Accelerated post traumatic osteoarthritis in a dual injury murine model.

OBJECTIVE: Joint injury involving destabilisation of the joint and damage to the articular cartilage (e.g., sports-related injury) can result in accelerated post-traumatic osteoarthritis (PTOA). Destabilised medial meniscotibial ligament (DMM) surgery is one of the most commonly used murine models and whilst it recapitulates Osteoarthritis (OA) pathology, it does not necessarily result in multi-tissue injury, as occurs in PTOA. We hypothesised that simultaneous cartilage damage and joint destabilisation would accelerate the onset of OA pathology. METHODS: OA was induced in C57BL/6 mice via (a) DMM, (b) microblade scratches of articular cartilage (CS) or (c) combined DMM and cartilage scratch (DCS). Mice were culled 7, 14 and 28 days post-surgery. Microcomputed tomography (µCT) and histology were used to monitor bone changes and inflammation. Dynamic weight bearing, an indirect measure of pain, was assessed on day 14. RESULTS: Osteophytogenesis analysis via µCT revealed that osteophytes were present in all groups at days 7 and 14 post-surgery. However, in DCS, osteophytes were visually larger and more numerous when compared with DMM and cartilage scratch (CS). Histological assessment of cartilage at day 14 and 28, revealed significantly greater damage in DCS compared with DMM and CS. Furthermore, a significant increase in synovitis was observed in DCS. Finally, at day 14 osteophyte numbers correlated with changes in dynamic weight bearing. CONCLUSION: Joint destabilisation when combined with simultaneous cartilage injury accelerates joint deterioration, as seen in PTOA. Thus, DCS provides a novel and robust model for investigating multiple pathological hallmarks, including osteophytogenesis, cartilage damage, synovitis and OA-related pain.

Moderate exercise protects against joint disease in a murine model of osteoarthritis.

Exercise is recommended as a non-pharmacological therapy for osteoarthritis (OA). Various exercise regimes, with differing intensities and duration, have been used in a range of OA rodent models. These studies show gentle or moderate exercise reduces the severity of OA parameters while high intensity load bearing exercise is detrimental. However, these studies were largely conducted in rats or in mouse models induced by severe injury, age or obesity, whilst destabilization of the medial meniscus (DMM) in mice has become a widely accepted model due to its lower variability, moderate progression and timescale. The present study was undertaken to provide insight into the effect of moderate exercise on early joint pathology in the DMM mouse model. Exercise was induced a week after induction by forced wheel walking for three or 7 weeks. Joints were analyzed by microcomputed tomography and histology. Assessment of skeletal parameters revealed that exercise offered protection against cartilage damage after 7 weeks of exercise, and a temporary protection against osteosclerosis was displayed after 3 weeks of exercise. Furthermore, exercise modified the metaphyseal trabecular microarchitecture of the osteoarthritic leg in both time points examined. Collectively, our findings corroborate previous studies showing that exercise has an important effect on bone in OA, which subsequently, at 8 weeks post-induction, translates into less cartilage damage. Thus, providing an exercise protocol in a surgical mouse model of OA, which can be used in the future to further dissect the mechanisms by which moderate exercise ameliorates OA.

Cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity.

