Decreased Nitric Oxide Levels Stimulate Osteoclastogenesis and Bone Resorption Both in Vitro and in Vivo on the Chick Chorioallantoic Membrane in Association with Neoangiogenesis



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.


Bone remodeling is a sensitively regulated dynamic process in which bone resorption by multinucleated osteoclasts (OC) is normally closely followed by osteoblast (OB)-mediated bone formation. Alterations in the homeostatic balance of this coupled bone resorption/formation cycle can lead to excessive bone loss such as that which occurs in postmenopausal osteoporosis, rheumatoid arthritis, periodontal disease, or tumor-associated osteolysis(1–3). Many systemic and local factors (hormones, cytokines, growth factors, matrix products, lipid metabolites, and ions) regulate bone remodeling under normal and pathological conditions through their effects on the recruitment, formation, differentiation, and function of OB and OC(4,5). Recently, the multifunctional signal molecule, nitric oxide (NO), has been shown to serve as an important intercellular signal modulator in bone and to exert profound effects on OB proliferation/survival, OC function, and bone remodeling processes(6–11). NO is formed from L-arginine in an oxidative reaction catalyzed by NO synthase (NOS) isoenzymes that are either constitutively expressed and calcium activated (endothelial eNOS and neuronal nNOS isoforms), or transcriptionally induced (inducible iNOS isoform) in response to inflammatory stimuli(12–14). Whereas low levels of NO are released in transient bursts upon activation of either of the constitutive NOS isoenzymes, prolonged higher levels of NO are typically released following iNOS induction by inflammatory signals. Both OB and OC express mRNA and protein for iNOS and can produce significant amounts of NO in a regulated fashion(15–20).

Elevated NO levels potently inhibit bone resorption both in vitro and in vivo. Thus, isolated rat or avian OC in culture rapidly contract and form fewer and smaller bone excavations when exposed to NO released by exogenous chemical donor molecules(21–23). Similarly, increased NO production by OB or bone organ cultures in response to inflammatory stimuli(24–26), or by OC cultures in response to raised intracellular calcium or protein kinase C activation(19), markedly inhibits OC bone resorption. In vivo, NO-generating compounds reduce OC bone resorption and thereby protect against estrogen deficiency-induced bone loss in ovariectomized animals(27). Conversely, OC-mediated bone resorption is typically enhanced either in vitro or in vivo when NO levels are suppressed by various NOS inhibitors, including the iNOS moderately selective substrate competitive inhibitor, aminoguanidine (AG)(18,19,22–28). However, in vitro studies have also been reported in which low levels of NO exerted favorable effects on basal OC-mediated bone resorption(23,29)

Previously, we showed that AG consistently increased basal as well as stimulated OC bone resorption, while diminishing NO production, in isolated avian OC cultures(19,22). Moreover, AG infusion into rats promoted in vivo bone resorption(22,28) and potentiated the bone loss caused by declining estrogen levels in ovariectomized rats(22). AG-induced osteopenia in growing rats was associated with significantly decreased urinary N02 and N03 levels, together with increased urinary excretion of collagen cross-links(28). The in vitro and in vivo findings therefore suggest that at least one mechanism by which AG stimulates bone resorption involves an increase in the resorptive activity of OC. Additional mechanisms may also be operative. For example, histological analyses of bone sections prepared from the AG infused osteopenic rats also revealed that OC were much more numerous than in matched control bone sections(22). This prompted us to ask whether AG also promoted osteoclastogenesis. If so, this could involve an increased recruitment and emigration of hematopoietic OC precursors from the vascular compartment and/or the stimulated formation and differentiation of OC, through direct or indirect actions of AG. Here, we report the results of our studies using two well-characterized OC developmental model systems, an in vitro avian bone marrow mononuclear cell culture system(30,31) and an in vivo chorioallantoic membrane (CAM) model(32–34), to address whether NO levels modulate osteoclastogenesis. The CAM system has also been successfully employed as a model for angiogenesis(35–37), and decreased NO levels have been shown to stimulate neoangiogenesis in this system(38,39). The present work represents the first time that the CAM has been used for the simultaneous purpose of modulating both neoangiogenesis and osteoclastogenesis at focal implantation sites. Our results demonstrate that AG-mediated suppression of endogenous NO production stimulates OC recruitment, formation, development, and bone resorptive activity in vitro and in vivo, coinciding with marked increases in neoangiogenesis on the CAM.


Chick chorioallantoic membrane (CAM) model of in vivo osteoclast development

Fertile White Leghorn chicken eggs (Kenroy Hatchery, Berger, MO or Spafas Inc., Roanoke, IL) were incubated upon arrival (day 0) in a horizontal position in a forced draft incubator at 37 C and 70% relative humidity. On day 3, the eggs were windowed by gentle sanding to expose a 3–4 cm2 opening on the chorioallantoic membrane (CAM), the opening was sealed with UV-sterilized adhesive tape, and the eggs were further incubated until day 7 as described previously(32–34). On day 7, agarose plugs and devitalized bovine cortical bone chips were prepared for implantation onto the CAM. Briefly, circular bone discs (5 mm2 diameter, 0.4 mm thickness) were prepared(19,22,40), further cut into quarters, alcohol rinsed, and extensively washed in sterile Hanks' balanced salt solution (HBSS, GIBCO BRL, Gaithersburg, MD) before use. Agarose implantation plugs were prepared by boiling a 3% sterile solution of agarose (Sigma Chemicals, St. Louis, MO) in HBSS, transferring 1 ml to a 35 mm tissue culture dish to solidify, punching out small plugs using a sterilized 30 mm2 cork borer, and impregnating each plug for 1 hr with 2.5 μl of HBSS with or without an appropriate dilution of a freshly prepared 2 mM aminoguanidine (AG, Sigma Chemical Co., St. Louis, MO) stock solution in HBSS. Optimal times and volumes were established in pilot studies using dye-based solutions to ensure full and reproducible impregnation of agarose plugs with AG. Smaller plugs were then punched out using a sterile 1 ml serological pipette, and 2 or 4 plugs (140 or 280 pg AG total, respectively) were implanted using sterilized tweezers onto the CAM directly adjacent to a bone chip placed on a lightly vascularized and slightly abraded site(32–34). Although relatively avascular regions are typically chosen for angiogenic (short-term) studies alone, such sites do not support sufficient OC recruitment, even in the presence of bone and angiogenic stimulators, to enable accurate assessments of either OC development or bone pit resorptive activity in this uniquely modified CAM assay model system. For this reason, lightly vascularized CAM regions were selected and these proved to be suitable for evaluating both angiogenic responses and OC formation and activity. The eggs were resealed and incubated until day 16 when CAMs were scored and photographed with a Nikon 55mm color camera to document angiogenenic responses. The number of eggs exhibiting substantially more angiogenesis at the implant site relative to control CAMs was scored for the surviving eggs (typically >90%) from each trial. Bone chips were removed from the CAMs, rinsed with HBSS, fixed in 1% paraformaldehyde/HBSS, stained for tartrate-resistant acid phosphatase (TRAP) activity as a marker of OC, and analyzed for OC formation and bone pit resorption. Because this study also reports the first use of small agarose plugs as an effective delivery vehicle for the focal application of test agents on the CAM, initial trials were performed to document that the relative levels of angiogenesis, OC formation, and OC-resorptive activity were unchanged by the presence of control agarose plugs (1, 2 or 4) when compared to CAMs implanted with a bone chip but lacking agarose plugs.

