Fracture risk based on HR-pQCT measures does not vary with age in older adults - the bone microarchitecture international consortium (BoMIC) prospective cohort study.

Fracture risk increases with lower areal BMD (aBMD); however, aBMD-related estimate of risk may decrease with age. This may depend on technical limitations of 2-dimensional (2D) DXA which are reduced with 3D high-resolution peripheral quantitative computed tomography (HR-pQCT). Our aim was to examine whether the predictive utility of HR-pQCT measures with fracture varies with age. We analyzed associations of HR-pQCT measures at the distal radius and distal tibia with two outcomes: incident fractures and major osteoporotic fractures. We censored follow-up time at first fracture, death, last contact or 8 years after baseline. We estimated hazard ratios (HR) and 95%CI for the association between bone traits and fracture incidence across age quintiles. Among 6835 men and women (ages 40-96) with at least one valid baseline HR-pQCT scan who were followed prospectively for a median of 48.3 months, 681 sustained fractures. After adjustment for confounders, bone parameters at both the radius and tibia were associated with higher fracture risk. The estimated HRs for fracture did not vary significantly across age quintiles for any HR-pQCT parameter measured at either the radius or tibia. In this large cohort, the homogeneity of the associations between the HR-pQCT measures and fracture risk across age groups persisted for all fractures and for major osteoporotic fractures. The patterns were similar regardless of the HR-pQCT measure, the type of fracture, or the statistical models. The stability of the associations between HR-pQCT measures and fracture over a broad age range shows that bone deficits or low volumetric density remain major determinants of fracture risk regardless of age group. The lower risk for fractures across measures of aBMD in older adults in other studies may be related to factors which interfere with DXA but not with HR-pQCT measures.

Prevalence of bone complications in young patients with sickle cell disease presenting low bone mineral density.

PURPOSE: Bone fragility in sickle cell disease (SCD) has been previously reported even in young patients, but the clinical consequences and specific management remain unclear. The objective of this study was to assess the prevalence of bone fragility in sickle cell patients and to evaluate the potential risk factors and associated complications. METHODS: We conducted a single-center cross-sectional study. Bone mineral densitometry (BMD) at the lumbar spine and the hip, Vertebral Fracture Assessment (VFA) and biological measurements were performed in patients aged between 20 and 40 years. RESULTS: One hundred and thirty-eight patients with sickle cell disease were included between June 2020 and December 2021. One hundred and one patients (73.2 %) were from Sub-Saharan Africa, 13 from North Africa (9.4 %), 11 from the Caribbean (7.9 %), 6 from the Indian Ocean. A Z-score < -2 was found in 43 patients (31.2 %) at the lumbar spine, in 4 patients (3 %) at the total hip, and in 5 patients (3.7 %) at the femoral neck. 59 patients (46.8 %) had vertebral deformities. Fragility fractures were recorded in 9 patients (10.8 %). Patients with low BMD had lower BMI (21.3 (19.0, 24.0) versus 24.0 (20.7, 26.1) Kg/m2, p = 0.003), lower osteonecrosis history (7 % versus 25.3 %, p = 0.011) and lower hemoglobin levels (9.0 (8.0, 10.0) versus 10.0 (9.0, 11.0) g/dL, p < 0.01). No association was found between history of fracture and low BMD. CONCLUSION: Young patients with SCD commonly have low BMD at the lumbar spine, but the prevalence of fragility fracture was low. Low BMD - specifically at the spine - may not be tantamount to bone fragility.

Quality of life in sarcopenia measured with the SarQoL questionnaire: A meta-analysis of individual patient data.

