Osteoclast Bone Resorption Activity Research Articles

Interleukin 1 Induces Multinucleation and Bone-Resorbing Activity of Osteoclasts in the Absence of Osteoblasts/Stromal Cells

Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors involved in bone loss associated with inflammation. We previously reported that IL-1 prolonged the survival of multinucleated osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts/stromal cells and bone marrow cells via the prevention of spontaneously occurring apoptosis. It was reported that macrophage colony-stimulating factor (M-CSF/CSF-1) prolongs the survival of OCLs without the help of osteoblasts/stromal cells. The present study was conducted to determine whether IL-1 also directly induces the multinucleation and activation of OCLs. Mononuclear osteoclast-like cells (prefusion osteoclasts; pOCs) were purified using the “disintegrin” echistatin from cocultures of murine osteoblastic cells (MB 1.8 cells) and bone marrow cells. Both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs through their respective receptors. However, actin ring formation (a functional marker of osteoclasts) by multinucleated cells was observed in the pOC cultures treated with IL-1, but not those treated with M-CSF. We previously reported that enriched multinucleated OCLs as well as pOCs placed on bone/dentine slices formed few resorption pits, but their pit-forming activity was greatly increased by the addition of osteoblasts/stromal cells. Here, pit-forming activity of both pOCs and enriched OCLs placed on dentine slices was induced by adding IL-1, even in the absence of osteoblasts/stromal cells. M-CSF failed to induce pit-forming activity in pOC and enriched OCL cultures. These results indicate that IL-1 induces the multinucleation and bone-resorbing activity of osteoclasts even in the absence of osteoblasts/stromal cells.

An immunohistochemical study of the behavior of cells expressing interleukin-1α and interleukin-1β within experimentally induced periapical lesions in rats

Cells expressing interleukin-1 (IL-1) were examined in rat periapical lesions using immunohistochemical methods. During the acute phase, both IL-1 alpha- and IL-1 beta-expressing cells were observed adjacent to many osteoclasts that contained large cytoplasms, and consequently the periapical lesions tended to expand. However, the number of IL-1 beta-expressing cells was about twice that of IL-1 alpha-expressing cells during the same period. On the other hand, there were significantly less IL-1 beta-expressing cells during the chronic phase and consequently a significant expansion of the periapical lesions was not seen. Furthermore, IL-1 alpha- and IL-1 beta-expressing cells were located far from the bone surfaces, whereas a fibroblastic cell layer lay between the IL-1-expressing cells and the bone surfaces. Both IL-1 alpha and IL-1 beta were mainly expressed by macrophages. In addition, after the percolated application of formocresol to experimentally induced periapical lesions, numerous macrophages expressing IL-1 beta became conspicuous within the vicinity of the extensive bone resorption sites. These results suggest that macrophages expressing IL-1 beta may play an important role in the activation and recrudescence of osteoclastic bone resorption, and this may be due to their localization close to the bone resorption sites noted within experimental periapical lesions.

Response of human osteoblasts to polymethylmetacrylate In vitro.

The effects of a polymethylmetacrylate (PMMA) powder with a diameter between 0.5 and 25 mu m have been studied in vitro on several human osteoblast populations obtained from different sources. Parameters of cell activity such as cell growth, collagen synthesis, osteocalcin, and interleukin-6 (IL-6) production have been evaluated. Cell proliferation and collagen synthesis were inhibited after exposure to bone cement, whereas osteocalcin and IL-6 production were stimulated. These results suggest that PMMA particles could affect osteoblast activity in a way that could contribute, together with other factors, to periprosthetic osteolysis through two different pathways: a reduced periprosthetic bone formation due to the reduced osteoblast proliferation and collagen synthesis, and an osteoblast-mediated activation of osteoclastic bone resorption as suggested by the increased osteocalcin and IL-6 synthesis. In fact, osteocalcin has been demonstrated to have a role in osteoclast recruitment to bone surfaces, and IL-6 is known to induce osteoclastogenesis and to directly stimulate bone resorption.

