Morphology of osteoclasts in resorbing fetal rat bone explants: effects of PTH and AIF in vitro.

Osteoclastic bone resorption was studied using 45Ca-labeled fetal rat bones cultured in the presence of parathyroid hormone (PTH) and an anti-invasion factor (AIF) derived from bovine hyaline cartilage which is enriched in a collagenase inhibitor. The specific morphological expressions of osteoclasts cultured in PTH and AIF were observed in both light and electron microscopy and analyzed cytometrically. Stimulation of bone resorption with PTH revealed significant increases in the numbers and activity of osteoclasts, whereas bones cultured in the presence of AIF showed significant decreases in numbers of osteoclasts and altered cell features including the loss of osteoclast contact with bone surfaces. These structural modifications were evaluated with 45Ca release data derived from matched-pair explants of fetal rat bones, revealing the existence of a relationship between resorptive states of the cultured bones and morphological expressions of osteoclastic activity.     

Mode of action of cortisol on bone resorption in fetal rat fibulae cultured in vitro

Several concentrations of cortisol (10^-8 to 10^-6 M) were tested for their ability to inhibit the spontaneous or parathyroid extract-induced (PTE) bone resorption in organ cultures of 19-day-old fetal rat fibulae. The criteria used for the assessment of bone resorption were: the release of ⁴⁵Ca from pre-labeled bone rudiments into the culture medium, the dry weight and the number of osteoclasts per bone. At a dose of 10^-6 M, cortisol diminished significantly the release of ⁴⁵Ca from fetal fibulae. This inhibition was accompanied by an increase in bone dry weight and by reduced numbers of osteoclasts. PTE, alone, caused extensive bone resorption with numerous osteoclasts and enhanced ⁴⁵Ca release, without weight gain. This resorptive action of PTE was partially suppressed by cortisol at a dose of 10^-6 M. Lower concentrations were ineffective. Radioautographic studies with ³H-thymidine revealed that cortisol restricts the recruitment of osteoclasts from osteoprogenitor cells, without decreasing the potential of precursor cells to form osteoblasts. PTE, however, stimulates the proliferation of progenitor cells and favors their differentiation towards osteoclasts. This effect can be suppressed by cortisol. It is concluded that cortisol inhibits bone resorption in vitro by limiting the ability of precursor cells to form osteoclasts. Moreover, our study shows that, in fetal rat bones cultured in vitro, osteoclasts arise from locally proliferating mononucleated precursor cells.     

Nuclear pockets and clefts in the lymphoid cell population of bone marrow and blood of children with acute lymphoblastic leukemia

Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for Walker tumor cells, the TE-85 osteosarcoma, and human breast carcinoma lines. These results suggest a mechanism by which bone resorption could promote the development of skeletal metastasis.     

Histomorphometry and autoradiography of cultured fetal rat long bones

The behavior of fetal rat long bones cultured in vitro according to Raisz's technique (1969) was studied by histomorphometry and autoradiography for a period of four days. The changes were recorded daily both on the trabecular and cortical bone by measuring the bone volume, the number of osteoclasts, and the number of nuclei per osteoclast. Radioactive calcium release was measured and compared to the changes in bone volume and in the number of osteoclasts. An autoradiographic study, using ³H-proline and ³H-thymidine in flash labeling in the medium and ³H-thymidine in follow-up labeling after one injection in vivo was performed to evaluate the bone formation, the cellular proliferation rate and cell differentiation. After four days in culture, an increase in total calcified bone volume was observed which correlatd with changes in the trabecular bone. No significant changes were recorded in the cortical bone. The results showed a good maintenance of the resorption and formation phenomena through an active process of cellular multiplication and differentiation. Undifferentiated cells were labeled in flash label and osteoblast, osteocyte and some osteoclast nuclei were labeled in follow-up studies.     