Age-related sarcopenia, resulting from a gradual loss in skeletal muscle mass and strength, is pivotal to the increased prevalence of functional limitation among the older adult community. The purpose of this meta-analysis of individual patient data is to investigate the difference in health-related quality of life between sarcopenic individuals and those without the condition using the Sarcopenia Quality of Life (SarQoL) questionnaire. A protocol was published on PROSPERO. Multiple databases and the grey literature were searched until March 2023 for studies reporting quality of life assessed with the SarQoL for patients with and without sarcopenia. Two researchers conducted the systematic review independently. A two-stage meta-analysis was performed. First, crude (mean difference) and adjusted (beta coefficient) effect sizes were calculated within each database; then, a random effect meta-analysis was applied to pool them. Heterogeneity was measured using the Q-test and I2 value. Subgroup analyses were performed to investigate the source of potential heterogeneity. The strength of evidence of this association was assessed using GRADE. From the 413 studies identified, 32 were eventually included, of which 10 were unpublished data studies. Sarcopenic participants displayed significantly reduced health-related quality of life compared with non-sarcopenic individuals (mean difference = -12.32; 95 % CI = [-15.27; -9.37]). The model revealed significant heterogeneity. Subgroup analyses revealed a substantial impact of regions, clinical settings, and diagnostic criteria on the difference in health-related quality of life between sarcopenic and non-sarcopenic individuals. The level of evidence was moderate. This meta-analysis of individual patient data suggested that sarcopenia is associated with lower health-related quality of life measured with SarQoL.

Dysregulation of MicroRNAs in Adult Osteogenesis Imperfecta: The miROI Study.

As epigenetic regulators of gene expression, circulating micro-RiboNucleic Acids (miRNAs) have been described in several bone diseases as potential prognostic markers. The aim of our study was to identify circulating miRNAs potentially associated with the severity of osteogenesis imperfecta (OI) in three steps. We have screened by RNA sequencing for the miRNAs that were differentially expressed in sera of a small group of OI patients versus controls and then conducted a validation phase by RT-qPCR analysis of sera of a larger patient population. In the first phase of miROI, we found 79 miRNAs that were significantly differentially expressed. We therefore selected 19 of them as the most relevant. In the second phase, we were able to validate the significant overexpression of 8 miRNAs in the larger OI group. Finally, we looked for a relationship between the level of variation of the validated miRNAs and the clinical characteristics of OI. We found a significant difference in the expression of two microRNAs in those patients with dentinogenesis imperfecta. After reviewing the literature, we found 6 of the 8 miRNAs already known to have a direct action on bone homeostasis. Furthermore, the use of a miRNA-gene interaction prediction model revealed a 100% probability of interaction between 2 of the 8 confirmed miRNAs and COL1A1 and/or COL1A2. This is the first study to establish the miRNA signature in OI, showing a significant modification of miRNA expression potentially involved in the regulation of genes involved in the physiopathology of OI. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A Fracture Risk Assessment Tool for High Resolution Peripheral Quantitative Computed Tomography.

Most fracture risk assessment tools use clinical risk factors combined with bone mineral density (BMD) to improve assessment of osteoporosis; however, stratifying fracture risk remains challenging. This study developed a fracture risk assessment tool that uses information about volumetric bone density and three-dimensional structure, obtained using high-resolution peripheral quantitative compute tomography (HR-pQCT), to provide an alternative approach for patient-specific assessment of fracture risk. Using an international prospective cohort of older adults (n = 6802) we developed a tool to predict osteoporotic fracture risk, called µFRAC. The model was constructed using random survival forests, and input predictors included HR-pQCT parameters summarizing BMD and microarchitecture alongside clinical risk factors (sex, age, height, weight, and prior adulthood fracture) and femoral neck areal BMD (FN aBMD). The performance of µFRAC was compared to the Fracture Risk Assessment Tool (FRAX) and a reference model built using FN aBMD and clinical covariates. µFRAC was predictive of osteoporotic fracture (c-index = 0.673, p < 0.001), modestly outperforming FRAX and FN aBMD models (c-index = 0.617 and 0.636, respectively). Removal of FN aBMD and all clinical risk factors, except age, from µFRAC did not significantly impact its performance when estimating 5-year and 10-year fracture risk. The performance of µFRAC improved when only major osteoporotic fractures were considered (c-index = 0.733, p < 0.001). We developed a personalized fracture risk assessment tool based on HR-pQCT that may provide an alternative approach to current clinical methods by leveraging direct measures of bone density and structure. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Spondyloarthritis and Sarcopenia: Prevalence of Probable Sarcopenia and its Impact on Disease Burden: The Saspar Study.