Osteoclast in vivo formation and bone pit resorption analysis

The number of bone-associated multinucleated TRAP stained OC was determined for a constant number of random fields per bone chip. Cells were subsequently removed and the bone pit resorptive activity quantified within these same fields using a computer-linked dark-field reflective light image analysis system(19,22,40). The total area of bone resorbed, number of pits formed, and size of each excavation were assessed. Results were also normalized to OC number in order to compare the mean area of bone resorbed per OC (area/OC), number of pits formed per OC (pits/OC), and size of individual pits (area/pit). Data are presented as means ± standard error of the mean (SEM) of at least 3 independent trials each having 3–6 replicate CAMs per condition.

Bone marrow-derived osteoclast-like cell development in vitro

Mononuclear cells were isolated from the bone marrow of White Leghorn hatchlings maintained 28 days on a low calcium diet as described previously(30,31). All animals were handled in accordance with the institutional Animal Care and Use Committee and standards approved by the NIH Guide for the Care and Use of Experimental Animals. Cells were plated on day 0 at a density of 7 × 106 per well of a 6-well cluster dish in phenol red-free α-MEM containing 10% fetal calf serum (FCS, GIBCO BRL, Gaithersburg, MD) and 1% antibiotic/antimycotic (GIBCO BRL). Medium was changed every other day, mononuclear cell fusion commenced on days 1–2 and peaked on days 2–4, and large multinucleated cells had formed and typically covered the dishes by day 6–8, at which time they were harvested in parallel with the culture conditioned medium for analysis. Developmental modulators are effective at promoting an OC-like phenotype in this bone marrow mononuclear cell system when given between days 0–2 prior to cell fusion, induction of antigens and other OC characteristics are optimally measured in the multinucleated cells that have formed by day 6–8 of culture, and differentiative properties persist upon withdrawl of the developmental modulator stimuli(30,31). Aminoguanidine (AG) and sodium nitroprusside (SNP, Sigma Chemical Co, St. Louis, MO) were each freshly dissolved in warm medium shortly before they were administered to cells directly on day 1 and again with refeeding on days 2 and 4, and the modulators were subsequently discontinued at the post-developmental day 6 refeeding of cultures. An analogous treatment regimen was employed for each of three other NO donors (all from Alexis Corporation, San Diego, CA) which were freshly prepared before each use: S-nitroso-N-acetyl-D,L-penicillamine (SNAP, in absolute EtOH), 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-l-triazene (NOC-12, in 1 mM NaOH/HBSS), and (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino] diazen-1-ium-1,2-diolate (NOC-15 or PAPA NONOate, in 1 mM NaOH/HBSS).

Enzyme-linked immunosorbent assay (ELISA), tartrate resistant acid phosphatase (TRAP) analysis, and nitrite determinations

Harvested cell pellets were fixed onto 96-well dishes and analyzed via a well-established ELISA protocol to determine the relative protein expression levels of a series of characteristic OC developmental markers reactive with monoclonal antibodies (Mab) 121F(30,32,40–42), 95H(41), 75B(41,43,44), and LM609 (Chemicon International, Temecula, CA) to the αvβ3 integrin receptor(45). Parallel cell lysates were also prepared for quantification of tartrate resistant acid phosphatase (TRAP) enzymatic activity levels by microtiter assay(30,40). ELISA and TRAP assay results were normalized for cell protein using bovine serum albumin (BSA) as a standard in the Lowry(46) or the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL), respectively. Relative NO levels in the culture supernatants were evaluated based on measuring nitrite levels (as a stable end product of NO) in the conditioned medium (CM, briefly centrifuged and stored at −20 C) harvested from cultures using the Greiss reagent in a microplate assay format(19,31,40). ELISA, TRAP, or nitrite measurements, which were assayed in duplicate from at least triplicate independent culture wells per trial and at least 2–4 independent trials, were corrected for nonspecific background values and expressed as the mean ± SEM.

Cytochemistry and scanning electron microscopy (SEM)

Bone marrow mononuclear cells were cultured on glass coverslips in the presence or absence of modulators as above until day 6–8, after which they were fixed in 1% paraformaldehyde (PF) in HBSS and either differentially stained using a 3-step Difquick (triphenylmethane dye, eosin Y, azure A and methylene blue) staining kit (Criterion Sciences, Riverdale, NJ) or stained for TRAP enzymatic activity(30,40). Cells were viewed and photographed using a Leitz Diaplan microscope (Ernst Leitz, Weflar, Germany). Cell spread areas of more than 100 Difquik stained multinucleated cells per condition on replicate coverslips were quantified using a computer-linked image analysis system and the number of nuclei within each multinucleated cell (which ranged from 2 to 38 nuclei) was simultaneously enumerated. SEM analysis was performed on glutaraldehyde fixed samples processed and viewed as described previously(32,47).

