Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo.

The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro.

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.

Locally delivered lovastatin nanoparticles enhance fracture healing in rats.

Statins stimulate bone formation in vitro and in vivo and, when given in large doses or by prolonged infusions, stimulate biomechanical strength of murine long bones with healing fractures. However, administration of statins by large oral doses or prolonged infusions to a fracture site is not a feasible therapeutic approach to hasten healing of human fractures. We administered lovastatin in biodegradable polymer nanobeads of poly(lactic-co-glycolide acid) to determine if lovastatin delivered in low doses in nanoparticles of a therapeutically acceptable scaffold could increase rates of healing in a standard preclinical model of femoral fracture. We found that these nanobeads: (1) stimulated bone formation in vitro at 5 ng/mL, (2) increased rates of healing in femoral fractures when administered as a single injection into the fracture site, and (3) decreased cortical fracture gap at 4 weeks as assessed by microcomputed tomography. These preclinical results suggest that lovastatin administered in a nanobead preparation may be therapeutically useful in hastening repair of human fractures.

Relationship between interleukin-1 and prostaglandins in resorbing neonatal calvaria.

The relationship between the cytokine interleukin-1 and prostaglandins in resorbing bone cultures is very confusing. In some reports, the effects of interleukin-1 are described as prostaglandin dependent, but in some they appear prostaglandin independent. Recent data suggest that events occurring during the preculture period may be important for understanding later effects of any factor on bone resorption in neonatal mouse calvaria. In this study, we examined neonatal mouse calvaria morphologically for indices of resorption to determine the importance of this preculture period to their subsequent response to interleukin-1. Our results show that after a preculture period of 24 h in control media, osteoclast numbers and resorption areas are markedly enhanced. The numbers of osteoclasts fall when the calvaria are transferred to fresh control media for the following 72 h. This increase in osteoclast formation was inhibited by the addition of indomethacin (10(-6) M) during the preincubation period. When interleukin-1 was added to bones after the preincubation period, osteoclast numbers present at this time were maintained and indomethacin had no effect on this response. Furthermore, the inhibitory effect of indomethacin on osteoclast formation during the preculture period could be reversed by adding prostaglandins of the E series in low concentrations together with indomethacin. The effects of interleukin-1 did not appear to be related to osteoclast precursor proliferation, since hydroxyurea (which inhibits DNA synthesis in these cultures) had no effect on the response of calvaria calavaria increases osteoclast to interleukin-1. These results indicate that prostaglandin production by mouse numbers during the preincubation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 receptor antagonist inhibits the hypercalcemia mediated by interleukin-1.

Recently, the effects of interleukin-1 (IL-1) on bone resorption in organ culture have been shown to be inhibited by an interleukin-1 receptor antagonist (IL-1RA), a novel monocyte cytokine in the IL-1 family. IL-1RA, which binds to IL-1 receptors and inhibits many of the effects of IL-1 alpha and beta, has been purified, cloned, and expressed. We used IL-1RA to investigate its effects on calcium homeostasis in vivo. After confirming that IL-1RA completely inhibited the effects of IL-1 on bone resorption in organ cultures, we tested the effects of IL-1RA on hypercalcemia mediated by IL-1 in normal mice and found that prolonged hypercalcemia provoked by IL-1 was completely inhibited by IL-1RA. The initial transient decrease in blood ionized calcium observed following an injection of IL-1 was also abrogated. IL-1RA had no effect alone on blood ionized calcium or on hypercalcemia mediated by parathyroid hormone (PTH) or PTH-related protein (PTHrP). These data suggest that antagonists to the IL-1 receptor may provide a useful therapeutic approach to osteoclastic bone resorption and hypercalcemia that is IL-1 dependent.

Systemic administration of acidic fibroblast growth factor (FGF-1) prevents bone loss and increases new bone formation in ovariectomized rats.

There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the transforming growth factor-beta superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor-1 and -2 (FGF-1 and FGF-2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF-1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post-OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF-1 and estrogen. However, FGF-1 also had an anabolic effect. In sham-operated rats, FGF-1 increased bone density to 2-fold, whereas estrogen had no effect. In rats 6 months post-OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF-1 induced extensive new woven bone formation with new trabecular-like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3-fold. FGF-1 and FGF-2 were also administered subcutaneously over the calvaria of mice in doses of 2-2000 microg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF-1 increases new bone formation and bone density, and systemic FGF-1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen-withdrawal.

Chinese hamster ovarian cells transfected with the murine interleukin-6 gene cause hypercalcemia as well as cachexia, leukocytosis and thrombocytosis in tumor-bearing nude mice.