To evaluate the prevalence of probable, confirmed, and severe sarcopenia in spondyloarthritis (SpA), according to the European Working Group on Sarcopenia in Older People 2019 (EWGSOP2) definition. A total of 103 patients (51% women) with SpA, mean age 47.1 ± 13.7 years, were included and compared to 103 age- and sex-matched controls. Grip strength was measured by dynamometry. Body composition was assessed by whole-body densitometry. In SpA patients gait speed was measured by the 4-m-distance walk test and quality of life was evaluated with a specific health-related questionnaire for sarcopenia (SaRQoL®). Twenty-two SpA patients (21%) versus 7 controls (7%) had a low grip strength, i.e., probable sarcopenia (p < 0.01), 15 SpA (15%) patients and 7 controls (7%) had low Skeletal Muscle mass Index (SMI) (ns), respectively, and 5 and 2% of SpA patients and controls had low grip strength and low SMI, i.e., confirmed sarcopenia (ns). All the sarcopenic SpA patients had a low gait speed, i.e., severe sarcopenia. Finally, probable sarcopenic SpA patients had significantly higher C-Reactive Protein (CRP, p < 0.001) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI score, p < 0.01), lower gait speed (p < 0.001), and SarQoL® score (p < 0.001) than SpA patients with normal grip strength. According to EWGSOP2 definition, the prevalence of probable sarcopenia was significantly higher in SpA patients compared to controls. Probable sarcopenia was associated with higher inflammation and disease activity, impaired muscle performance, and quality of life. These results suggest that muscle strength may be a salient hallmark in SpA.

Bone Microarchitecture Phenotypes Identified in Older Adults Are Associated With Different Levels of Osteoporotic Fracture Risk.

Prevalence of osteoporosis is more than 50% in older adults, yet current clinical methods for diagnosis that rely on areal bone mineral density (aBMD) fail to detect most individuals who have a fragility fracture. Bone fragility can manifest in different forms, and a "one-size-fits-all" approach to diagnosis and management of osteoporosis may not be suitable. High-resolution peripheral quantitative computed tomography (HR-pQCT) provides additive information by capturing information about volumetric density and microarchitecture, but interpretation is challenging because of the complex interactions between the numerous properties measured. In this study, we propose that there are common combinations of bone properties, referred to as phenotypes, that are predisposed to different levels of fracture risk. Using HR-pQCT data from a multinational cohort (n = 5873, 71% female) between 40 and 96 years of age, we employed fuzzy c-means clustering, an unsupervised machine-learning method, to identify phenotypes of bone microarchitecture. Three clusters were identified, and using partial correlation analysis of HR-pQCT parameters, we characterized the clusters as low density, low volume, and healthy bone phenotypes. Most males were associated with the healthy bone phenotype, whereas females were more often associated with the low volume or low density bone phenotypes. Each phenotype had a significantly different cumulative hazard of major osteoporotic fracture (MOF) and of any incident osteoporotic fracture (p < 0.05). After adjustment for covariates (cohort, sex, and age), the low density followed by the low volume phenotype had the highest association with MOF (hazard ratio = 2.96 and 2.35, respectively), and significant associations were maintained when additionally adjusted for femoral neck aBMD (hazard ratio = 1.69 and 1.90, respectively). Further, within each phenotype, different imaging biomarkers of fracture were identified. These findings suggest that osteoporotic fracture risk is associated with bone phenotypes that capture key features of bone deterioration that are not distinguishable by aBMD. © 2021 American Society for Bone and Mineral Research (ASBMR).

Development and Validation of a Predictive Model of Hypovitaminosis D in General Adult Population: SCOPYD Study.

The worldwide global increase in serum 25-hydroxyvitamin D (25(OH)D) measurements has led some countries to restrict reimbursement for certain clinical situations only. Another approach could consist in providing physicians with screening tools in order to better target blood test prescription. The objective of the SCOPYD study was to identify the best combination of predictors of serum VitD concentration among adults aged 18-70 years. Potential risk factors for VitD deficiency were collected using a comprehensive self-administered questionnaire. A multivariable linear regression was used to build a predictive model of serum 25(OH)D concentration. Among 2488 participants, 1080 (43.4%) had VitD deficiency (<50 nmol/L) and 195 (7.8%) had severe deficiency (<25 nmol/L). The final model included sunlight exposure in the preceding week and during the last holidays, month of blood sampling, age, sex, body mass index, skin phototype, employment, smoking, sport practice, latitude, and VitD supplementation in preceding year. The area under the curve was 0.82 (95% CI (0.78; 0.85)) for severe deficiency. The model predicted severe deficiency with a sensitivity of 77.9% (95% CI (69.1; 85.7)) and a specificity of 68.3% (95% CI (64.8; 71.9)). We identified a set of predictors of severe VitD deficiency that are easy to collect in routine that may help to better target patients for serum 25(OH)D concentration determination.