Interferon-gamma (IFN-gamma) has been shown to inhibit interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) stimulated bone resorption by strongly stimulating nitric oxide (NO) synthesis. Here we studied the mechanisms underlying this inhibition. Osteoclasts were generated in 10-day cocultures of mouse osteoblasts and bone marrow cells and the effect of cytokine-induced NO on osteoclast formation and activity was determined. Stimulation of the cocultures with IL-1 beta, TNF-alpha and IFN-gamma markedly enhanced NO production by 50- to 70-fold, and this was found to be derived predominantly from the osteoblast cell layer. When high levels of NO were induced by cytokines during early stages of the cocultures, osteoclast formation was virtually abolished and bone resorption markedly inhibited. Cytokine stimulation during the latter stages of coculture also resulted in inhibition of bone resorption, but here the effects were mainly due to an inhibitory effect on osteoclast activity. At all stages, however, the inhibitory effects of cytokines on osteoclast formation and activity were blocked by the NO-synthase inhibitor L-NMMA. Further investigations suggested that the NO-mediated inhibition of osteoclast formation was due in part to apoptosis of osteoclast progenitors. Cytokine stimulation during the early stage of the culture caused a large increase in apoptosis of bone marrow cells, and these effects were blocked by L-NMMA and enhanced by NO donors. We found no evidence of apoptosis in osteoclasts exposed to high levels of cytokine-induced NO at any stage in the culture, however, or of apoptosis affecting mature osteoclasts exposed to high levels of NO, suggesting that immature cells in the bone marrow compartment are most sensitive to NO-induced apoptosis. In summary, these studies identify NO as a potentially important osteoblast-osteoclast coupling factor which has potent inhibitory effects on bone resorption. These actions, in turn, are mediated by inhibition of osteoclast formation probably due to NO-induced apoptosis of osteoclast progenitors and by inhibition of the resorptive activity of mature osteoclasts.

Evidence for a pathogenic role of nitric oxide in inflammation-induced osteoporosis.

Inflammatory disease is associated with increased production of nitric oxide (NO) and activation of the inducible nitric oxide synthase (iNOS) pathway. Several studies have addressed the role of NO as a mediator of cytokine effects on bone cell activity in vitro. Stimulatory and inhibitory actions have been found, however, depending on the concentrations produced and model system used. In view of this, it has been difficult to predict whether increased production of NO during inflammation is likely to increase bone loss or prevent it. We have investigated the pathogenic role of NO in an animal model of inflammation-induced osteoporosis (IMO). NO production was increased in IMO when compared with controls (+344%; p < 0.01), and this was accompanied by activation of inducible NOS (iNOS) in the bone marrow space. Bone mineral density (BMD) was reduced in IMO when compared with controls (-64%; p < 0.01), and this was found to be associated with reduced osteoblast numbers (-44%; p < 0.05) and increased osteoclast numbers (+38%; p < 0.01). The NOS inhibitor L-NMMA reversed the deleterious effects of IMO on bone mass and bone turnover, but L-NMMA had no effect on bone mass in control animals. This study has important implications for many inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, and inflammatory bowel disease which are associated with increased NO production and osteoporosis. Our data not only suggest that iNOS activation and increased NO production contribute to the pathogenesis of osteoporosis in these situations, but also suggest that NOS inhibitors could be of therapeutic value in the prevention and treatment of such bone loss.

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling.

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.

The flurbiprofen derivatives HCT1026 and HCT1027 inhibit bone resorption by a mechanism independent of COX inhibition and nitric oxide production.

Prostaglandins and nitric oxide both modulate bone resorption and bone formation. We previously reported that a nitrosylated derivative of flurbiprofen, termed HCT1026, exerted inhibitory effects on osteoclastic bone resorption, which could not be reproduced by combining the parent compound with nitric oxide (NO) donors. The aim of this study was to investigate the mechanism by which HCT1026 inhibits bone resorption. We compared the effects of flurbiprofen and HCT1026 on osteoclast and osteoblast activity with those of HCT1027--an analogue of HCT1026, which lacks an NO-donating moiety. We found that HCT1026 and HCT1027 inhibited bone resorption in interleukin (IL)-1-stimulated murine osteoblast-bone marrow cocultures, with half-maximal effects (IC50) at 20 +/- 5 microM for HCT1026 and 25 +/- 6 microM for HCT1027 compared with 399 +/- 25 microM for flurbiprofen (P < 0.0001). These differences were unrelated to cyclooxygenase (COX) inhibition since HCT1026 and HCT1027 were about seven to eight times less potent than flurbiprofen at inhibiting COX-1 activity and half as potent at inhibiting COX-2 activity. Further studies showed that HCT1026 and HCT1027 activated caspase-3 in rabbit osteoclasts and promoted osteoclast apoptosis, as assessed by nuclear morphology and TUNEL assays. We conclude that HCT1026 and HCT1027 inhibit osteoclast formation and activity by a mechanism that is independent of NO production and COX inhibition. This raises the possibility that both compounds interact with a novel molecular target expressed on osteoclasts to promote apoptosis and inhibit bone resorption. This demonstrates that HCT1026 and derivatives could represent a novel class of antiresorptive drugs with therapeutic value in the treatment of bone diseases associated with accelerated bone loss due to osteoclast activation.