The effects of interleukin-6 (IL-6) in vivo were assessed by inoculating Chinese hamster ovarian (CHO) cells which were transfected with the murine IL-6 gene in nude mice. Nude mice bearing CHO cells expressing IL-6 developed hypercalcemia. Tumor-bearing mice also showed increases in white cell count, platelet count, and decreases in body weight. In nude mice carrying CHO tumors which had not been transfected with the IL-6 gene, there were no changes in these parameters. These results suggest that increased circulating concentrations of IL-6 in patients with malignant disease may contribute to a number of paraneoplastic syndromes including hypercalcemia, cachexia, leukocytosis and thrombocytosis.

5-Lipoxygenase metabolites inhibit bone formation in vitro.

The leukotrienes and peptido-leukotrienes are 5-lipoxygenase (5-LO) metabolites of arachidonic acid that appear to have unique effects on bone, distinct from those of the prostaglandins. Application of exogenous leukotrienes in vitro and in vivo results in increased osteoclast formation and bone resorption. While 5-LO metabolites of arachidonic acid clearly stimulate osteoclastic bone resorption, little is known concerning their effects on osteoblastic bone formation. We examined the effects of the 5-LO metabolites 5-HETE, the leukotriene LTB4 and, as representative of the peptido-leukotrienes, LTD4 on the formation of mineralized nodules of fetal rat calvarial cells in the presence of dexamethasone and recombinant human bone morphogenetic protein-2 (rhBMP-2). We also examined the effects of these 5-LO metabolites on alkaline phosphatase activity and cell proliferation in these cultures and the effects of 5-HETE and LTB4 on cultured explants of neonatal murine calvariae. We found that the bone-forming capacity of osteoblasts was impaired when cells were cultured in the presence of 5-LO metabolites. These data indicate that metabolites of the 5-LO pathway are negative regulators of bone formation. The continued presence of these metabolites in the bone environment might account, in part, for the bone loss associated with chronic inflammatory conditions.

Effects of interleukin-1 on bone turnover in normal mice.

Interleukin-1 (IL-1) is a potent stimulator of osteoclastic bone resorption in vitro and causes hypercalcemia and increased osteoclastic resorption when infused into normal mice for 72 h. However, its longer term or local effects on bone turnover in vivo are unknown. To study these, we injected IL-1 alpha once daily for 3 days into the sc tissue over the calvariae of normal mice and examined its effects on calvarial bone morphology during the subsequent 4 weeks using quantitative histomorphometry. Increased bone resorption inside the calvariae and elevated plasma calcium concentrations were present 24 h after the last IL-1 injection. These early systemic effects were not prevented by indomethacin. During the following 3-4 weeks most of the bone on the injected side of the calvariae was resorbed by osteoclasts and was subsequently replaced by increased amounts of new bone. These longer term local effects on bone turnover were prevented by indomethacin. However, indomethacin did not prevent the formation of new bone inside the calvariae at sites of resorption induced by IL-1 independent of prostaglandin production. These findings indicate that IL-1 stimulates bone turnover systemically, independent of prostaglandin production, and that it has profound long term local effects on bone turnover that are mediated through prostaglandins.

A noncyclical analog of salmon calcitonin (N alpha-propionyl Di-Ala1,7,des-Leu19 sCT) retains full potency without inducing anorexia in rats.

A new analog of salmon calcitonin (N alpha-propionyl Di-Ala1,7,des-Leu19 sCT; RG-12851; here termed CTR), which lacks the ring structure of native calcitonin, was tested for biological activity in several in vitro and in vivo assay systems. The analog (CTR) and salmon calcitonin (sCT) stimulated kidney cell adenylate cyclase activity and inhibited bone resorption in organ cultures of fetal rat long bones with similar potencies and efficacies. Furthermore, CTR and sCT, at similar doses, induced comparable hypocalcemic responses in mice following sc injection or infusions. However, unlike sCT, CTR did not induce anorexia and weight loss in rats following sc injection. These data suggest that the ring structure of sCT may be important for the anorexigenic effect but is not required for effect on bone resorption or calcium homeostasis. Clinical studies appear warranted as, potentially, CTR might induce fewer side effects than does sCT.

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Statins and bone formation.