大鼠破骨细胞体外分离培养和鉴定

采用机械分离方法，从新生大骨长管骨分离破骨细胞获得成功。分离的破骨细胞具有如下特性：１．在体外迅速贴附于盖玻片或骨片；２．多核（一般为３￣１０个细胞核）；３．伪足运动活跃，降钙素或降钙素基因相关肽可以抑制其伪足运动；４．酸性磷酸酶染色强阳性，酸性醋酸酯酶弱阳性，ＰＡＳ中等阳性；５．细胞悬液与骨磨片共同培养，可以形成骨吸收陷窝。由此可见，本研究分离的细胞，符合破骨细胞的公认特性，故本法分离培养破骨细     

Effect of dichloromethylene diphosphonate (Cl2-MDP) on bone resorption induced by PHA-stimulated human mononuclear cells in organ culture

Human mononuclear cells stimulated by PHA caused resorption of fetal rat bones in tissue culture. Non-adherent cells alone failed to produce substantial bone resorbing activity. The mixed culture of adherent and non-adherent cells (1:40) produced significant bone mineral resorption in vitro. The bone resorption induced by human mononuclear cells stimulated by PHA required the presence of living bone tissue in culture. The bone resorbing effect caused by PHA-stimulated cells was blocked by dichloromethylene diphosphonate at 10(-5) M concentration, but mononuclear cells preincubated in diphosphonate containing media were capable to resorb fetal rat bone in tissue culture.     

PTH对破骨细胞骨吸收功能的影响及成骨细胞介导作用

采用分离、培养兔破骨细胞和成骨细胞的方法，体外研究甲状旁腺激素（ＰＴＨ）对破骨细胞骨吸收功能的影响，以及成骨细胞和破骨细胞之间的相互作用。结果表明，ＰＴＨ对破骨细胞的骨吸收功能无直接影响，但在成骨细胞参与下，ＰＴＨ对破骨细胞性骨吸收有明显的促进作用。说明成骨细胞在ＰＴＨ调节破骨细胞功能活动中有着重要的介导作用。     

OSTEOCLASTS ARE CAPABLE OF PARTICLE PHAGOCYTOSIS AND BONE RESORPTION

Osteoclasts are multinucleated cells specialized for the function of lacunar bone resorption. Although they are known to be capable of phagocytosis of inert particles, it is not known whether this abolishes their ability to respond to hormones or to form resorption lacunae. Human and rat osteoclasts were isolated from giant cell tumours of bone and rat long bones, respectively, and cultured on coverslips and cortical bone slices, both in the presence and in the absence of particles of latex (1 μm diameter) and polymethylmethacrylate (PMMA) (<50 μm). By light microscopy, it was evident that osteoclasts which had phagocytosed both latex and PMMA particles remained responsive to calcitonin. Osteoclast phagocytosis of particles was also evident on scanning electron microscopy, where it could also be seen that these cells were associated with the formation of resorption lacunae. These findings underline the fact that the osteoclast is a true member of the mononuclear phagocyte system and that phagocytosis does not abrogate either its hormonal response to calcitonin or its highly specialized function of bone resorption. That osteoclasts which have phagocytosed biomaterial particles such as PMMA are still able to carry out lacunar bone resorption is of interest in clinical conditions such as aseptic loosening, where a heavy foreign body particle load is often associated with extensive bone resorption. © 1997 John Wiley & Sons, Ltd.     

Alveolar mononuclear cells can develop into multinucleated osteoclasts: an in vitro cell culture model

Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood. The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding alveolar mononuclear cells to new-born rat calvaria osteoblasts in vitro. To utilize osteoclast-free bone, fetal calvariae were obtained from newborn Wistar-rats and cultured in DMEM medium for 14 days. On the day of osteoblast culture, alveolar mononuclear cells were isolated from newborn Wistar rats with a serial washing method and then co-cultured with the calvarial osteoblasts. Bone resorption characteristics were observed both with light and scanning electron microscopy. When alveolar mononuclear cells were cultured for 14 days on the calvarial osteoblasts in response to 1 alpha, 25-dihydroxyvitamin D3, they formed tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells. Resorption pits were seen in the 7-14 days long-term cultures. These results indicate that osteoclasts can be derived from alveolar mononuclear cells in vitro when a suitable microenvironment is provided by calvarial osteoblasts and vitamin D(3).     

Potassium effects on bone: comparison of two model systems

Effects of elevated potassium on bone resorption and on the inhibition by ouabain of parathyroid hormone (PTH)-stimulated resorption were studied in neonatal mouse calvaria, fetal mouse limb bones, and fetal rat limb bones. Ouabain inhibited PTH-stimulated resorption, and K at least partially reversed the inhibition by ouabain in all three systems. However, in contrast to calvaria, neither limb bone system was stimulated to resorb by increased K. Although the reversal of ouabain inhibition in all three systems was likely mediated by an effect on Na-K-ATPase, the resorptive effect of K alone must occur by a different mechanism because it was seen only in the calvaria. The production of prostaglandins may play a partial role in the mechanism of the stimulation of Ca release from calvaria by K. Potassium (35 mM) stimulated production of PGE2 by calvaria but not by limb bones. Indomethacin inhibited the increase in PGE2 in calvaria and partially blocked the stimulated bone resorption observed in response to K. The fetal rat limb bone cultures also differed from the mouse calvaria in being more readily inhibited by increased osmolarity. Thus, secondary effects may be responsible for the variant responses of different bone tissues to certain stimuli.     