Cell viability, apoptosis and necrosis measures

The number of adherent, nonadherent and total cells in day 2 cultures treated with or without SNP, and the number of adherent multinucleated cells, mononuclear cells, and total cells in day 7–8 cultures was determined by hemacytometer counting after mild trypsin/EDTA release of adherent cells(47). Adherent or nonadherent cell viability was assessed (without cell detachment) by trypan blue dye exclusion(40), active uptake and acidification of fluorescein diacetate (FDA, Sigma Chemicals, St. Louis, MO) combined with ethidium bromide (EtBr, Sigma Chemicals) nuclear staining(40), or annexin V FITC fluorescent staining of apoptotic cell membranes and propidium iodide nuclear staining to distinguish necrotic cells using a commercial kit (Boehring Mannheim, Indianapolis, IN) as recommended.

Statistical analysis

Data are presented as the mean ± SEM of 2 to 6 independent trials having at least 3 replicates per condition and assayed at least in duplicate. Differences between treatments were analyzed using single-factor ANOVA. For simultaneous comparisons between multiple treatments, significant differences were determined using the post-ANOVA Bonferroni test. Differences were considered significant for P < 0.05.


AG promotes localized angiogenesis on the CAM

Focal application of the iNOS selective inhibitor AG onto the chick CAM led to marked increases observed 9 days later in the degree of angiogenesis proximal to the implantation site (Fig. 1E-G) when compared to control CAMs not receiving AG (Fig. 1B-D). Thus, the proportion of CAMs exhibiting substantial angiogenic sprouting from vessels in the immediate vicinity of the implantation site significantly increased by 60% and 90% relative to control CAMs in response to the lower and higher doses of AG, respectively, (Fig. 1A). The CAM region encircling the site of AG application often overgrew so far as to have entirely enveloped the implanted bone slice; in all cases the outgrowing CAM tissue was also highly vascularized (Fig. 1G). Such overgrowth of CAM tissue to enclose the bone chip was never observed in over 50 control CAMs implanted with bone that did not receive AG treatment (Fig. 1B-D).

AG stimulates the recruitment, formation, and development of resorptive OC on bone slices implanted onto the CAM

Parallel to the induction of angiogenesis, AG dose-dependently increased the number of OC that formed in vivo on bone slices harvested from the CAM (Fig. 2A). Thus, the number of hematopoietic precursors recruited to the implant site that fused and differentiated into TRAP-positive multinucleated OC was significantly elevated by 50% and 60% in response to the lower and higher doses of AG, respectively, when compared to the number of OC that formed on implanted bone in control CAMs (Fig. 2A). OC formed on bone in AG CAMs were as intensely stained for TRAP enzymatic activity as those on control CAMs and both populations were capable of excavating typical lacunar resorption pits on bone (not shown). AG-induced OC were frequently situated at interior regions of the bone slice as well as the usual peripheral locations for OC in control CAMs, presumably due to the enhanced angiogenesis elicited by AG at the implant site. In addition, OC formed under the influence of AG appeared to be larger and more active in forming resorption lacunae than OC formed on control bone implants, findings that were quantitatively confirmed below.

AG stimulates the bone resorptive activity of OC formed in vivo on bone slices implanted onto the CAM

AG not only stimulated angiogenesis and OC formation in vivo on the CAM, but also enhanced OC function in vivo by significantly and dose-dependently raising OC-mediated bone pit resorptive activity both overall and on a per cell basis (Figs. 2 and 3). At the higher dose of AG (280 pg) the mean area of bone resorbed per OC increased a remarkable 2-fold (Fig. 2B), the mean number of pits formed per OC increased by 60% (Fig. 2C), and the mean size of individual resorption pits excavated by OC increased by 25% over those on the control CAM bone implants (Fig. 2D). Thus, AG stimulated the in vivo bone matrix degradative activity of individual OC. Since the resorptive activity of an OC (total area of bone resorbed per OC) is a function of both the number and size of the pits that it forms, and AG increased the number of pits formed per OC to a greater degree than the mean size of pits formed, AG stimulated in vivo OC bone resorptive activity primarily by causing OC to initiate more resorption sites during the experimental incubation period. Finally, because AG (280 pg) exerted dual actions in vivo to increase both the number of OC formed on the CAM bone implants (1.6-fold, Fig. 2A) and the bone pit resorptive activity of individual OC (2-fold, Fig. 2B), the net overall effect of AG on in vivo bone resorption was nearly a 3-fold stimulation over the levels of bone resorption occurring in the absence of AG (Fig. 3).

AG enhances the in vitro fusion of chick bone marrow mononuclear cells to form large OC-like multinucleated cells

Increased OC formation in vivo on bone implants of AG treated CAMs could be due to a stimulated recruitment of OC precursor cells as a consequence of enhanced angiogenesis. However, it is also possible that AG might exert additional direct osteoclastogenic effects on precursor cells. This was investigated using a well-characterized in vitro chick bone marrow mononuclear cell developmental culture system in which precursor cells fuse to form multinucleated cells (MNC) by day 6–8 that exhibit induced levels of multiple phenotypic and functional OC characteristics if differentiation promoting agents have been administered (eg. day 1) to the marrow cultures prior to cell fusion (days 2–4). TRAP-positive MNC arose by day 4 in both control (Fig. 4A) and AG treated cultures (Fig. 4B), and MNC became more numerous in both sets of cultures by day 8 (Fig. 4D, E). MNC formed under the influence of AG appeared to be generally larger in size when examined by TRAP staining (Fig. 4B, E), Difquik staining (Fig. 4H) or scanning electron microscopy (not shown) at either day 4 or day 8 in comparison with MNC in the corresponding control samples (Fig. 4A, D, G). No other differences were noted in cell shape, general morphology, membrane ruffling, or association of small cells with MNC for AG versus control cultures. AG did not affect in vitro bone marrow mononuclear cell proliferation prior to cell fusion since the total number of cells (predominantly nonadherent) was unchanged after 24 h (from day 1 to day 2) of AG treatment (Table 1). However, AG significantly decreased within 24 h of its administration the number of bone marrow mononuclear cells that became adherent to the dishes, without altering the number of nonadherent cells present in the cell cultures (Table 1). This suggested that AG may have enhanced bone marrow mononuclear cell fusion into multinucleated cells (MNC) leading to fewer, but larger, MNC adherent to the dishes than in the control cultures. Such an effect agrees with the microscopic observations from day 4 and day 8 cultures in which larger MNC appeared to be generated following AG treatment (Fig. 4B, E, H). Image analysis quantification of MNC cell spread areas in control and AG treated cultures was therefore performed. Whereas similar numbers of MNC formed by day 8 in control and AG treated cultures (Table 1), the average cell spread area was significantly greater by 45% for MNC formed in the presence of AG (Fig. 5A). Increased cell spread areas were not simply due to enhanced spreading of the cells to yield a flatter morphology, but also must have involved increased cell fusion, because the average number of nuclei per MNC was significantly increased by 34% for MNC formed in the presence of AG, without any appearance of mitotic figures (Fig. 5B).

