A role for G protein-coupled receptor 137b in bone remodeling in mouse and zebrafish.

G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba-deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba-/- mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba-deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba-/- fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation.

Clcn7F318L/+ as a new mouse model of Albers-Schönberg disease.

Dominant negative mutations in CLCN7, which encodes a homodimeric chloride channel needed for matrix acidification by osteoclasts, cause Albers-Schönberg disease (also known as autosomal dominant osteopetrosis type 2). More than 25 different CLCN7 mutations have been identified in patients affected with Albers-Schönberg disease, but only one mutation (Clcn7G213R) has been introduced in mice to create an animal model of this disease. Here we describe a mouse with a different osteopetrosis-causing mutation (Clcn7F318L). Compared to Clcn7+/+ mice, 12-week-old Clcn7F318L/+ mice have significantly increased trabecular bone volume, consistent with Clcn7F318L acting as a dominant negative mutation. Clcn7F318L/F318L and Clcn7F318L/G213R mice die by 1month of age and resemble Clcn7 knockout mice, which indicate that p.F318L mutant protein is non-functional and p.F318L and p.G213R mutant proteins do not complement one another. Since it has been reported that treatment with interferon gamma (IFN-G) improves bone properties in Clcn7G213R/+ mice, we treated Clcn7F318L/+ mice with IFN-G and observed a decrease in osteoclast number and mineral apposition rate, but no overall improvement in bone properties. Our results suggest that the benefits of IFN-G therapy in patients with Albers-Schönberg disease may be mutation-specific.

Bone histomorphometry revisited.

Bone histomorphometry is defined as a quantitative evaluation of bone micro architecture, remodelling and metabolism. Bone metabolic assessment is based on a dynamic process, which provides data on bone matrix formation rate by incorporating a tetracycline compound. In the static evaluation, samples are stained and a semi-automatic technique is applied in order to obtain bone microarchitectural parameters such as trabecular area, perimeter and width. These parameters are in 2D, but they can be extrapolated into 3D, applying a stereological formula. Histomorphometry can be applied to different areas; however, in recent decades it has been a relevant tool in monitoring the effect of drug administration in bone. The main challenge for the future will be the development of noninvasive methods that can give similar information. In the herein review paper we will discuss the general principles and main applications of bone histomorphometry.

Evaluation of bone mechanical strenght and fracture risk assessment (Frax) in patients with hip joint replacement surgery.

BACKGROUND: Fracture risk assessment tools are useful to calculate the long term probability of osteoporotic fracture. However, how it reflects bone quality is unknown. The aim of this study was to correlate the WHO clinical fracture risk assessment tool, FRAX, with bone mechanical properties. METHODS: Six patients submitted to hip replacement surgery, either due to osteoporotic fractures or to osteoarthritis, were evaluated. Bone samples were collected and the mechanical properties assessed by compression tests. Patients' data regarding the presence of clinical risk factors for fracture were registered. Laboratorial assessment of bone metabolic parameters and a dual X-ray absorptiometry(DXA) were done. RESULTS: Analysis of the load-displacement curves showed that patients with fragility fractures (n=4) had low values of elastic modulus, yield load and energy absorbed until yield point. Osteoarthritis patients tend to have a better biomechanical performance.Femoral neck DXA scan was also performed in 3 patients. Fragility fracture patients had a lower bone mineral density than the patients with osteoarthritis. FRAX algorithm was applied and a positive relation was found between FRAX results and biomechanical parameters. Blood bone metabolic markers were within the normal range for all the subjects. CONCLUSIONS: The worse mechanical properties observed in the fragility fracture patients were related to high probability of fracture given by FRAX. These observations, in a very small sample, need further confirmation. However, they suggest that the fracture risk assessment tool, FRAX, may reflect the current mechanical bone behavior of the patient.

An analysis of joint replacement in patients with systemic lupus erythematosus.

A small but important group of patients in our lupus cohort has needed total joint replacement (TJR). Arthritis was identified in 94% of our lupus patients. We have determined how many of our patients needed TJR, explored the risk factors for this procedure in patients with SLE and reviewed the outcome for these patients. Records of the cohort of patients with SLE who have attended our lupus clinic at University College of London Hospital/Middlesex from 1978 to 2008 were reviewed and patients who underwent TJR were identified. We recorded demographic data, other major systemic manifestations of SLE, autoantibody profile, previous use of steroids, other major systemic illnesses, smoking and alcohol habits. Nineteen patients with SLE from our cohort of 500 were found to have at least one TJR. Avascular necrosis (AVN) or concomitant rheumatoid arthritis (RA) was present in the majority of these patients. In contrast, age at disease onset, the presence of anti-cardiolipin antibodies, Raynaud's phenomenon and smoking habits were not found to be contributing factors for the need to replace joints. Four of our 19 patients (21.1%) had complications of the joint replacement: two of them had infections of the replaced joint, one had a large haematoma immediately after the surgery requiring surgical evacuation and the other had a deep vein thrombosis. None of the patients so far has required joint re-replacement. In conclusion, 4% of SLE patients in our cohort have one or more joints replaced, the majority because of AVN or RA.