Effect of MALP-2, a lipopeptide from Mycoplasma fermentans, on bone resorption in vitro

Mycoplasmas may be associated with rheumatoid arthritis in various animal hosts. In humans, mycoplasma arthritis has been recorded in association with hypogammaglobulinemia. Mycoplasma fermentans is one mycoplasma species considered to be involved in causing arthritis. To clarify which mycoplasmal compounds contribute to the inflammatory, bone-destructive processes in arthritis, we used a well-defined lipopeptide, 2-kDa macrophage-activating lipopeptide (MALP-2) from M. fermentans, as an example of a class of macrophage-activating compounds ubiquitous in mycoplasmas, to study its effects on bone resorption. MALP-2 stimulated osteoclast-mediated bone resorption in murine calvaria cultures, with a maximal effect at around 2 nM. Anti-inflammatory drugs inhibited MALP-2-mediated bone resorption by about 30%. This finding suggests that MALP-2 stimulates bone resorption partially by stimulating the formation of prostaglandins. Since interleukin-6 (IL-6) stimulates bone resorption, we investigated IL-6 production in cultured calvaria. MALP-2 stimulated the liberation of IL-6, while no tumor necrosis factor was detectable. Additionally, MALP-2 stimulated low levels of NO in calvaria cultures, an effect which was strongly increased in the presence of gamma interferon, causing an inhibition of bone resorption. MALP-2 stimulated the bone-resorbing activity of osteoclasts isolated from long bones of newborn rats and cultured on dentine slices without affecting their number. In bone marrow cultures, MALP-2 inhibited the formation of osteoclasts. It appears that MALP-2 has two opposing effects: it increases the bone resorption in bone tissue by stimulation of mature osteoclasts but inhibits the formation of new ones.     

Osteoprotegerin Abrogated Cortical Porosity and Bone Marrow Fibrosis in a Mouse Model of Constitutive Activation of the PTH/PTHrP Receptor