Figure FIG. 1..

AG stimulates focal angiogenesis around the implant site on the CAM. Small agarose plugs with or without AG were implanted onto the CAM immediately adjacent to a small bone chip situated a short distance away from a blood vessel intersection, and the eggs were incubated for 9 days as described in the Methods. The relative extent of AG-induced localized angiogenesis was evaluated in comparison with control CAMs implanted with bone and agarose plugs lacking AG. A. AG significantly and dose-dependently increased the proportion of eggs exhibiting substantially greater angiogenesis surrounding the CAM implantation site compared to CAMs lacking AG. Results were obtained from 9 to 12 eggs per condition from at least 3 independent trials and are expressed as mean ± SEM percent increase in the proportion of AG treated CAMs per trial exhibiting substantially greater focal angiogenesis than in control CAMs lacking AG. Significant AG-induced increases over control angiogenic levels are denoted by **, p < 0.01 and ****, p < 0.001. B-D. Photographs taken at harvest after 9 days of incubation of control CAMs implanted with bone (arrows) and agarose plugs lacking AG. Note the relatively moderate level of angiogenic sprouting from nearby larger vessels in the vicinity of the implantation site. E-G. Photographs taken at harvest after 9 days of incubation of CAMs implanted with bone (arrows) and agarose plugs containing AG (280 pg total). Note the substantial increase in focal angiogenic sprouting toward the implantation site in AG treated CAMs compared to control CAMs (B-D). Overgrowth of the CAM tissue to envelop the bone and agarose plugs was often seen in response to AG (G), but never observed in control CAMs (B-D).

Figure FIG. 2..

AG stimulates OC precursor recruitment, formation, and bone pit resorptive activity on bone implanted onto the CAM. Bone chips harvested from control or AG treated CAMs were stained for TRAP activity, analyzed across a constant number of random fields for the number of TRAP-positive OC that had formed, and subsequently analyzed within those same fields for the number and size of resorption pits excavated and the total areas of bone resorbed as described in the Methods. A. AG significantly and dose-dependently elevated the number of multinucleated TRAP-positive OC formed on the implanted bones. B-D. AG significantly and dose-dependently increased the total area of implanted bone that was resorbed in vivo per OC formed on the CAM (B), the number of lacunar excavations initiated per OC (C) and, to a lesser degree, the mean size of individual resorption pits formed (D). Results were obtained from 9 to 15 eggs per condition from at least 3 independent trials, encompassing between 740 and 1130 OC and their associated pits analyzed per conditon. OC formation data is expressed as mean ± SEM percent number of AG-induced TRAP-positive OC formed relative to the mean number (49.3 ± 6.0, set at 100%) of OC that formed on bone implants of control CAMs lacking AG. Resorption data is expressed as mean ± SEM percent of mean control levels (set at 100%) for each of the three resorption parameters. In control CAMs lacking AG, the actual mean ± SEM of area of bone resorbed per OC was was 269.4 ± 40.7 μm2, area per pit was 487.6 ± 40.5 μm2, and pits per OC was 0.55 ± 0.07. Significant differences in AG-induced increases in OC formation and resorption parameters relative to control levels in the absence of AG are denoted by *, p < 0.05, **, p < 0.01, ***, p < 0.005, and ****, p < 0.001.

AG promotes the in vitro differentiation of OC-like cells formed from chick bone marrow mononuclear precursor cells

The OC-like nature of multinucleated cells (MNC) that formed in the presence or absence of AG was also monitored by quantifying the TRAP biochemical activity and relative protein expression levels of multiple characteristic OC markers recognized by monoclonal antibodies (Mab) LM609, 121F, 75B, and 95H by ELISA (Fig. 6A). AG treatment of chick bone marrow precursor populations led to significant dose-dependent increases in the expression levels of all four antigenic differentiation markers assayed in MNCs which developed by day 7 (Fig. 6A). Concurrently, AG significantly and dose-dependently reduced the corresponding nitrite levels measured in the conditioned medium (CM) collected from the critical day 0 to day 2 developmental period of the prefusion cultures (Fig. 6B). Nitrite levels in the culture CM from the subsequent day 4 to 6 period were similarly reduced as a function of increasing AG, although changes in these low nitrite levels attained statistical significance only at 500 μM AG (Fig. 6B). No changes in overall cell-associated TRAP biochemical activity (normalized for cell protein) were measured in response to AG treatment of the bone marrow cultures (Fig. 6A).

Figure FIG. 3..

AG markedly stimulates the net overall in vivo bone resorptive response observed on the CAM as a combined function of increasing both OC numbers (angiogenic recruitment and development) and OC bone resorptive function. Focal application of 140 or 280 pg AG significantly and dose-dependently raised the overall amount of implanted bone resorbed in vivo by two or three-fold, respectively, relative to control levels. This was due to significant increases in both OC formation (OC numbers) and the resorptive capacity of individual OC (area of bone resorbed per OC). Results were obtained from 9 to 15 eggs per condition from at least 3 independent trials and are expressed as mean ± SEM percent of control levels (as in Figure 2). Significant differences from control levels are denoted by *, p < 0.05 and ***, p < 0.005.