A Useful Method to Evaluate Bone Resorption Inhibitors, Using Osteoclast-like Multinucleated Cells

Since excessive bone resorption in postmenopausal osteoporosis is a pathological manifestation of the disease, it is important to control bone resorption activity of osteoclasts as a means of controlling the disease. To screen bone resorption inhibitors, a simple and quantitative method to assay bone resorption is needed. However, it has been difficult to prepare a lot of osteoclasts required for the screening of many compoundsin vitro.We used the method for the preparation of mouse osteoclast-like multinucleated cellsin vitroand developed a useful method in which osteoclast-like multinucleated cells were placed on dentin slices and the calcium released from dentin into the culture medium was measured. Under the optimal conditions, the increase of the calcium concentration caused by bone resorption activity of the osteoclast-like cells was significant and inhibited by calcitonin in a dose-dependent manner. We also examined the inhibitory effect of calcitonin on the pit area caused by bone resorption and found that the decrease of the calcium released from dentin was correlated with that of the pit area. Furthermore, several known bone resorption inhibitors such as bisphosphonate, bafilomycin A1, and herbimycin A showed inhibitory effects on the calcium release from dentin slices. Thus, this simple method provides us a useful screening system to find bone resorption inhibitors with novel mechanisms.

Intestinal delivery of calcitonin in pig

Salmon calcitonin (sCT) is a 32 amino-acid peptide hormone which has efficacy in the treatment of osteoporosis and Paget's disease by virtue of a direct inhibitory effect on the bone resorption activity of osteoclasts. In clinical practice the peptide is administered by sub-cutaneous injection, or in some cases intranasally, because the oral availability is very low. The formulation employed, known here as Bridgelock, is a solid-dose lipid-based preparation in which the peptide is embedded directly in the oil phase. We report here results of administering this lipidic formulation containing salmon calcitonin, and demonstrate that significant quantities of this hormone pass across the intestinal wall intact to exert a biological effect in pigs. In a surgically manipulated pig model it has been found that the bio-availability of calcitonin could be increased when calcitonin is administered in conjunction with an emulsion-based carrier, giving rise to an order of magnitude increase in pharmacodynamic response as measured by fall in plasma calcium. At the time of peak fall of calcium, the inter-subject variability for the formulation was comparable with that seen for sub-cutaneous administration. In addition, a dose response is demonstrated for the model between 1250 and 5000 iu per pig. Repeatability within the model was confirmed testing the same batch of formulation x months apart in a second group of pigs.

Regulation of osteoclast function.

Regulation of osteoclast function.

Concanavalin A Directly Stimulates Bone-Resorbing Activity of Osteoclasts and Their Gene Expression of Cathepsin K/OC-2

Concanavalin A (Con A), a plant lectin that recognizes cell-surface glycoproteins, up-regulates osteoclastic bone-resorbing activity of cultured isolated rabbit pure osteoclasts as well as unfractionated bone cells. The effect of Con A was blocked by α-methyl mannopyranoside. Several Con A-binding proteins were detected in the plasma membranes from osteoclasts. Furthermore, Con A increased the levels of transcripts of cathepsin K/OC-2, one of the proteases responsible for osteoclastic bone-resorbing activity. These results indicate that Con A directly enhances the function of osteoclasts by the association with surface glycoproteins of osteoclasts.

卵巣摘出ラットにおける上顎骨，下顎頭および大腿骨の構造変化に関する比較組織学的研究

Postmenopausal estrogen deficiency results in bone loss (osteoporosis) in humans and experimental animals. The acute and significant loss of trabecular bone in the ovariectomized (OVX) rat provides a useful experimental model of postmenopausal osteoporosis. Five months after ovariectomy of 3-month-old female rats, the mid and distal femurs, maxillae, and mandibular condyles were dissected and processed for light and backscattered electron microscopic examinations. Net trabecular bone values of femurs in OVX ats showed significant loss of trabecular (but not cortical) bones compared to those in sham-operated controls. Net bone volume in the maxillae of OVX rats was similar to that of controls. In mandibular condyles, OVX caused prominent bone loss similar to that in femurs. Throughout the observations, active osteoclastic bone resorption was rarely recognized, presumably due to the fact that the rats had been in the statistic post-OVX phase. These results suggested that, in this animal model of postmenopausal steoporosis, the bone loss is more prominent in trabecular bone areas formed by endochondral ossification such asmetaphyses of femurs and mandibular condyles than cortical bone areas formed by intramembranous ossification such as mid-diaphyses of femurs and maxillae.