A new method for the quantification of aortic calcification by three-dimensional micro-computed tomography.

To gain a better understanding of the mechanisms that underpin aortic calcification, rodent models have been previously utilised. Regions of calcium and phosphate deposition are commonly visualised using labor-intensive two-dimensional histomorphometric techniques. In this study, we developed a novel micro-computed tomography (µCT) imaging protocol to quantify calcification in vascular tissues using high resolution three-dimensional (3D) reconstructions of aortae derived from the well-established Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 knockout (Enpp1-/-) mouse model of medial aortic calcification. A dual-contrast method was employed for specimen preparation and the application of corn oil as a submersion medium for the samples during scanning, which allowed the definition and quantification of soft tissue. 3D µCT was utilised to produce reconstructions of calcified and non-calcified aortae. A highly accurate quantification of a standardized region of calcium deposition was undertaken on these reconstructions. An excellent correlation between images obtained from µCT and those obtained with Alizarin red staining, of whole aortae for calcium deposition, was observed. This imaging protocol provides a powerful tool for studying the development of aortic calcification and potential therapeutic approaches for clinical intervention.

Nitric oxide and bone.

Nitric oxide (NO) is a free radical which has important effects on bone cell function. The endothelial isoform of nitric oxide synthase (eNOS) is widely expressed in bone on a constitutive basis, whereas inducible NOS is only expressed in response to inflammatory stimuli. It is currently unclear whether neuronal NOS is expressed by bone cells. Pro-inflammatory cytokines such as IL-1 and TNF cause activation of the iNOS pathway in bone cells and NO derived from this pathway potentiates cytokine and inflammation induced bone loss. These actions of NO are relevant to the pathogenesis of osteoporosis in inflammatory diseases such as rheumatoid arthritis, which are characterized by increased NO production and cytokine activation. Interferon gamma is a particularly potent stimulator of NO production when combined with other cytokines, causing very high concentrations of NO to be produced. These high levels of NO inhibit bone resorption and formation and may act to suppress bone turnover in severe inflammation. The eNOS isoform seems to play a key role in regulating osteoblast activity and bone formation since eNOS knockout mice have osteoporosis due to defective bone formation. Other studies have indicated that the NO derived from the eNOS pathway acts as a mediator of the effects of oestrogen in bone. eNOS also mediates the effects of mechanical loading on the skeleton where it acts along with prostaglandins, to promote bone formation and suppress bone resorption. Pharmacological NO donors have been shown to increase bone mass in experimental animals and preliminary evidence suggests that these agents may also influence bone turnover in man. These data indicate that the L-arginine/NO pathway represents a novel target for therapeutic intervention in the prevention and treatment of bone diseases.

Nitric oxide is a mediator of apoptosis in the rheumatoid joint.

OBJECTIVE: To study the role of nitric oxide (NO) derived from the inducible nitric oxide synthase (iNOS) pathway in the induction of apoptosis in the rheumatoid joint. METHODS: Joint tissue was obtained from four rheumatoid arthritis (RA) patients, three osteoarthritis patients and two patients with a fractured neck of the femur (NOF#), and apoptotic cells were identified in cryosections using the TUNEL (terminal dUTP nick end labelling) assay. Expression of iNOS was determined using immunohistochemistry. NO synthesis and the effect of NOS inhibitors on apoptosis levels were studied in explant cultures of RA cartilage and synovium. RESULTS: Numbers of apoptotic cells were greatly increased in rheumatoid synovium and articular cartilage compared with NOF# and osteoarthritic synovium. Immunohistochemistry showed co-localization of iNOS staining and apoptosis in the synovial lining layer and articular cartilage. The NOS inhibitor L-NMMA (L-N(G)-monomethylarginine) strongly inhibited apoptosis in explant cultures of synovium and cartilage, and this was reversed by the NO donor S-nitroso-acetyl-penicillamine. CONCLUSION: This study indicates that NO acts as a mediator of apoptosis in RA and suggests that NOS inhibitors reverse this process.