The main therapy needed most in the bone field is an anabolic agent for the treatment of osteoporosis. Current drugs on the market, which included bisphosphonates, calcitonin, estrogen and related compounds, vitamin D analogues trabecular microarchitecture. Therefore, it would be desirable to have a satisfactory and universally and iprifalvone, are essentially bone resorption inhibitors that mainly act to stabilize bone mass. Patients with established osteoporosis have lost more than 50% of their bone mass at critical sites in the skeleton, and more over have marked disruption of acceptable drug that would stimulate new bone formation and correct this disturbance of trabecular microarchitecture characteristic of established osteoporosis. Recently inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which controls the first step in the biosynthesis of cholesterol, have been shown to stimulate bone formation in rodents both in vitro and in vivo. The effect is associated with an increased expression of the bone morphogenetic protein-2 (BMP-2) gene in bone cells. These statins drugs are widely used agents for lowering cholesterol and reducing heart attacks, however they are also known to elicit numerous pleiotropic effects including inhibition of proliferation and migration of smooth muscle cells, inhibition of tumor growth and anti-inflammatory activity. Some of these effects have been attributed to not only to the reduction of cholesterol synthesis by inhibition of the HMG-CoA reductase enzyme but also by the concurrent reduction in downstream metabolites of the mevalonate pathway such as mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. The findings that statins are capable of increasing bone formation and bone mass in rodents suggests a potential new action for the statins, which may be beneficial in patients with established osteoporosis where marked bone loss has occurred. Recent clinical data suggests that they may reduce the risk of fracture in patients taking these drugs. However, their precise role can only be determined by appropriate randomized clinical trials, which demonstrate their efficacy in this regard in patients.

A murine model of human myeloma bone disease.

Myeloma causes a devastating and unique form of osteolytic bone disease. Although osteoclast activation is responsible for bone destruction, the precise mechanisms by which myeloma cells increase osteoclast activity have not been defined. An animal model of human myeloma bone disease would help in clarification of these mechanisms. Multiple myeloma occurs spontaneously in aging C57 BL/KaLwRij mice and has all of the features of the disease in humans, including the characteristic bone lesions. The disease can be induced in normal C57 BL/KaLwRij mice by inoculation of fresh marrow-derived cells from mice with myeloma, but this model is difficult to study because of variability in the number of myeloma cells in marrow-derived preparations. To develop a better animal model of human myeloma bone disease, we have established and subcloned a cell line from this murine myeloma and found that it causes osteolytic bone lesions in mice characteristic of human myeloma bone disease. The cell line produces interleukin-6, but grows independent of exogenous interleukin-6. Mice inoculated intravenously with the cultured cells predictably develop an identical disease to the mice injected intravenously with fresh bone-marrow-derived myeloma cells, including monoclonal gammopathy and radiologic bone lesions. We found that some of the mice became hypercalcemic, and the bone lesions are characterized by increased osteoclast activity. We found identical results when we inoculated Nu/Bg/XID mice with cultured murine myeloma cells. Because we can inoculate mice with precise numbers of cells and predict accurately when the mice will develop bone lesions, become hypercalcemic, and die, this should be a convenient model for determining the mechanisms by which the myeloma cells cause osteoclast activation in this model of human myeloma bone disease.

MEPE has the properties of an osteoblastic phosphatonin and minhibin.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo, (33)PO(4) uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 microg kg(-1) 31 h(-1)), similar to mice given the phosphaturic hormone PTH (80 microg kg(-1) 31 h(-1)). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE [65.0% (P < 0.001)] and PTH groups [53.3% (P < 0.001)] relative to the vehicle group [28.7% (SEM 3.97)]. In addition, Hu-MEPE significantly inhibited (33)PO(4) uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (V(max) 53.4% inhibition; K(m) 27.4 ng/ml, and V(max) 9.1% inhibition; K(m) 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50-800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serine-aspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail.

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.

Anabolic agents and the bone morphogenetic protein pathway.

A major unmet need in the medical field today is the availability of suitable treatments for the ever-increasing incidence of osteoporosis and the treatment of bone deficit conditions. Although therapies exist which prevent bone loss, the options are extremely limited for patients once a substantial loss of skeletal bone mass has occurred. Patients who have reduced bone mass are predisposed to fractures and further morbidity. The FDA recently approved PTH (1-34) (Teriparatide) for the treatment of postmenopausal osteoporosis after both preclinical animal and clinical human studies indicated it induces bone formation. This is the only approved bone anabolic agent available but unfortunately it has limited use, it is relatively expensive and difficult to administer. Consequently, the discovery of low cost orally available bone anabolic agents is critical for the future treatment of bone loss conditions. The intricate process of bone formation is co-ordinated by the action of many different bone growth factors, some stored in bone matrix and others released into the bone microenvironment from surrounding cells. Although all these factors play important roles, the bone morphogenetic proteins (BMPs) clearly play a central role in both bone cartilage formation and repair. Recent research into the regulation of the BMP pathway has led to the discovery of a number of small molecular weight compounds as candidate bone anabolic agents. These agents may usher in a new wave of more innovative and versatile treatments for osteoporosis as well as orthopedic and dental indications.