Chronic arthritis directly induces quantitative and qualitative bone disturbances leading to compromised biomechanical properties.

OBJECTIVES: Rheumatoid arthritis (RA) is associated with an increased risk of fragility fractures. In RA patients, the direct effect of inflammation on bone is difficult to study because their skeleton is also affected by medication with corticosteroids and other drugs as well as aging and menopause, which contribute to bone fragility. This study used an animal model of chronic arthritis to evaluate the direct impact of chronic inflammation on biomechanical properties and structure of bone. METHODS: In the SKG mouse chronic arthritis model three point bending tests were performed on femoral bones and compression tests on vertebral bodies. Collagen structure was analysed using second-harmonic generation (SHG) imaging with a two-photon microscope, ultramorphology by scanning electron microscopy (SEM) coupled with energy dispersive x-ray spectroscopy (EDS) and bone density using water pycnometer. RESULTS: Arthritic bones had poor biomechanical quality compared to control bones. SHG, SEM and pycnometry disclosed variable signs of impaired collagen organization, poor trabecular architecture and low bone density. CONCLUSION: Present data demonstrate for the first time that chronic inflammation per se, without confounding influence of drugs and aging, leads to impairment of bone biomechanics in terms of stiffness, ductility and ultimate strength (fracture).

Biomechanical effects of inflammatory diseases on bone-rheumatoid arthritis as a paradigm.

Inflammatory diseases, such as rheumatoid arthritis (RA), influence the bone remodelling process and increase the risk of fracture. Bone can be viewed as a composite material comprising of two phases: the organic phase, constituted predominantly by collagen type I, and the mineral phase, composed primarily by calcium phosphate, in the form of mineral crystals. The mineral component confers bone with strength and stiffness while the organic phase is responsible for bone toughness and ductility and acts as a scaffold for the mineralisation process. The efficacy of bone as a structural material depends on the balance between these different bone components and their biomechanical properties. The main determinants of mechanical properties of bone are the amount of mineral, the collagen content, the orientation of the collagen fibers and minerals and the accumulation of microcracks in the bone matrix. In a mice model of arthritis mechanical testing has shown that arthritic femurs have a significantly lower Young's modulus, yield stress and work until ultimate stress. This evidence suggests that one of the major explanations for the increased fracture risk in RA is related to the changes on bone components induced by inflammation that result in compromised biomechanical properties.

Osteoimmunology--the hidden immune regulation of bone.

Osteoimmunology is an emerging field of research dedicated to the investigation of the interactions between the immune and skeletal systems. These interactions are not only mediated by the release of cytokines and chemokines but also by direct cell-cell contact. Recently, it was proposed that immunoreceptors found in the immune cells are also an essential signal for osteoclasts activation, along with receptor activator NF-kappaB (RANK) ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). In addition, adipose tissue also produces several factors (adipokines) that are known to interfere with the immune system and bone homeostasis. Chronic inflammation strongly influences osteoimmunology determining profound metabolic, structural and functional changes in bone.

Delayed neutrophil apoptosis in very early rheumatoid arthritis patients is abrogated by methotrexate therapy.

OBJECTIVES: To analyse the activation state and apoptosis of circulating neutrophils in untreated very early rheumatoid arthritis (VERA) and after exposure to low dose corticosteroids and methotrexate (MTX). METHODS: Neutrophils were isolated from the peripheral blood of VERA patients at 3 different times: before any treatment was started, 2 weeks after starting a low dose of prednisone (5-10 mg) and 4 months after reaching more than 20mg/week of MTX. The expression of different activation markers (CD11b, CD64, CD86 and CD69) in freshly isolated neutrophils was analysed by flow cytometry. Apoptosis was measured by the loss of DNA content, which was analysed by flow cytometry using propidium iodide. RESULTS: Compared to neutrophils from healthy controls, we have found a delayed neutrophil apoptosis within 6 h and 22 h of cultured polymorphonuclear leukocytes (PMN) derived from VERA patients without any treatment or treated with corticosteroids. The delay of PMN apoptosis was restored to control levels after treatment with MTX. CONCLUSION: The treatment of VERA patients with corticosteroids did not affect the delay of neutrophil apoptosis. However, delayed apoptosis was restored to control levels after treatment with low dose MTX, which highlights the importance of early RA treatment with MTX.