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Circulating pre‐OCs may be recruited to locally inflamed sites through specific interactions with activated microvasculature. We found that HMVECs stimulated the adhesion and TEM of circulating pre‐OCs, in an ICAM‐1‐ and CD44‐dependent manner, leading to greater RANKL‐induced OC formation and bone pit resorption.

Chemoattractants that recruit OC precursors to locally inflamed sites of resorption are not well known. A chemokine receptor, CCR1, was expressed in OC precursors and elevated in mature OCs, and its ligands promoted OC precursor recruitment, RANKL development, and OC motility. Cytokines induced OB release of such chemokines, which may therefore significantly contribute to inflammatory bone loss.

Signals targeting OCs to bone and resorption sites are not well characterized. A chemoattractant receptor (CXCR4), highly expressed in murine OC precursors, mediated their chemokine (SDF‐1)‐induced chemoattraction, collagen transmigration, and MMP‐9 expression. Thus, bone vascular and stromal SDF‐1 may direct OC precursors into bone and marrow sites for development and bone resorption.

Increased local osteoclast (OC)‐mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor‐associated osteolysis and inflammatory‐related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre‐OC to emigrate from the circulation and develop into bone‐resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo. Angiogenesis elicited by bFGF on the CAM was accompanied by increased OC formation and bone pit resorption (both overall and on a per OC basis) on the bone implants in vivo. In complementary in vitro assays, bFGF did not directly stimulate avian OC development from bone marrow mononuclear cell precursors, consistent with their low mRNA expression of the four avian signaling FGF receptors (FGFR)‐1, FGFR‐2, FGFR‐3, and FGFR‐like embryonic kinase (FREK). In contrast, bFGF activated isolated avian OC bone pit resorption via mechanisms inhibited by a selective cyclo‐oxygenase (COX)‐2 prostaglandin inhibitor (NS‐398) or p42/p44 MAPK activation inhibitor (PD98059), consistent with a relatively high expression of FGFR‐1 by differentiated avian OCs. Thus, bFGF may sensitively regulate local bone resorption and remodeling through direct and indirect mechanisms that promote angiogenesis and OC recruitment, formation, differentiation, and activated bone pit resorption. The potential for bFGF to coinduce angiogenesis and OC bone remodeling may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. Alternatively, inhibiting such bFGF‐dependent processes may aid in the treatment of inflammatory‐related or metastatic bone loss.

The receptor activator of NF-κB (RANKL) is the essential signal required for full osteoclast (OC) development, activation, and survival. RANKL is highly expressed in areas of trabecular bone remodeling and inflammatory bone loss, is increased on marrow stromal cells or osteoblasts by osteotropic hormones or cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy receptor also crucial for preventing arterial calcification. Vascular endothelial cells (VEC) are critically involved in bone development and remodeling and influence OC recruitment, formation, and activity. Although OCs develop and function in close association with bone VEC and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-α and interleukin-1α elevate RANKL and OPG expression 5–40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the surface of tumor necrosis factor-α-activated HMVEC. Cytokine-activated HMVEC promoted the formation, fusion, and bone resorption of OCs formed in co-cultures with circulating human monocytic precursors via a RANKL-mediated mechanism fully antagonized by exogenous OPG. Furthermore, paraffin sections of human osteoporotic fractured bone exhibited increased RANKL immunostaining in vivoon VEC located near resorbing OCs in regions undergoing active bone turnover. Therefore, cytokine-activated VEC may contribute to inflammatory-mediated bone loss via regulated production of RANKL and OPG. VEC-derived OPG may also serve as an autocrine signal to inhibit blood vessel calcification.

High nitric oxide (NO) levels inhibit osteoclast (OC)‐mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen‐deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC‐like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose‐dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular‐independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC‐like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose‐dependently inhibited in vitro OC‐like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC‐mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC‐mediated bone remodeling via AG or other pro‐angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.

Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg‐laying hen maintained on a low‐calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1)The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low‐calcium diet. A corresponding decrease in bone‐associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast‐directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (>99%) nonviable, but the third population contains 8‐fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25‐fold in comparison with the first two populations. The osteoclast‐like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody‐reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