Exogenous administration of an NO donor inhibits in vitro OC-like cell development in chick bone marrow cell cultures

In contrast to the stimulatory effects of AG on osteoclastogenesis in association with reduced endogenous NO levels, raising NO levels via administration of the exogenous NO donor sodium nitroprusside (SNP) inhibited OC-like cell development in chick bone marrow mononuclear cell cultures. Thus, SNP significantly and dose-dependently reduced the already low uninduced protein expression levels of all four OC antigenic markers levels monitored by ELISA as well as the TRAP biochemical activity of MNC which developed in the presence of this agent in vitro (Fig. 6C). Parallel dose-dependent increases in nitrite levels in the CM of SNP treated cultures were observed for both the critical prefusion day 0 to day 2 and post-fusion day 4 to day 6 periods (Fig. 6D). MNC formed under the influence of SNP resembled MNC formed in control cultures in cell size and shape (Fig. 4C, F, I), but the former stained less intensely for TRAP (Fig. 4C, F), frequently exhibited an unusual peripheral ring arrangement of their multiple nuclei (Fig. 4I), and demonstrated a more complex membranous network at their outer cell margins by SEM (not shown). Although SNP (150 μM) did not affect the total number of cells adherent to the dishes on day 8 (Table 1), the number of MNC formed by day 8 was significantly decreased, while the number of nonadherent mononuclear cells present in the day 2 cultures was significantly elevated, over control levels (Table 1). This suggested that fewer precursor cells may have attached to the dishes and been available for fusion into MNC in the presence of SNP. Whether the increased number of day 2 nonadherent cells reflected SNP effects on cell viability was assessed by trypan blue dye exclusion or fluorescein diacetate/ethidium bromide staining. In either case, cell viability of the day 2 nonadherent mononuclear (but not adherent) cells was reduced by 15% and 20% following 24 h treatment with 50 μM and 150 μM SNP, respectively (not shown). Moreover, apoptosis measured by annexin-V FITC labeling demonstrated 1.5-fold increases in the proportion of day 2 nonadherent cells that exhibited early stages of apoptosis after 24 h of exposure to SNP relative to control cultures lacking SNP (control = 10%, 50 μM SNP = 17%, and 150 μM SNP = 16%). In addition, SNP caused 2 to 3-fold increases in the proportion of day 2 adherent cells that were apoptotic (control = 7%, 50 μM SNP = 12%, and 150 μM SNP = 18%). No change in the very low number of necrotic (propidium iodide stained) cells (1–2%) present in the day 2 nonadherent cell population occurred following SNP treatment, and no adherent cells were necrotic.

Other NO donors similarly inhibit OC-like cell development in chick bone marrow cell cultures

SNP releases NO via a process thought to involve cell contact and cyanide is co-produced along with NO. Therefore, other non-cytotoxic NO donors, having different mechanisms and rates of spontaneous NO release, were also tested for their ability to inhibit OC-like cell development in the chick bone marrow cell culture system. Like SNP, both SNAP and NOC-12 significantly inhibited the developmental expression of OC markers reactive with Mabs 121F and LM 609 (Table 2). Nitrite levels in the CM derived from the day 0 to day 2 prefusion and day 4 to day 6 postfusion periods were dose-dependently raised in parallel by these NO donors (Table 2). At comparable levels of NO released by 100 μM concentrations of either SNP or NOC-12, a similar inhibition of OC marker expression was achieved. This suggests that SNP-derived NO, rather than cyanide, may be responsible for the osteoclastogenesis inhibitory actions of this donor. Delayed administration of SNAP until after the critical prefusion regulatory period resulted in no inhibition of OC developmental markers (not shown), an expected outcome if NO donor repression of OC marker expression represents a developmental effect. In contrast to the inhibitory effects obtained using the NO donors SNP, SNAP, and NOC-12, each of which releases NO over a relatively prolonged time period (t1/2 of several hours or more at 37 C), similar or higher levels of NO released as a more rapid transient burst in the presence of NOC-15 (t1/2 of 10–15 min. at 37 C) did not influence OC-like cell development in these cultures (Table 2). Therefore, NO generated from various exogenous NO donors inhibits osteoclastogenesis in chick bone marrow mononuclear cell cultures. Furthermore, a relatively extended period of NO production appears to be required during the prefusion developmental period to achieve such inhibition.

Figure FIG. 4..

Microscopic evaluation of in vitro MNC cells formed in isolated bone marrow mononuclear cell cultures in the presence or absence of NO modulators. A-F. MNC forming by day 4 (A-C) or fully formed by day 8 (D-F) in control (A, D), 250 μM AG (B, E), or 100 μM SNP (C, F) treated bone marrow mononuclear cell cultures were fixed and stained for TRAP enzymatic activity. Note the more diffuse localization of TRAP activity in MNC of control and SNP treated cultures compared to the more focal subcellular localization of TRAP in MNC of AG treated cultures. Mags. A-C = × 78, D-F = × 196. G-I. MNC formed in parallel control (G), 250 μM AG (H), or 100 μM SNP (I) treated cultures by day 8 were differentially stained with Difquik to better visualize cell margins and individual nuclei. In repeated viewing of such fields across multiple trials, the AG treated cultures (H) were noted to generally contain larger MNC than were seen in the control (G) or SNP (I) treated cultures. An unusual peripheral arrangement of the multiple nuclei was also consistently observed in nearly all of the MNC formed under the influence of SNP (I) that was not evident in MNC formed in either the control (G) or AG (H) treated cultures. Mags. G-I = × 314.

Table Table 1.. AG and SNP Effects on Chick Marrow Mononuclear Cell Numbers and Their Fusion into MNC
 Number of cells (% of C)a
 AG: 0 μM10 μM250 μM
Day 2
 nonadherent cells100 ± 7100 ± 10103 ± 10
 [1.07 ± 0.08]  
 adherent cells100 ± 685 ± 676 ± 5
 [0.53 ± 0.03]  
  total cells100 ± 1199 ± 897 ± 7
 [1.60 ± 0.01]  
Day 8
 adherent MNC100 ± 792 ± 892 ± 12
 [0.06 ± 0.01]  
 Total adherent cells100 ± 6118 ± 10106 ± 11
 [0.54 ± 0.03]  
 SNP: 0 μM50 μM100 μM150 μM
  1. aChick bone marrow mononuclear cells were cultured in the presence or absence of AG or SNP and the number of adherent, nonadherent, multinucleated, and total cells was determined for prefusion day 2 and postfusion day 8 cultures as described in the Methods. Data was derived from at least 2 wells per condition, each of which was assayed in duplicate, from a minimum of 3 independent bone marrow cultures. Results are expressed as the mean ± SEM percentage of the corresponding control cell population (C). The numbers in brackets are the actual mean ± SEM total cell numbers (×106) for control cell populations per well of a 24-well cluster dish.

  2. *P < 0.05, P < 0.01, P < 0.005.