Effects of calcitonin and parathyroid hormone on the distribution of F-actin in the clear zone of osteoclasts in vivo.

To elucidate the relation between the distribution of F-actin bands in the clear zone and the bone-resorbing activity of osteoclasts in vivo, the endocranial surfaces of calvariae from 7-day-old Wistar rats were stained with F-actin-specific fluorescein isothiocyanate-labeled phalloidin (FITC-phalloidin) with and without the intraperitoneal injection of the calcium-regulating hormones, calcitonin (CT), and parathyroid hormone (PTH). Some specimens were double-stained with FITC-phalloidin and a monoclonal antibody, ED1, which stained monocytes and macrophages. In normal rats, almost 80% of the osteoclasts showed ring-shaped F-actin bands in the clear zone, and the remaining 20% showed arch- or line-shaped F-actin bands. From 15 min to 1 h after the injection of CT (salmon, 10 mg/kg), the number of osteoclasts with ring-shaped F-actin bands decreased significantly. At 1 h, few F-actin bands were detected in osteoclasts, which were still stained by ED1. However, these F-actin bands recovered to the normal level at 6 h. On the other hand, from 15 min to 6 h after the injection of PTH (bovine, 50 mg/kg), the number of osteoclasts with arch- or line-shaped F-actin bands decreased significantly. These results indicate that osteoclasts with arch- or line-shaped F-actin bands in the clear zone had lower bone-resorbing activity than those with ring-shaped F-actin bands, and that the formation of bands could be controlled by calcium-regulating hormones over the course of a few hours. Observation of the endocranial surfaces of calvariae might be useful for examining factors which affect the activities of osteoclasts in vivo.

C-type Natriuretic Peptide Increases Bone Resorption in 1,25-Dihydroxyvitamin D3-stimulated Mouse Bone Marrow Cultures

Most agents that regulate osteoclast bone resorption exert their effects indirectly, through the osteoblast. Nitric oxide, which stimulates soluble guanylyl cyclase, has been reported to inhibit osteoclast bone resorption directly, by a cGMP-independent mechanism (1). In this report, we demonstrate that C-type natriuretic peptide (CNP), an activator of membrane-bound guanylyl cyclase, stimulates bone resorption by osteoclast-containing 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated mouse bone marrow cultures. Quantitative reverse transcription polymerase chain reaction assays and anti-CNP immunocytochemistry were used to demonstrate that CNP is expressed in mouse marrow cells cultured in the presence, but not the absence, of, 1,25-(OH)2D3. mRNA for guanylyl cyclase type B, the receptor for CNP, was expressed in cultures independent of 1,25-(OH)2D3. CNP (1 and 10 microM) elevated cGMP production in marrow cultures to 350 and 870%, respectively, of control values. 10 microM CNP increased osteoclast bone resorptive activity, measured by the resorption area on whale dentine wafers, or by the NH4Cl-inhibitable release of [3H]proline from radiolabeled bone chips, to 214 and 557% of control, respectively, without affecting osteoclast formation. Bone resorption by the marrow cultures was inhibited by 7F9.1, a monoclonal antibody raised against CNP, but not by control antibodies. These results indicate that CNP is a potent activator of osteoclast activity and may be a novel local regulator of bone remodeling.

The Effect of Hepatocyte Growth Factor on the Behavior of Osteoclasts

Hepatocyte growth factor (HGF) stimulates the growth, motility and morphogenesis of a variety of cell types, including hemopoietic progenitors. We found that HGF is a potent inhibitor of bone resorption by isolated rat osteoclasts. However, in the presence of the osteoblastic cell line UMR 106, it stimulated osteocIastic resorption. HGF also increased osteoclastic motility and spread area, over a similar concentration range. We detected no effect on osteoclast formation or survival. Our data suggest that HGF may be involved in the recruitment of osteoclasts to sites of bone resorption, but that during migration resorptive functions are suppressed. Once on resorptive sites, the osteoclast response to HGF is modulated by osteoblastic cells and the bone resorptive activity of osteoclasts may be stimulated accordingly.