Transdermal application of lovastatin to rats causes profound increases in bone formation and plasma concentrations.

INTRODUCTION: Statins are drugs that inhibit HMG Co-A reductase and have been shown to enhance bone formation in vitro and in vivo in rodents. However, the statins currently used for cholesterol-lowering have been selected for their capacity to target the liver where their effects on cholesterol synthesis are mediated and they undergo first pass metabolism. When given in lipid-lowering doses, these agents do not likely reach sufficient blood concentrations to reliably cause substantial increases in bone formation in humans. Moreover, statins are inactivated by cytochrome P450 enzymes, resulting in even less peripheral distribution of the biologically active moieties beyond the liver. METHOD: To investigate whether an alternate method of administration might produce beneficial effects on bone formation, we administered lovastatin by dermal application to rats to circumvent the first-pass effects of the gut wall and liver. RESULTS: We found that the statin blood levels measured by HMG Co-A reductase activity were higher, maintained longer and less variable following transdermal application than those following oral administration. Also the increased circulating statin levels were associated with significantly enhanced biological effects on bone. After only 5 days of administration of transdermal lovastatin to rats, there was a 30-60% increase in trabecular bone volume, and 4 weeks later, we observed more than a 150% increase in bone formation rates. There was also a significant increase in serum osteocalcin, a marker of bone formation. We also found that lovastatin administered transdermally produces these profound effects at doses in the range of 1% of the oral dose, without any evidence of the hepatotoxicity or myotoxicity that can occur following oral statin administration. Several doses (0.01-5 mg kg(-1) day(-1)) and dosage schedules were examined, and collectively the data strongly suggest a powerful anabolic effect but with an unusually flat dose-response curve. CONCLUSION: These results show transdermal application of statins produces greater beneficial effects on bone formation than oral administration does.

Inhibition of osteoporosis in autoimmune disease prone MRL/Mpj-Fas(lpr) mice by N-3 fatty acids.

OBJECTIVE: Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease involving the breakdown of cartilage and juxta-articular bone, which is often accompanied by decreased bone mineral density (BMD) and increased risk of fracture. Anti-inflammatory omega-3 fatty acids may prevent arthritis and bone loss in MRL/lpr mice model of arthritis and in humans. METHODS: In this study, the effect of long term feeding of 10% dietary n-3 (fish oil (FO)) and n-6 (corn oil (CO)) fatty acids begun at 6 weeks of age on bone mineral density (BMD) in different bone regions in an MRL/lpr female mouse model of RA was measured at 6, 9, and 12 months of age by dual energy x-ray absorptiometry (DEXA). After sacrificing the mice at 12 months of age, antioxidant enzyme activities were measured in spleen, mRNA for receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) was measured by RT-PCR in lymph nodes, and synovitis was measured in leg joints. RESULTS: At 6, 9 and 12 months of age, BMD was significantly higher (p < 0.05) in distal femur, proximal tibia, and lumbar spine of FO fed mice than those of CO fed mice. Spleen catalase (CAT) and superoxide dismutase (SOD) activities were also significantly higher (p < 0.01) in FO fed mice than in CO fed mice. Histology of knee joints revealed mild synovitis in CO fed mice, which was not present in FO fed mice. RT-PCR analysis of lymph nodes revealed decreased RANKL mRNA (p < 0.001) expression and enhanced OPG mRNA expression (p < 0.01) in FO fed mice compared to CO fed mice. CONCLUSIONS: These results suggest beneficial effects of long-term FO feeding in maintaining higher BMD and lower synovitis in this mouse model. These beneficial effects may be due, in part, to increased activity of antioxidant enzymes, decreased expression of RANKL, and increased expression of OPG in FO fed mice thereby altering the RANKL/OPG ratio. These significant beneficial effects on BMD suggest that FO may serve as an effective dietary supplement to prevent BMD loss in patients with RA.

Lanthanide-induced calcergy.

The subcutaneous injection of solutions of chloride salts of the rare earths is followed by calcification of the dorsal fascia in the mouse. The concentration of salt required to produce a constant, predictable response varies from salt to salt. The macroscopic outline of the reaction is generally irregular and therefore a quantitative radiological method has been developed to allow an accurate assessment of the dose/response relationship. This with the majority of salts is best described as a log regression and with three as a linear regression.