Day 2
 nonadherent cells100 ± 693 ± 12130 ± 7134 ± 10
 [1.15 ± 0.07]   
 adherent cells100 ± 1399 ± 18136 ± 2881 ± 11
 [0.37 ± 0.05]   
 total cells100 ± 4103 ± 17122 ± 4*133 ± 8*
 [1.52 ± 0.06]   
Day 8
 adherent MNC100 ± 5109 ± 1746 ± 234 ± 3
 [0.07 ± 0.01]   
 total adherent cells100 ± 3109 ± 1084 ± 1497 ± 8
 [0.37 ± 0.01]   
Figure FIG. 5..

AG promotes the in vitro fusion of avian bone marrow mononuclear cells to form larger multinucleated OC-like cells containing more nuclei per cell than in control cultures. Cells cultured on glass coverslips in the presence or absence of 250 μM AG were fixed on day 8 and Difquik stained as described in the Methods. Cell spread areas were quantified by light microscopy using a computer-linked image analysis program and the number of nuclei within individual MNC was counted. Data was obtained from at least 3 independent culture wells for each condition, encompassing more than 100 MNC analyzed per condition. Results are presented as distribution histograms of the number of MNC exhibiting various cell spread areas (A) and the number of nuclei present within MNC (B) for control (light bars) or AG (dark bars) treated cultures. Insert graphs depict the mean ± SEM of cell spread areas (A) or number of nuclei per MNC (B) for these populations. Significant differences from control cultures lacking AG are denoted by ****, p < 0.001.

Figure FIG. 6..

AG promotes the developmental expression of characteristic OC phenotypic and functional markers in MNC cells that form in vitro from avian bone marrow mononuclear cell precursors. A. AG significantly and dose-dependently increased the expression of OC developmental antigens recognized by monoclonal antibodies 121F, LM-609, 95H, and 75B in MNC harvested on day 8 that were analyzed by ELISA as described in the Methods. AG did not elevate the TRAP enzymatic activity of the OC-like cells that formed in vitro. B. AG significantly and dose-dependently reduced the nitrite levels measured in the culture conditioned medium obtained from cells during the critical prefusion period (day 0 to day 2) of time in which developmental modulators are effective for promoting osteoclastogenesis in the avian bone marrow mononuclear cell culture system. AG also inhibited, but less dramatically, the lower basal nitrite levels produced in these cultures post-fusion (day 4 to day 6). C. An exogenous nitric oxide donor, SNP, significantly and dose-dependently reduced the expression of all four of these OC developmental antigens analyzed by ELISA in MNC that formed by day 8. SNP also significantly and dose-dependently reduced the TRAP enzymatic activity of the MNC that formed in vitro. D. SNP significantly and dose-dependently raised the nitrite levels measured in the conditioned medium obtained from cell cultures during both the critical prefusion (day 0 to day 2) as well as post-fusion (day 4 to day 6) periods. Results were obtained from at least 4 independent cultures having 3 or more replicate wells per treatment condition, each of which was assayed at least in duplicate for the relevant antigen, TRAP activity, or nitrite levels in the conditioned medium. Significant differences from control cultures lacking AG or SNP are denoted by *, p < 0.05, **, p < 0.01, ***, p < 0.005, and ****, p < 0.001.

Nitrite levels in the CM negatively correlate with the developmental expression of characteristic OC phenotypic and functional markers in MNC cells formed in vitro

Higher nitrite levels measured in the CM for the critical day 0 to day 2 prefusion developmental period of avian bone marrow mononuclear cell cultures were linearly negatively correlated with the mean developmental expression levels of the four differentiation markers in MNC cells that formed in either AG or SNP treated cultures. Thus, AG-induced declines in nitrite concentrations, from an endogenous level of ∼4 μM to as little as 1.4 μM in the presence of 500 μM AG (Fig. 6B), were significantly (all P < 0.05) correlated with the mean increased expression of the OC differentiation markers recognized by Mabs 121F (r = 0.89), LM-609 (r = 0.95), 75B (r = 0.88) and 95H (r = 0.99) induced in parallel by AG. AG reduction of basal nitrite below control levels was therefore associated with greater developmental antigen expression and, conversely, higher nitrite levels were correlated with lesser developmental antigen expression. SNP elevation of nitrite levels, which reached as high as 22.6 μM in the presence of 150 μM SNP, were also linearly negatively correlated with the mean expression levels of all four of these OC developmental markers (r values ranging from 0.79 to 0.98). However, all four antigenic markers exhibited a more modest decline (20 to 50% from control levels) as SNP-mediated nitrite levels increased 5-fold from 4.4 to 22.6 μM, in comparison with the more pronounced rise in antigen expression (50 to 100% over control levels) that occurred over a 3-fold AG-mediated reduction in nitrite levels from 4.4 to 1.4 μM (Fig. 7).

Table Table 2.. Effects of NO Donors on the Developmental Expression of OC Antigens 121F and αvβ3 Relative to Medium Nitrite Levels
  OC marker (% of C)Nitrite (μM)
NO donorDonor concentration (μM)121Fαvβ3Day 0–2Day 2–4
  1. Chick bone marrow mononuclear cells were cultured in the presence or absence of various doses of NO donors and the MNC that formed by day 7–8 were analyzed for protein expression levels of the OC developmental markers reactive with MAbs 121F and LM609 (αvβ3) by ELISA as described in the Methods. Data was derived from 4–8 wells per condition, each of which was assayed in replicates of 2–4, from 2 or more independent bone marrow cultures. Antigen results are expressed as the mean ± SEM percentage of marker expression in MNC formed in control cell populations not exposed to NO donors. Vehicle controls appropriate for each NO donor were run in parallel and antigen levels in these MNC were indistinguishable from the levels determined in control MNC formed without vehicle additions. Nitrite (μM) was measured in the CM obtained from these same bone marrow cell cultures over the critical prefusion day 0 to day 2 and postfusion day 4 to day 6 periods as described in the Methods. Significant differences in OC-marker expression or CM nitrite levels from control MNC populations lacking NO donors are denoted by: *p < 0.05, p < 0.01, p < 0.005, and §p < 0.001.