Stem cell factor stimulates chicken osteoclast activity in vitro.

Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally differentiated osteoclasts. Osteoclast formation was analyzed in long-term cocultures of osteoblasts and nonadherent, osteoclast-depleted bone marrow cells. Osteoclast activity was studied in short-term (48 h) cultures of bone marrow cell populations enriched for osteoclasts, on dentine slices. SCF strongly enhanced osteoclast differentiation. The IL-6-related chicken myelomonocytic growth factor (cMGF) had a similar effect, and the effects of SCF and cMGF were additive. SCF, but not cMGF, also stimulated the bone-resorbing activity of existing osteoclasts. As osteoblasts have been found to regulate osteoclast activity and formation, chicken osteoblasts were tested for their ability to express and secrete SCF. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that osteoblasts express SCF mRNA and that parathyroid hormone increases expression levels about fourfold. SCF did not accumulate in the culture medium, but remained cell (osteoblasts) surface associated.

Induction of osteoclast characteristics in cultured avian blood monocytes; modulation by osteoblasts and 1,25-(OH)2 vitamin D3.

It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

Activation of the inducible nitric oxide synthase pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis.

OBJECTIVE: Osteoporosis is a major clinical problem in chronic inflammatory diseases such as rheumatoid arthritis. The mechanism of bone loss in this condition remains unclear, but previous studies have indicated that depressed bone formation plays a causal role. Since cytokine-induced nitric oxide (NO) production has been shown to inhibit osteoblast growth and differentiation in vitro, this study was undertaken to investigate the role of the inducible NO synthase (iNOS) pathway in the pathogenesis of inflammation-mediated osteoporosis (IMO) by studying mice with targeted inactivation of the iNOS gene (iNOS knockout [iNOS KO] mice). METHODS: IMO was induced in wild-type (WT) and iNOS KO mice by subcutaneous injections of magnesium silicate. The skeletal response was assessed at the tibial metaphysis by measurements of bone mineral density (BMD), using peripheral quantitative computed tomography, by bone histomorphometry, and by measurements of bone cell apoptosis. RESULTS: NO production increased 2.5-fold (P < 0.005) in WT mice with IMO, but did not change significantly in iNOS KO mice. Total BMD values decreased by a mean +/- SEM of 14.4+/-2.0% in WT mice with IMO, compared with a decrease of 8.6+/-1.2% in iNOS KO mice with IMO (P < 0.01). Histomorphometric analysis confirmed that trabecular bone volume was lower in WT mice with IMO compared with iNOS KO mice with IMO (16.2+/-1.5% versus 23.4+/-2.6%; P < 0.05) and showed that IMO was associated with reduced bone formation and a 320% increase in osteoblast apoptosis (P < 0.005) in WT mice. In contrast, iNOS KO mice with IMO showed less inhibition of bone formation than WT mice and showed no significant increase in osteoblast apoptosis. CONCLUSION: Inducible NOS-mediated osteoblast apoptosis and depressed bone formation play important roles in the pathogenesis of IMO.

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption.

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFkappaB and in NFkappaB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFkappaB in osteoclast precursors.

A quantitative analysis of the effect of insulin-like growth factor-1 infusion during mandibular distraction osteogenesis in rabbits.