The effect of interleukin-6 and soluble interleukin-6 receptor protein on the bone resorptive activity of human osteoclasts generated in vitro.

The role of interleukin-6 (IL-6) in the regulation of bone resorption is controversial and has not been studied using human tissue in vitro. This study exploits a recently described in vitro model, whereby osteoclasts, defined as cells that resorb bone, can be generated from human bone marrow, and investigated the effect of IL-6 and its soluble receptor on bone resorption, in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2 vitamin D3]. Human bone marrow was cultured to form a confluent stroma, sedimented onto devitalized bone slices, and recharged with non-adherent bone marrow cells. 1,25(OH)2 vitamin D3 increased bone resorption, whereas IL-6 failed to induce a similar stimulatory effect. Both IL-6 at 100 ng/ml and soluble IL-6 receptor protein in the absence of exogenous IL-6 inhibited the stimulatory effect of 1,25(OH)2 vitamin D3. Bone resorption was never observed when non-adherent haemopoietic cells were cultured in the absence of stroma but in the presence of IL-6, which indicates that IL-6 cannot replace the stromal factor(s) required for the formation of cells capable of resorbing bone. These results suggest that IL-6 at high concentrations is not a critical cytokine in stimulating osteoclastic bone resorption.

Detection of interleukin-1β mRNA in rat periapical lesions

Cells expressing interleukin-1 beta (IL-1 beta) mRNA were demonstrated by in situ hybridization in rat periapical lesions. A great number of osteoclasts with significant tartrate-resistant acid phosphatase activity were observed on the bone surfaces, and numerous IL-1 beta mRNA-expressing cells were widely distributed in the periodontal ligaments. IL-1 beta mRNA-expressing cells were mainly observed around the blood vessels in the vicinity of the bone resorption sites and occasionally found near the osteoblasts. Immunohistochemistry and enzyme histochemistry assays showed that IL-1 beta mRNA-expressing cells were not bone cells, but that they had the characteristic features of macrophages. These results suggested that macrophages may contribute to the production of IL-1 beta and play an important role in activation of osteoclastic bone resorption.

A histochemical study of the behavior of macrophages during experimental apical periodontitis in rats

The behavior of macrophages from experimentally induced periapical lesions of rats was studied in paraffin sections using nonspecific esterase and a monoclonal antibody, ED1. Macrophages were seen near the regularly arranged osteoblasts in controls and the detached osteoblasts at the initiation phase of bone resorption. In addition, numerous macrophages were widely distributed throughout the periodontium at the activation phase of bone resorption. On the other hand, macrophages were rarely seen near the bone formation surfaces, but large numbers of macrophages were localized in microabscess at the activation phase of bone formation. It is suggested that macrophages may play an important role in activation of osteoclastic bone resorption and inhibition of complete bone repair in bone remodeling during experimental apical periodontitis.

Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.

We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.

Establishment of a rapid bone resorption in vitro assay using previiously frozen mouse unfractionated bone cells pretreated with parathyroid hormone

We established a useful assay system for evaluating osteoclast-mediated bone resorption based on the use of unfractionated bone cells obtained from 10- to 11-day-old mice. When cells from 10 to 11 mice were treated for 7 days with rat parathyroid hormone (rPTH, 10 −8 M), a total of 4 to 5 × 10 7 cells could be obtained from the culture by treatment with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells. When the harvested cells were cultured on dentine slices without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone rsorption. Eel calcitonin (eCT) decreased the number of pits in a dose-dependent manner, and its half maximal inhibition dose (ID 50) was 1.08 × 10 −11 M. Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable of forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclaslic cell line for evaluating bone resorption is available at present, this system can provide a useful, practical means for assaying osteoclastic bone-resorbing activity.