Control0100 ± 5100 ± 51.4 ± 0.11.2 ± 0.3
SNP5077 ± 4‡§107 ± 612.3 ± 0.2§21.9 ± 0.2§
 10065 ± 4§88 ± 6*17.8 ± 0.4§32.4 ± 0.6§
 15052 ± 3*80 ± 3*22.6 ± 0.4§37.5 ± 0.8§
SNAP1074 ± 6*71 ± 6*1.4 ± 0.11.9 ± 0.2*
 5087 ± 768 ± 6*2.8 ± 0.4§6.8 ± 0.1§
 10068 ± 7*58 ± 47.0 ± 1.3§13.6 ± 0.1§
NOC-1210103 ± 10101 ± 102.7 ± 0.1§4.2 ± 0.2§
 5082 ± 485 ± 76.6 ± 0.4§16.0 ± 0.3§
 10076 ± 8*76 ± 5*15.6 ± 1.1§32.0 ± 0.3§
NOC-1510116 ± 497 ± 102.0 ± 0.2§2.6 ± 0.1§
 5095 ± 4103 ± 1110.2 ± 0.4§13.2 ± 0.3§
 100111 ± 44105 ± 626.1 ± 1.6§29.6 ± 0.6§
 250108 ± 13135 ± 1191.4 ± 4.6§85.2 ± 1.7§
 50092 ± 1399 ± 12155.7 ± 3.1§170.0 ± 1.7§
Figure FIG. 7..

Differential sensitivity of OC developmental marker expression relative to medium nitrite level changes above or below endogenous culture levels in response to SNP or AG. Correlational data generated from the results shown in Figure 6 were co-plotted here to reveal the similar trends (slopes) for all four OC differentiation antigenic markers relative to changes in medium nitrite levels, their marked increases in parallel with small declines in nitrite levels, and their contrasting modest inhibition in relation to larger nitrite level changes above endogenous basal nitrite concentrations. Note the apparent switch that occurs at 8–12 μM nitrite in the relative sensitivity of OC developmental marker expression as a function of medium nitrite levels.


Here, we have demonstrated for the first time that AG, an iNOS selective inhibitor previously shown to potentiate OC-mediated bone resorption in vitro and bone loss in vivo(18,19,22,28), also acts to promote bone resorption by exerting stimulatory influences on: 1) OC precursor recruitment via enhanced neoangiogenesis and 2) OC development via increased fusion, formation, and differentiation of bone marrow precursor cells. NO therefore appears capable of influencing multiple aspects of OC development and function. The present findings therefore highlight a central role for endogenous NO levels in regulating normal bone physiology. Moreover, they emphasize the close relationship between vascularity and OC formation/resorption, events that are crucial for normal bone development(48) but are linked to pathological consequences when inappropriately stimulated(49).

Angiogenesis involves a complex series of highly regulated events in which endothelial cells proliferate, migrate, differentiate, and organize into new blood vessels and capillary networks during such physiological processes as embryonic development, the reproductive cycle, and wound healing. Pathological formation of new blood vessels may significantly contribute to tumor progression and to tissue (and bone) destruction in chronic inflammatory disorders such as rheumatoid arthritis and periodontal disease(1–3,49). Although NO is clearly a potent vasodilator, its role in angiogenesis is still controversial with evidence supporting both stimulatory and inhibitory actions for NO in regulating neovessel formation(50,51). Our in vivo studies utilized a novel adaptation of the chick CAM model, heretofore employed to independently investigate either angiogenesis(35–37) or OC formation and resorption(32–34). Small agarose plugs impregnated with AG were placed directly adjacent to the implanted bone chip on which OC would subsequently develop and excavate resorption pits. While it is possible that the induction of angiogenesis and ectopic OC formation in the modified CAM assay might differ in some respects from that which occurs in the bone compartment, this design allowed for the focal application of a stimulus at the bone implantation site, simultaneous assessment of both angiogenesis and OC development/activity in response to AG, and exploration of these interrelationships using a readily manipulated in vivo system. Localized angiogenesis in vivo on the CAM was potently and dose-dependently stimulated by AG. Based on parallel CAM studies, AG-induced neoangiogenesis actually appeared to exceed the microcapillary formation elicited by two other well-known angiogenic stimulators, basic fibroblast growth factor (bFGF) or interleukin-8 (IL-8)(52). Neoangiogenic responses on the CAM have also been evoked by more general inhibitors of NO production (L-NAME, L-NMMA), and endogenous NO has been proposed to function in vivo to maintain the non-angiogenic status of the vascular endothelium(38). Conversely, angiogenenesis on the CAM has been inhibited by agents that release NO, induce its synthesis, or prevent its destruction(38,53), and therapeutically useful NO donors have inhibited tumor growth and metastasis in association with reducing angiogenesis in a mouse tumor model(53). This and much other evidence indicate that NO serves both as the primary vasodilatory signal and as a potent autocrine regulator of neoangiogenesis and vasculogenesis under physiological and pathological conditions.

AG has previously been shown to exert pro-resorptive actions both in vitro in isolated OC cultures and in vivo in rats infused with this agent(18,19,22,28). OC were not only more active, but also more numerous, on the trabecular surfaces of bone sections from AG treated animals(22). We therefore further investigated if AG stimulated osteoclastogenesis in addition to mature OC function. Here, AG-induced neoangiogenesis on the CAM was accompanied by an increased recruitment, formation, and differentiation of resorptive OC on the bone implants. Presumably, increased angiogenesis facilitated OC precursor mobilization into the vicinity of the bone implant, accounting for both the increase in OC numbers and their location at interior sites of the bone as opposed to their formation solely at the outer peripheral bone margins in control CAMs. In addition to promoting OC formation, AG also stimulated the bone resorptive capability of OC in vivo on the CAM as evidenced by significantly increased areas of bone resorbed per OC, numbers of pits formed per OC, and slightly larger excavations formed in vivo on AG bone implants. Analogous to these in vivo results, AG has also stimulated isolated OC bone pit resorption in vitro by increasing the area of bone resorbed per OC, primarily via increasing the number of pits formed per OC rather than size of pit lacunae(19,22). Owing to the combined stimulatory effects of AG on OC recruitment, formation, and activity on the CAM, overall bone resorption in vivo was potently stimulated 3-fold over control CAMs lacking angiogenic stimulation. The present CAM findings therefore support and extend the results obtained previously from in vivo rat studies by directly demonstrating that AG stimulates the formation in vivo of greater numbers of OC having an enhanced bone resorptive capability. Furthermore, the data suggest that pathological conditions involving enhanced angiogenesis may favor increased OC formation and activity, thereby contributing to a localized osteopenia.