Intracortical porosities and marrow fibrosis are hallmarks of hyperparathyroidism and are present in bones of transgenic mice expressing constitutively active parathyroid hormone/parathyroid hormone-related protein receptors (PPR*Tg). Cortical porosity is the result of osteoclast activity; however, the etiology of marrow fibrosis is poorly understood. While osteoclast numbers and activity are regulated by osteoprotegerin (OPG), bisphosphonates suppress osteoclast activity but not osteoclast numbers. We therefore used OPG and bisphosphonates to evaluate the extent to which osteoclasts, as opposed to bone resorption, regulate marrow fibrosis in PPR*Tg mice after treatment of animals with vehicle, OPG, alendronate, or zoledronate. All three agents similarly increased trabecular bone volume in both PPR*Tg and control mice, suggesting that trabecular bone resorption was comparably suppressed by these agents. However, the number of trabecular osteoclasts was greatly decreased by OPG but not by either alendronate or zoledronate. Furthermore, intracortical porosity and marrow fibrosis were virtually abolished by OPG treatment, whereas alendronate and zoledronate only partially reduced these two parameters. The greater reductions in cortical porosity and increments in cortical bone mineral density with OPG in PPR*Tg mice were associated with greater improvements in bone strength. The differential effect of OPG versus bisphosphonates on marrow fibrosis, despite similar effects on trabecular bone volume, suggests that marrow fibrosis was related not only to bone resorption but also to the presence of osteoclasts.In bone remodeling, a balance between bone formation by osteoblasts, which are cells of mesenchymal origin, and bone resorption by osteoclasts, which are cells of hematopoietic origin, allows for maintenance of skeletal and mineral homeostasis. Failure to coordinate the processes of formation and resorption results in porotic¹ or sclerotic bone.^2,3,4 Parathyroid hormone (PTH) is a major regulator of bone homeostasis that positively modulates bone formation and bone resorption. Hyperparathyroidism is characterized by increased bone turnover, cortical porosity, and expansion of fibroblastoid cells in bone (marrow fibrosis).^5,6,7 Cortical porosity is the result of intracortical bone resorption, which is increased with hyperparathyroidism,⁸ and also with intermittent⁹ or continuous¹⁰ PTH administration. Increased cortical porosity is associated with decreased bone strength, and may contribute to the increased fracture risk in patients with hyperparathyroidism. The mechanisms leading to the expansion of fibroblastoid cells in the bones of patients with hyperparathyroidism are not well understood. Expansion of fibroblastoid cells in trabecular areas is also observed in other conditions of high bone turnover such as Paget’s disease¹¹ and fibrous dysplasia.^12,13 Transgenic mice expressing constitutively active PTH/parathyroid hormone-related protein (PTHrP) receptors on cells of the osteoblast lineage (PPR*Tg)¹⁴ also have severe cortical porosity and expansion of fibroblastoid cells in trabecular bone areas, with increased number of osteoclasts in both trabecular and cortical areas. Of note, the marrow fibrosis described in conditions of high bone turnover is not related to the fibrotic process observed in some hematological diseases and typically characterized by the accumulation of silver staining positive fibers.¹⁵Osteoclasts differentiate from precursors belonging to the monocyte/macrophage lineage. Macrophage colony stimulating factor and receptor activator of nuclear factor kappa B ligand (RANKL) are essential for osteoclast differentiation and are produced by cells of the osteoblast lineage.¹⁶ These same cells also secrete a glycoprotein called osteoprotegerin (OPG),¹⁷ a soluble member of the tumor necrosis factor receptor superfamily that acts as a decoy receptor for RANKL and prevents its interaction with the cognate receptor RANK expressed on precursor and mature osteoclasts. RANKL is essential for osteoclast formation, function and survival,¹⁸ and each of these activities are prevented by OPG. The dependence of osteoclasts on RANKL for their differentiation and survival probably explains why OPG and other RANKL inhibitors can markedly reduce osteoclast numbers.¹⁹ Bisphosphonates such as alendronate (ALN) and zoledronic acid (ZOL) are potent inhibitors of bone resorption that selectively accumulate on mineral surfaces, are subsequently internalized by osteoclasts, and impair osteoclast activity.²⁰ Despite clear evidence for antiresorptive effects of bisphosphonates in animal and human studies, bisphosphonates frequently do not reduce and can even increase osteoclast numbers.^{19,21,22,23,24} We sought to exploit the differential effects of OPG versus bisphosphonates on osteoclast numbers to determine whether osteoclasts per se, or their resorptive activity, are related to the pathophysiology of bone marrow fibrosis. This question was addressed by treating PPR*Tg mice with the RANKL inhibitor OPG, or with the bisphosphonates ALN or ZOL for 3 months. This study design also provided the opportunity to compare for the first time the effect of these agents on cortical porosity and bone strength in a murine model of high bone turnover downstream of the PTH/PTHrP receptor.     

流体剪应力对大鼠破骨细胞骨吸收活性的影响

探讨流体剪应力对大鼠破骨细胞骨吸收活性的影响。我们采用低温离心法获取6月龄健康雌性SD大鼠椎骨骨髓细胞,以1.6×106细胞密度种植于血盖片上,采用1,25-(OH)2维生素D3体外诱导获取破骨细胞。于破骨细胞诱导的第7d取出细胞爬片,置于流体剪应力装置中,分别加载5.97、11.36、16.08、20.54dyne/cm2大小的流体剪应力,持续30min,以未加载流体剪应力的破骨细胞为对照组。实验结束时,以2.5%戊二醛固定细胞爬片,置于0.25mol/L氢氧化铵液超声处理10min,清除细胞爬片上的破骨细胞,经1%硪酸固定,梯度酒精脱水,醋酸异戊酯置换酒精,CO2临界点干燥,喷金后扫描电镜观察骨吸收陷窝并计数,图像分析法测定骨吸收陷窝的面积;同时分别收集每次灌流液10ml,冻干,用1ml复溶后,紫外分光光度仪检测抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatase,TRAP)活性的变化。结果显示:在本实验中所选用的流体剪应力可增强破骨细胞TRAP活性,而骨吸收陷窝的数量和面积也增加,尤其是剪应力在16.08dyne/cm2时,破骨细胞TRAP活性增强以及骨吸收陷窝的数量和面积增高最为明显。结果表明,一定范围内的流体剪应力可以增强破骨细胞的骨吸收活性。     

