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 NOgenerating 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.Bone-remodeling disorders such as osteoporosis, osteoarthritis, and periodontal disease are frequently associated with perturbations in the interplay between local and systemic bone-remodeling regulatory pathways. Inflammatory cytokines and arachidonic acid derivatives have been implicated as intercellular messengers involved in humoral-mediated and local osteopenia (1, 2). Postmenopausal bone loss associated with diminished estrogen levels is correlated with increased levels of interleukin 1 and stromal cell-derived interleukin 6, cytokines known to stimulate osteoclast activity and development (3, 4). Estrogen also directly inhibits osteoclast-mediated resorption (5,6) Howard (13) reported that NO-generating compounds may increase cGMP levels in isolated chicken osteoclasts. Furthermore, sodium nitroprusside (SNP) has been shown to inhibit the parathyroid hormone or 1,25-(OH)2-vitamin D3 stimulation of resorption in the 19-day fetal rat limb resorption assay system, with concomitant increases in cGMP (14).The current study was designed to investigate the role of NO in both an isolated in vitro avian osteoclast system and an in vivo rat osteoporosis model system using a NOgenerating agent and selective NOS inhibitors. These findings demonstrate that NO regulates osteoclast bone-resorption activity in vitro and in vivo and that similar effects are seen in birds and mammals.MATERIALS AND METHODS Animals. Three-month-old female Sprague-Dawley rats (250-300 g) from Charles River Breeding Laboratories were used in all in vivo experim...

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the organic and inorganic components of bone matrix. The functional and developmental relationship between osteoclasts and foreign body giant cells is unclear. The osteoclast plasma membrane ruffled border juxtaposed to the bone surface is a unique morphologic characteristic of active osteoclasts. In the studies reported here giant cell formation was induced in response to a variety of materials implanted onto the richly vascularized chick chorioallantoic membrane. Light and electron microscopic techniques were used to examine the morphologic characteristics of the giant cells. In addition, immunohistochemical methods were used to demonstrate the appearance of a 150 kD cell surface antigen on chicken osteoclasts recognized by monoclonal antibody 121F. Giant cells that formed in response to mineralized bone particles exhibited ruffled borders and stained positively with the 121F antibody. Many giant cells that formed in response to hydroxyapatite possessed ruffled borders similar to but not as extensive as those observed on giant cells formed on bone. Immunohistochemical localization of the 121F antigen on these cells suggested that the antigen was present, but staining intensity was reduced compared to that of bone-associated giant cells. The formation of mineral matrix complexes by the adsorption to hydroxyapatite of bone extract or osteocalcin enhanced ruffled borders and the presence of the 121F antigen on elicited giant cells. In contrast, giant cells that formed on non-resorbable materials, such as Sepharose beads, mica, and methacrylate, lacked ruffled borders and were negative for the 121F antigen. It appears that expression of the 121F osteoclast antigen correlates with the appearance and extent of ruffled membranes on giant cells. Furthermore, it appears that giant cell ruffled membrane development and the presence of the 121F osteoclast antigen are related to giant cell formation in response to resorbable materials that are subject to extracellular dissolution. Expression of this antigen may be indicative of the developmental and/or functional state of giant cells (osteoclasts) that form on resorbable substrates. In addition, components of the bone matrix, including osteocalcin, in association with bone mineral, lead to elevated levels of this osteoclast antigen.

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The present report describes the first in a series of studies designed to identify the factor or factors responsible for eliciting osteoclast differentiation. Particles of mineralized and demineralized bone, hydroxyapatite (HA), and eggshell were grafted onto the chorioallantoic membranes (CAMs) of chick embryos. After 3 of 6 days, portions of CAMs with associated grafts were harvested, processed for light and electron microscopy, and examined for the presence of multinucleated giant cells with the morphological characteristics of osteoclasts. Light microscopic examination revealed that, within only 3 days, many particles of mineralized materials had become surrounded or engulfed by multinucleated giant cells. Ultrastructurally, all such cells possessed a vacuolated and mitochondria-enriched cytoplasm, but they differed in the nature of the contacts formed at the cell-particle interface. With eggshell, the cells developed filopodia but lacked clear zones and ruffled membranes. With HA, clear zones were evident but cytoplasmic extensions and membrane ruffling were absent. Implants of mineralized bone, however, elicited the formation of giant cells with prominent clear zones and ruffling of the plasma membrane like that observed in bonafide osteoclasts. In contrast, grafts of demineralized bone did not evoke giant cell formation but rather recruited two cell types morphologically akin to either fibroblasts or macrophages. We conclude that the factor(s) responsible for osteoclast differentiation resides specifically within bone matrix and is intimately associated with the mineral phase. Further, in response to such a factor(s), osteoclast differentiation can occur ectopically, outside of the developing vertebrate body.