Complementary in vitro studies were performed to learn whether AG also influenced osteoclastogenesis via other mechanisms that might not directly involve angiogenesis. Using a well-characterized in vitro avian OC-like cell developmental system(30,31), AG dose-dependently stimulated OC-like cell development in the absence of vascular cells. Thus, AG promoted the fusion of mononuclear cells to form larger multinucleated cells that contained more nuclei per cell and exhibited increased expression of multiple OC phenotypic and functional markers(40–45) than in control cultures. Increased OC-like cell developmental marker expression was significantly correlated with declining endogenous NO (nitrite) production in the AG early prefusion cultures. Somewhat unexpectedly, AG did not co-induce TRAP activity AG in these cultures. Whether there is a differential regulation of this sensitively redox-regulated iron-containing enzyme as a consequence of the AG-mediated inhibition of basal NOS activity is a subject for further investigation. Conversely, SNP inhibited in vitro OC-like cell development in parallel with rising NO (nitrite) levels in the prefusion cell cultures. Inhibition was manifested in a reduced viability of the precursor bone marrow cell population, decreased attachment of prefusion cells to the culture dish, fewer multinucleated cells that formed, and lesser expression of OC differentiation markers (and TRAP activity) in those multinucleated cells that formed in vitro. The latter inhibition was particularly remarkable since very little basal OC-like cell development takes place in these cultures in the absence of differentiation promoting agents(30,31). Other slow-release NO donors, such as SNAP and NOC-12, that are nontoxic and exhibit different mechanisms and rates of spontaneous NO release(54,55), also inhibited in vitro osteoclastogenesis. Differences observed between SNP, SNAP, and NOC-12 in their dose responsiveness and inhibitory effectiveness may reflect the fact that biological responses to NO donors are highly complex functions of the specific properties of these compounds, dependent upon the mode and rates of NO released, particular redox forms involved, reactivity with other biomolecules, and other considerations(55). It is therefore of interest that similar levels of OC marker suppression were achieved with NO donor concentrations of SNP and NOC-12 that yielded comparable levels of nitrite in the media. Moreover, NO release was only inhibitory for osteoclastogenesis if it occurred during the early prefusion developmental period and if such release was relatively prolonged (hours) as opposed to a transient burst (minutes) generated by the fast-release NO donor NOC-15.

Consistent with our findings, NO inhibitors (including AG) have stimulated, whereas elevated NO levels inhibited, mouse in vitro OC-like cell development(56). A rise in NO levels (100 μM SNP) has also suppressed vitamin D3 stimulated mouse(57) and avian (our unpublished observations) OC-like cell development. In contrast, some in vitro organ or cell co-culture studies have reported that basal NO levels were required for OC development and/or activity under certain experimental conditions(18,23,26,29). Possibly, such differences relate to specific culture conditions, types of other cells present, redox status of the cultures, and/or other factors that influence OC formation and function in these systems. However, our in vitro and in vivo findings agree well with the in vivo studies performed in rats which have shown that NOS inhibitors increase OC numbers and reduce bone mass(22,27,28) while, conversely, NO donors attenuate OVX-induced bone loss(27). Together, the findings indicate that endogenous NO production has an important role in regulating bone remodeling through restraining basal OC formation and bone resorption. Recently, this relationship has been extended to humans. Postmenopausal women were shown to have lower serum NO levels along with reduced circulating estrogen levels and increased bone loss, estrogen replacement therapy raised serum NO levels while inhibiting such bone loss, and intermittent nitrate use by postmenopausal women protected against estrogen-deficient bone loss(58). Thus, a deficiency in NO may contribute to the bone loss that occurs following menopause and NO donors may prove to be effective pharmacological agents for the prevention of postmenopausal osteoporosis. The fact that these same bone-sparing therapeutic NO donors(58) also exhibit potent anti-angiogenic actions in vivo(53) again highlights the close alliance between OC-mediated bone resorption and vascularity.

OC development in vitro was differentially sensitive to higher or lower NO levels. Thus, substantial increases in OC development were promoted by small declines in NO below basal levels, whereas only modest developmental changes occurred in response to larger elevations in NO above endogenous levels. The inflection point in this differential sensitivity was in the vicinity of 8 to 12 μM nitrite (10 to 50 μM SNP). This is the level that generally corresponds to the demarcation between basal NO changes that occur under normal physiological conditions (<10 μM) and the more pronounced elevations in NO (>10 μM) that are generated under pathological inflammatory stimulated conditions. It is also the level at which differential NO effects have been observed for in vitro bone resorption in mouse neonatal calvarial organ cultures: resorption increased with cytokine-mediated NO elevations in the 0–8 μM range but declined towards basal as NO concentrations exceeded 12 μM(25,26). Furthermore, in mixed co-cultures of fetal mouse osteoblasts with mouse bone marrow cells, NO augmented OC generation at low to moderate SNP concentrations (3–30 μM) but inhibited OC formation at high concentrations (100 μM SNP)(57). Our findings using an in vitro avian bone marrow culture system suggest that OC development may be very efficiently modulated by the relatively small fluctuations in basal NO levels that occur under normal physiological conditions, whereas high NO production may exert relatively little influence (i. e. only a mild inhibition) on osteoclastogenesis. In the latter case, this might enable other locally co-produced stimulatory signals (eg. cytokines, prostanoids) to prevail in promoting the increased OC formation and bone resorption seen in various inflammatory disorders(25,26).

In conclusion, we have shown here for the first time that NO can sensitively regulate multiple aspects of OC-mediated bone resorption in vitro and in vivo via effects on OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorption capability. Reduced basal NO levels generally favored OC formation and promoted OC bone resorption in conjunction with neoangiogenesis, whereas elevated NO levels inhibited these processes. Potential in vivo regulation of local bone metabolism via modulating physiological NO levels and angiogenesis at specific sites may have numerous clinical applications. Thus, the ability of AG to locally promote neoangiogenesis and initiate OC-mediated bone remodeling might provide an effective therapeutic approach for treating the avascular necrosis of bone which commonly occurs following radiation therapy, chemotherapy treatment, or steroid regimens(59,60), and its use as an aid in fracture repair(61) or alternative to vascularized bone grafting in skeletal reconstructive surgery(62,63) merits investigation.


The authors wish to thank Mr. Niraj Patel, Ms. Rafiya Agha, and Ms. Kelley Wear for initial preliminary studies related to the current project. This work was supported by NIH grants DK46547 to PC. O and AR32927, AR42715, and DE06891 to PO.