Osteoclastic Invasion and Mineral Resorption of Fetal Mouse Long Bone Rudiments are Inhibited by Culture Under Intermittent Compressive Force

To study the effect of low-magnitude mechanical stimuli on mineralized matrix metabolism, fetal mouse long bone rudiments were cultured for 5d in the absence or presence of intermittent (0.3 Hz) compressive force (ICF) of 132 g/cm². ICF treatment stimulated mineralization of the diaphyseal bone collar as well as hypertrophic cartilage, but inhibited the release of ⁴⁵Ca from prelabeled rudiments. ICF also inhibited the migration of osteoclasts and their precursors from the periosteum into the diaphysis and the subsequent excavation of a primitive marrow cavity.

These data suggest that osteoclasts are sensitive to mechanical stimuli. Mechanical stimulation seems to protect the bone rudiment against osteoclastic attack and has a strong anabolic effect on mineral metabolism.     

A discrepancy between measurements of bone resorption in vivo and in vitro in newborn osteopetrotic rats

Bone resorption in newborn osteopetrotic rats and their normal littermates was measured in vivo and in vitro with different results. The resorption measured in vivo was that induced by injection of parathyroid extract after labeling bone matrix with ³H. Resorption was measured in vitro, by a commonly used method, as the release into the culture medium of ⁴⁵Ca and ³H previously incorporated in vivo into bone mineral and matrix respectively. In osteopetrotic rats resorption induced by parathyroid extract in vivo was 34 percent less than in littermate controls, whereas the resorption measured in vitro was 26 percent greater in bone from osteopetrotic rats. In addition, the hypercalcemia induced by injections of parathyroid extract was only half as great in osteopetrotic rats as in normal littermates. Acid phosphatase appears to be important in bone resorption; osteoclasts are rich in the enzyme and it is released from them during bone resorption. Newborn osteopetrotic rats have more osteoclasts which are richer in acid phosphatase than normal littermates. Therefore, it is proposed that the enhanced resorption found in bone from osteopetrotic rats in vitro is the result of release of this excessive acid phosphatase from osteoclasts under the conditions of culture and does not truly reflect resorption in vivo.     

The effects of calcium regulating hormones on bone resorption by isolated human osteoclastoma cells

Cells were disaggregated from osteoclastomas, and the response of the giant cells to calcium-regulating hormones, prostaglandin (PG)E1 and dibutyryl cyclic AMP (dbcAMP) was observed by phase-contrast time-lapse video microscopy. The pattern and nature of their response was very similar to that previously found to be characteristic of osteoclasts: calcitonin (CT), PGE1 and dbcAMP induced cytoplasmic quiescence, while parathyroid hormone (PTH) showed no influence on cytoplasmic motility or behaviour. The cells were also cultured on slices of devitalized cortical bone for 5 or 18 h. After this time the giant cells were associated with the appearance in the scanning electron microscope of characteristic resorption pits, the volume of which was calculated by computer-assisted morphometric and stereophotogrammetric techniques after removal of cells. Calcitonin caused a dramatic reduction in the volume of bone resorbed by these isolated cells compared with control cultures, while PTH was without significant effect. This result supports the view that PTH does not increase bone resorption in intact bone through a direct effect on osteoclasts. PGE1, which stimulates bone resorption when added to intact bone, paradoxically reduced resorption in our cultures. It thus appears possible that PGE1 acts as a direct inhibitor of osteoclastic bone resorption but has an additional effect on other cells in bone, which are induced by PGE1 to cause osteoclastic stimulation.     

PGE2 stimulates both resorption and formation of bone in vitro: differential responses of the periosteum and the endosteum in fetal rat long bone cultures

The ability of PGE2 to stimulate bone resorption in vitro and in vivo is well established but the effects of this compound on bone formation are still controversial. Recent clinical reports have suggested that long-term infusion of PGE in infants with cyanotic heart diseases led to a stimulation of periosteal bone formation and to hyperostosis. In the present report, we describe the effects of PGE2 (10(-5) M) in bone organ cultures on bone resorption, measured by the release of 45Calcium and the number of osteoclasts in sections of cultured bones, and bone volume, by measuring separately medullary and cortical areas. PGE2 induced a marked increase in 45Ca release and in cortical and medullary osteoclast numbers over 4 days in vitro; despite this increase in bone resorption, cortical bone volume remained constant, indicating a parallel increase in bone resorption and formation at this site. Morphological and quantitative data demonstrated a higher extent of osteoblastic surface along the periosteum of PGE2-treated bones when compared with control cultures. Medullary bone volume, on the other hand, decreased sharply during the culture period, demonstrating a lack of parallel increase in bone formation at this site. It is concluded that, under these experimental conditions, prostaglandin E2 stimulated both resorption and formation along the periosteum and only bone resorption along the endosteum of the cultured bones. The overall effect of PGE2 on bone as a whole, however, was net bone loss.     