High turnover osteoporosis in acro-osteolysis (Hajdu-Cheney syndrome)

Acro-osteolysis with diffuse osteoporosis in the absence of other associated diseases is named Hajdu-Cheney syndrome. Reduced bone formation rather than enhanced bone resorption has been indicated as the mechanism of osteoporosis. On the assumption that in this syndrome the active bone resorption which produces distal osteolysis must also predominate in generalized osteoporosis, we investigated bone histology, calcium kinetics, calciotropic hormones and bone markers in a patient suffering from sporadic Hajdu-Cheney syndrome. A radius bone biopsy taken far from the osteolytic lesions showed severe osteoporosis with a marked increase in osteoclastic bone resorption and reduced bone formation. Total body calcium clearance, performed through an analysis of the kinetics of calcium infusion, was 2.8 times higher than in normal controls, indicating the presence of active osteoclastic bone resorption. Serum parathormone, 1,25-dihydroxycholecalciferol, alkaline phosphatase and urinary hydroxiproline were in the normal range. These data indicate that in Hajdu-Cheney syndrome trabecular osteoporosis is produced by the same mechanism that induces distal osteolysis, which suggests that it may be sustained by local acting factors stimulating osteoclastic resorption.

A new approach for calcitonin determination based on target cell responsiveness

We report the development and validation of three microbioassays for calcitonin based on calcitonin-induced inhibition of the activity of isolated osteoclasts. Having precisely quantified osteoclast motility, spreading and bone resorptive activity, we have applied stringent analytical procedures to define assay characteristics. We have found that the appropriately transformed responses significantly regress on log dose of the peptides. Furthermore, potency estimates obtained using calcitonins from three species (human, salmon and a synthetic analogue of eel calcitonin) have been found to be consistent with those obtained using conventional calcitonin bioassays. In addition, the assays are remarkably sensitive (detection limit 10(-15) M), highly specific and precise. We have determined plasma levels of bioactive calcitonin on samples from patients with medullary thyroid carcinoma; these are several-fold lower than those obtained using our routine calcitonin radioimmunoassay. Our study thus, forms the basis of an entirely new approach for the determination of 'biologically active' calcitonin, and we envisage that such target cell-specific assays could become useful microanalytical methods.

Glucocorticoids impair bone resorptive activity and viability of osteoclasts disaggregated from neonatal rat long bones.

It is generally believed that glucocorticoids cause osteoporosis through a combination of decreased bone formation and increased bone resorption. However, the direct effect of glucocorticoids on osteoclasts has not been determined. We therefore tested the effects of hydrocortisone and dexamethasone on bone resorption by osteoclasts disaggregated from neonatal rat long bones. Hydrocortisone and dexamethasone caused a dose-dependent inhibition of osteoclastic bone resorption in the range 10(-7) to 10(-5) M, and 10(-9) to 10(-6) M, respectively, at concentrations likely to occur during therapy and disease. Inhibition of bone resorption was found to be associated with impaired osteoclast survival: osteoclast numbers were reduced to approximately 25% of control values by 10(-6) M hydrocortisone and 10(-7) M dexamethasone. Osteoclast cytotoxicity by glucocorticoids was completely antagonized by progesterone, which itself had no effect on osteoclast survival. Analysis of the time course of these inhibitory effects showed a nonsignificant reduction in survival by 6 h and marked inhibition of survival by 12 h. We could detect no specific changes in osteoclast morphology in association with this impaired viability. The relative potencies of the glucocorticoids for impairment of osteoclast viability was similar to their relative affinities for binding the glucocorticoid receptor, and this, together with inhibition by progesterone, suggests a receptor-mediated mechanism. Such a receptor-mediated cytotoxic action of glucocorticoids has only previously been reported with lymphoid cells. The sensitivity of osteoclasts to the lethal effects of glucocorticoids suggests that glucocorticoids may have a role in physiology as inhibitors of osteoclastic bone resorption.

Rapidly progressive osteopenia in the oophorectomized and nutrition deprived rats

Eight-week old healthy female Wistar rats were oophorectomized and placed on a low calcium-low protein diet. Histological analysis showed evidence of osteopenia after 7 days. The bone ash calcium content was reduced at 4 weeks. This rapidly progressive bone loss was apparently related to active osteoclastic bone resorption following a nutrition-related secondary hyperparathyroidism. Abnormal large osteoid and osteitis fibrosa were absent. These skeletal alterations resemble findings in case of osteopenia in postmenopausal women.