AIM: To quantify, by histomorphometry, the effects of local insulin-like growth factor-1 (IGF-1) during mandibular distraction at various rates. METHODOLOGY: Mature rabbits underwent bilateral mandibular corticotomy and distraction lengthening. Recombinant IGF-1 was administered to two groups of rabbits via osmotic infusion pumps. Distraction regimes were as follows: Group A, 1 mm/day for 15 days; Group B, as for A plus IGF-1; Group C, 3 mm/day for 5 days; Group D, as for C plus IGF-1; and Group E, sham-operated controls. After a 28-day consolidation period, rabbits were sacrificed and bone deposition quantified using DEXA scanning, three-point bending, histological examination and sampled for histomorphometric analysis. RESULTS: DEXA scanning and three-point bending failed to detect any effect of distraction rate or of IGF-1 infusion. Histological and histomorphometric analysis suggested 1 mm/day to be the ideal distraction rate, as this was associated with greater osteoblastic activity and consistent bony union. However, IGF-1 infusion significantly enhanced osteoblastic activity at both distraction rates and resulted in bony union when distraction was performed at 3 mm/day. CONCLUSIONS: Distraction osteogenesis at a rate of 1 mm/day provides greater osteogenic stimulus than 3 mm/day. Exogenous IGF-1 has a positive influence on osteoblastic activity during distraction. Its effect is probably minimised by high levels of endogenous IGF-1.

Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis.

Nitric oxide has been implicated as a mediator of inflammatory arthritis, and recent work has shown that pro-inflammatory cytokines stimulate NO production in vitro by activation of the inducible nitric oxide synthase (iNOS) pathway. In order to identify the cellular sources of NO production within the joint, we have used immunohistochemical techniques to study the distribution of iNOS in synovium and cartilage from normal and diseased joints. iNOS was most strongly expressed in the synovial lining layer, subsynovium, vascular smooth muscle and chondrocytes from patients with rheumatoid arthritis (RA). Analysis of serial sections, coupled with double immunofluorescent staining, showed that the CD68+ macrophages in the synovial lining layer and, to a lesser extent, fibroblasts were the predominant source of iNOS within synovium, whereas T cells, B cells and neutrophils were negative. A similar pattern of iNOS staining was seen in osteoarthritis, but fewer cells were iNOS positive and the intensity of staining, particularly in cartilage, was much weaker than in RA. In contrast, no evidence of iNOS was detected in non-inflammatory synovium or in cartilage derived from normal joints (fractured neck of femur). In conclusion, these data support the hypothesis that synovium and cartilage are important sources of increased NO production in patients with inflammatory arthritis. Localization of iNOS at these sites within the inflamed joint raises the possibility that increased local production of NO may contribute to the pathogenesis of inflammatory arthritis by increasing synovial blood flow and by modulating cellular function within synovium and articular cartilage.

Inhibition of bone resorption in vitro and prevention of ovariectomy-induced bone loss in vivo by flurbiprofen nitroxybutylester (HCT1026).

OBJECTIVE: Inhibitors of prostaglandin production, such as nonsteroidal antiinflammatory drugs (NSAIDs), and pharmacologic nitric oxide (NO) donors, such as organic nitrates, have been suggested to protect against bone loss in both humans and experimental animals. Recently, a new class of nitrosylated NSAID (known as NO-NSAIDs) has been developed, which combines the properties of a NO donor with those of a cyclooxygenase (COX) inhibitor. This study investigated the effects of one of these compounds, flurbiprofen nitroxybutylester (HCT1026), on bone metabolism in vitro and in vivo. METHODS: The effects of HCT1026 on osteoclast formation and resorption were determined in vitro using cocultures of primary mouse osteoblasts and osteoclasts. The effect of HCT1026 in vivo was assessed using a mouse model of ovariectomy-induced bone loss. RESULTS: HCT1026 was significantly more efficacious than the parent compound, flurbiprofen, at inhibiting osteoclast formation and bone resorption in vitro, and these effects could not be reproduced by combinations of flurbiprofen with a variety of NO donors. Studies in vivo showed that HCT1026 protected against ovariectomy-induced bone loss by inhibiting osteoclastic bone resorption, whereas flurbiprofen at similar concentrations was ineffective. CONCLUSION: These data indicate that HCT1026 is a potent inhibitor of bone resorption in vitro and protects against ovariectomy-induced bone loss in vivo by a novel mechanism that appears to be distinct from its NO donor properties and from its inhibitory effects on COX activity. We conclude that HCT1026 may be of clinical value in the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, which are characterized by joint inflammation as well as periarticular and systemic bone loss.