The osteopetrotic rabbit: Skeletal cytology and ultrastructure

The lethal, autosomal recessive osteopetrotic mutation in the rabbit, osteosclerosis (os/os), has recently been made available for experimental investigation. We have examined the cytology and ultrastructure of skeletal cells in mutants and report abnormalities in osteoblasts, osteocytes, and osteoclasts. Mutant osteoclasts lack a well-defined ruffled border and show few morphological signs of bone resorption. Osteoblasts in mutants form bone in neonatal life but show signs of degeneration by 2 weeks after birth. Mutant osteoblasts and osteocytes contain large, electron-dense cytoplasmic inclusions. External surfaces of mutant long bones show no evidence of bone resorption by scanning electron microscopy, and fibrosis of intertrabecular spaces is a prominent feature in mutants. These data, considered with recent evidence that the functions of osteoblasts and osteoclasts are interrelated, suggest that reduced bone resorption, a characteristic feature of osteopetrosis, may be related to osteoblast incompetence in this mutation.     

The effects of calcitonin and colchicine on the cellular response to diphosphonate.

The mechanism by which diphosphonates inhibit bone resorption remains speculative; however, the osteoclast appears to be selectively affected by the drug. This study examined the effects off diphosphonate on osteoclasis in bones treated concomitantly with agents which reduce the osteoclast ruffled border, calcitonin and colchicine. Dichloromethylene diphosphonate, calcitonin, and colchicine inhibited parathyroid-hormone-stimulated bone resorption, as indicated by a significantly reduced release of 45Ca from prelabelled long bones. Quantitative and qualitative histological analyses of osteoclasts indicated differences among effects resulting from agents. Only bones treated with diphosphonate demonstrated a majority of osteoclasts with degenerative appearance; colchicine-treated bones exhibited many spherulated cells with cytoplasmic blebs. The number of normal osteoclasts in calcitonin-treated bones was the same as in controls and decreased with diphosphonate and colchicine treatment. All inhibitory agents reduced the number of nuclei per osteoclast; furthermore, colchicine effected an additional significant reduction, as compared with diphosphonate and calcitonin. The number and frequency of abnormal osteoclasts was increased by diphosphonate, but unaffected by colchicine and calcitonin. The addition of calcitonin, but not colchicine, to diphosphonate-treated bones decreased the incidence of abnormal osteoclasts. Although both calcitonin and colchicine are known to reduce the osteoclast ruffled border, this study has shown differences between the morphological effects of the 2 agents which presumably reflect differences in their mode of action and their interaction with diphosphonate.     

N-acetyl muramyl dipeptide stimulation of bone resorption in tissue culture.

N-Acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), a structurally defined fragment of bacterial peptidoglycan, stimulated significant release of previously incorporated 45Ca from fetal rat bones in tissue culture over the concentration range of 0.1 to 10.0 micrograms/ml. MDP-Stimulated bone resorption was not inhibited by the addition of the prostaglandin synthetase inhibitor indomethacin to the culture medium. MDP was neither mitogenic for nor stimulated the release of osteoclast-activating factor from cultured human peripheral blood mononuclear cells. Thus, MDP-stimulated bone resorption in vitro is mediated by a mechanism which is not dependent upon prostaglandins or osteoclast-activating factor. 6-O-Stearoyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, a lipophilic analog of MDP, was slightly more potent than MDP. Two diastereomers of MDP, N-acetyl-muramyl-L-alanyl-L-isoglutamine and N-acetyl-muramyl-D-alanyl-D-isoglutamine, which are inactive as adjuvants, were at least 1,000 times less active than MDP in stimulating bone resorption. The stereochemical specificity for bone-resorptive activity paralleled that required for adjuvant activity, macrophage activation, and activation of the reticuloendothelial system.