Osteoclasts generate high levels of superoxide anions during bone resorption that contribute to the degradative process, although excessive levels of this free radical may be damaging. One mechanism for their removal is via superoxide dismutase (SOD), a protective superoxide scavenging enzyme. We have previously described a novel developmentally regulated 150 kDa plasma membrane glycoprotein of avian osteoclasts which is reactive with the osteoclast‐specific monoclonal antibody (Mab) 121F and is related immunologically, biochemically, and in protein sequence to mitochondrial Mn2+/Fe2+SOD. We hypothesized that this unusual osteoclast surface component may be involved in protection against superoxides generated during active bone resorption. Increasing concentrations of monovalent Fab fragments prepared from Mab 121F, but not those from another antiosteoclast Mab designated 29C, markedly inhibited both bone particle and bone pit resorption by avian osteoclasts, while reducing tartrate‐resistant acid phosphatase activity and causing the morphological contraction of osteoclasts on bone. Thus, the SOD‐related membrane antigen may be essential for osteoclast bone resorption. Osteoclast superoxide production, monitored kinetically by cytochrome c reduction and histochemically by nitroblue tetrazolium reduction staining, was significantly greater in the presence of 121F, but not 29C, Fab treatment. Furthermore, the release of another free radical known as nitric oxide, which is produced by osteoclasts, can scavenge superoxides, and acts to potently inhibit osteoclast bone resorption, was dose‐dependently increased by 121F Fab in resorbing osteoclast cultures. Therefore, Mab 121F binding may block the potential protective function of the osteoclast plasma membrane SOD‐related glycoprotein, leading to a rapid elevation of superoxide levels and a subsequent rise in osteoclast nitric oxide release, feedback messages which may be sensed by the osteoclast as signals to cease active bone resorption.

Osteoclast differentiation from hematopoietic precursors into multinucleated cells uniquely capable of removing the organic and inorganic components of bone matrix occurs in a multistep process, during which osteoclasts acquire the specialized characteristics necessary for bone resorptive activity and physiological regulation. Among those traits is a novel plasma membrane glycoprotein, reactive with the anti‐osteoclast monoclonal antibody 121F, which is expressed during the course of osteoclast differentiation, shares structural and functional homologies with Mn2+/Fe2+superoxide dismutase, and has been hypothesized to protect the osteoclast from the damaging effects of superoxide radicals generated during active bone resorption. We have reported previously that the expression of this membrane antigen is induced on multinucleated giant cells when the profusion marrow mononuclear cells are cultured in conditioned medium from avian calvaria. The studies reported here were designed to investigate the relationship between expression of the 121F antibody‐reactive osteoclast membrane antigen and tartrate resistant acid phosphatase levels, bone resorptive activity, calcitonin responsiveness, and ultrastructural features of avian bone marrow‐derived multinucleated giant cells formed either in the presence or absence of diffusible osteoblast secreted factors. Parallel analyses of in vivo formed osteoclasts isolated from the same animals were performed for direct comparisons. In this report we demonstrate: (1) that the 121F monoclonal antibody‐reactive osteoclast membrane antigen is stably induced in giant cells by soluble osteoblast‐derived factors in a species nonrestricted but concentration‐ and temporal‐dependent manner; (2) that osteoblast‐mediated antigen induction is reflected in both increased numbers of cells and elevated expression of individual cells that are reactive with the 121F antibody, as determined by ELISA and histomorphometry; (3) that osteoblast conditioned medium, in addition to inducing this antigen in bone marrow cells, also elevates other defining osteoclast characteristics in these avian giant cells including their TRAP activity, cell retraction from the bone surface in response to calcitonin, bone resorptive function, and expression of a series of additional osteoclast antigenic markers; and (4) that secreted osteoblast products alone do not raise the levels of these traits for in vitro formed marrow giant cells to the extent associated with in vivo formed osteoclasts. Therefore, osteoblast soluble factors alone appear unable to promote the full differentiation of bone marrow cells in vitro into mature bone‐resorbing osteoclasts. This inductive bone marrow model system, in conjunction with the ability to monitor an expanded profile of osteoclastic markers afforded by specific monoclonal antibodies, may therefore serve as a valuable tool for investigating intermediate stages of osteoclast cytodifferentiation and for identifying signals responsible for their partial or complete development into unique bone‐resorbing cells.

Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast‐directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody‐coated beads to mixed cell preparations derived from marrow‐depleted, collagenase‐ and/or trypsin‐treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead‐cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody‐coated bead binding to osteoclasts pretreated with the soluble antiosteoclast antibody and also by the absence of binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100‐fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody‐coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody‐coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody‐coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified poplations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.

Osteoclast cell function relates to bone resorption. Isolation and characterization of these cells from in vivo sources remain difficult. The aim of this study was to show the feasibility of using flow cytometry to identify and characterize human mature osteoclasts obtained from bone tissues. Bone femoral heads obtained as discarded surgical material were used. To check the nature of 121F⁺ (a monoclonal antibody specific for human osteoclasts) cells by flow cytometry, we used laser scanning cytometry to analyze simultaneously the immunophenotype and DNA cell content of osteoclast-like cell-enriched bone samples. Results were compared with conventional morphologic and cytochemical studies. The percentage of cells that showed both cytochemical (tartrate-resistant acid phosphatase [TRAP]⁺) and immunophenotypic (121F⁺) osteoclast-associated characteristics was very similar (12.5 ± 6.2 versus 14.7 ± 11.7; P = 0.46). Laser scanning cytometry showed that 121F⁺ cells were bigger (P = 0.04) and they had a higher DNA cell content (P = 0.04) and more nuclei per cell (P = 0.04) than the 121F^- cells present in the same sample. This study relied on the combined use of the 121F⁺ antibody and different cytometry-based techniques to characterize the osteoclast populations from human bone. Cytometry (Clin. Cytometry) 49:261–266, 2002. © 2002 Wiley-Liss, Inc.

Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung. Gal-3TM1 and Gal-3TR1 messages both contain an open reading frame encoding a predicted 70-amino acid TM1 sequence inserted between the N-terminal Gly/Pro repeat domain and the carbohydrate recognition domain (exons 3 and 4). Gal-3TR1 mRNA contains an additional 241-bp sequence, which encodes a truncated open reading frame between the 4th and 5th exons, and, whose translation is expected to terminate within the carbohydrate recognition domain encompassing exons 4, 5, and 6. Immunoblotting and affinity chromatography showed that purified osteoclast preparations and intestinal homogenates contained a 36-kDa lactose-binding galectin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses on chymotryptic peptides from the 36-kDa lectin confirmed its identity as Gal-3TM1. The TM1 insert contains a single transmembrane-spanning region and a leucine zipper-like stalk domain that is predicted to position the intact carbohydrate recognition domain of Gal-3TM1 on the exterior surface of the plasma membrane. Immunofluorescent staining of chicken osteoclasts confirmed the expression of Gal-3TM1 at the plasma membrane. Gal-3TM1 is the first example of a galectin superfamily member capable of being expressed as a soluble protein and as a transmembrane protein.

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.

Studies on the origin, identification, and characterization of osteoclasts have been difficult. This is in part due to a lack of definitive osteoclast markers and the similarity of these cells in form and function to cells of the mononuclear phagocyte system. To solve this problem, we inoculated isolated chick osteoclasts into mice to generate osteoclast-specific monoclonal antibodies. Supernatants from growth-positive hybridomas were screened by indirect immunofluorescent methods against cultured osteoclasts, monocyte-derived multinucleated giant cells, cultured monocytes, fibroblasts, and limb mesenchyme. Select hybridomas were cloned to produce 375 clones, which were analyzed as described above. Antibody from select clones was also reacted with paraffin sections of bone. In addition, two clones have been analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody binding from an osteoclast-specific clone and a clone reactive with osteoclasts, giant cells, and cultured monocytes (as determined by immunohistochemical assay) was confirmed by antibody-binding and titration curves quantitated by ELISA. The above studies demonstrate that osteoclast specific antigens exist, and that osteoclasts, giant cells, and cultured monocytes share common determinants not found on other cells screened.

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin.

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for α_V integrin subunit was observed. The α_Vβ₃ integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation. Copyright 1995, 1999 Academic Press

Osteoblasts have been shown to modulate osteoclast activity, but the reverse process has not been investigated. In the current study conditioned medium (CM) was collected from osteoclasts and osteoclast‐like cells and its effects on osteoblast alkaline phosphatase (ALPase) activity and collagen synthesis ([3H]proline hydroxylation) were determined. In primary chick osteoblasts, cultured chick embryo frontal bones, and UMR‐106‐01 cells, collagen synthesis and ALPase activity, but not [3H]thymidine incorporation, were inhibited by CM from chick marrow‐derived giant cells, which possess some of the phenotypic characteristics of osteoclasts. However, collagen synthesis in chick embryo fibroblasts was not affected by giant cell CM. CM collected from cultures of chicken osteoclasts and human osteoclastoma cells and marrow‐derived giant cells inhibited collagen synthesis in UMR‐106‐01 cells, but the effects on ALPase activity varied with the cell type. In contrast, mononuclear cell and fibroblast CM did not alter collagen synthesis. Initial characterization studies demonstrate that the inhibitor is a heat‐labile factor with a molecular weight greater than 3500. In summary, authentic osteoclasts, tumor osteoclast‐like cells, and chicken and human multinucleated giant cells produce a soluble factor that alters osteoblast collagen synthesis, suggesting that osteoclasts play a role in the modulation of osteoblast activity.

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.

Based on reports of electrically induced bone formation and findings that some materials used to promote bone ingrowth are stimulatory in bead form, the osteogenic potential of beads with different surface charges was examined. In this preliminary study, three types of Sephadex beads were injected into chick femora: type I, DEAE beads, positively charged; type II, CM beads, negatively charged; type III, G‐25, uncharged. Beads were injected into the femoral midshaft, and after 3 days, 4 days, and 1 week, birds were sacrificed and femora were processed for histology. Type I beads: at 3 days, were surrounded by multinucleated giant cells; by 4 days, patches of bead‐associated new bone were present along with giant cells; after 1 week, occasional bead‐associated multinucleated cells were seen, but now most beads were surrounded by new intramedullary bone, forming an extensive bead‐bone lattice. With bead types II and III, bead‐associated new bone was seen at 3 days and 4 days only when beads lodged near the endosteum or in the metaphysis. At 7 days, no bone was seen with either of these two bead types. The response to the type I beads may be likened to a remodeling phenomenon with large numbers of giant cells at 3 days, new bone and giant cells at 4 days, and evidence only of bone formation at 7 days.