentiation from hemopoietic cells, on bone slices in the presence of 1,25-dihydroxyvitamin D3. After incubation, bone resorption was assessed by scanning electron microscopy. No resorption was induced in cells derived from single-lineage colonies, but resorptive cells differentiated in 17% of granulocyte-macrophage (GM) colonies and 38% of multilineage colonies. Since only a minority of GM colonies contained osteoclastic precursors, this suggests that the GM colonies that contained osteoclasts were not typical GM colonies but may have been a form of multilineage colony analagous to other multilineage colonies that contain granulocytes, macrophages, and a third cell type. No resorptive cells were formed when IL3-derived colonies were incubated on bone slices without ts8 cells. The results suggest that osteoclasts are derived from a multilineage precursor, upon which IL3 acts to generate cells capable of osteoclastic differentiation, which form resorptive cells upon incubation with bone marrow stromal cells in the presence of 1,25-dihydroxyvitamin D3.

Animals↗