A Signature of Circulating miRNAs Associated With Fibrous Dysplasia of Bone: the mirDys Study.

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis-related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real-time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR-25-3p, miR-93-5p, miR-182-5p, miR-324-5p, miR-363-3p, and miR-451a) were significantly overexpressed in serum, with FDR <0.05. The expression level of these six miRNAs was not associated with the FD severity. In conclusion, we identified a signature of circulating miRNAs associated with FD. These miRNAs are potential negative regulators of gene expression in bone cell progenitors, suggesting their activity in FD by interfering with osteoblastic and osteoclastic differentiation to impair bone mineralization and remodeling processes. © 2020 American Society for Bone and Mineral Research.

Genetic testing is useful in adults with limited phenotypes of genetic skeletal conditions.

We show the value of genetic screening in 3 adults with limited phenotypes of three bone sclerosing genetic disease (GD): osteopetrosis (OPT), Camurati-Engelmann disease (CED) and pycnodysostosis. INTRODUCTION: OPT, CED and pycnodysostosis are three rare bone diseases often diagnosed in childhood. However, some atypical phenotypes raise the problem of delayed diagnosis in adults. Genetic tests may then be useful to establish a formal diagnosis. METHODS: We report 3 cases of adult patients with symptomatic or asymptomatic bone sclerosing lesions for whom the clinical, radiological and biological explorations were atypical and did not allow a formal diagnosis. These unusual descriptions led to the search for genetic mutations. RESULTS: These 3 cases of limited phenotypes were associated with unknown or poorly described variants of 3 rare bone genetic diseases. CONCLUSIONS: Genetic tests proved useful to establish the diagnosis and manage the condition of adults with rare bone sclerosing GD.

Osteoporosis prevention: Where are the barriers to improvement in French general practitioners? A qualitative study.

BACKGROUND: Osteoporosis prevention, diagnosis and treatment remain suboptimal. OBJECTIVES: We conducted a qualitative study to understand barriers towards care initiation and levers to improve awareness and management of osteoporosis among general practitioners (GPs). METHODS: Semi-structured face-to face interviews were conducted with 16 GPs in the Rhône area of France to explore their knowledge and representations regarding osteoporosis. A thematic analysis of transcripts was performed to identify GPs' perceptions on osteoporosis diagnosis, prevention, treatment and patients' expectations. RESULTS: Interviewed GPs considered osteoporosis far less important than other chronic diseases. They questioned whether osteoporosis was a disease or normal aspect of ageing. They associated osteoporosis with fragility fractures, female sex, menopause, and old age but rarely with male sex. They regarded bone mineral density as the reference diagnostic test, but certain GPs indicated that they had difficulties to interpret the results and to know when to prescribe. Biphosphonates were mentioned as the reference treatment but some GPs expressed distrust about osteoporosis medications. Most of them did not think to screen for osteoporosis risk factors in their patients in a preventive medical approach. They mentioned the lack of time to implement prevention and were expecting clear and pragmatic guidelines, as well as information campaigns in general population to increase awareness on osteoporosis. CONCLUSION: GPs tended to underestimate the salience of osteoporosis. Clear recommendations, better awareness of GPs and the general population could improve osteoporosis prevention and treatment.

Cortical and trabecular bone microarchitecture as an independent predictor of incident fracture risk in older women and men in the Bone Microarchitecture International Consortium (BoMIC): a prospective study.

BACKGROUND: Although areal bone mineral density (aBMD) assessed by dual-energy x-ray absorptiometry (DXA) is the clinical standard for determining fracture risk, most older adults who sustain a fracture have T scores greater than -2·5 and thus do not meet the clinical criteria for osteoporosis. Importantly, bone fragility is due to low BMD and deterioration in bone structure. We assessed whether indices of high-resolution peripheral quantitative CT (HR-pQCT) were associated with fracture risk independently of femoral neck aBMD and the Fracture Risk Assessment Tool (FRAX) score. METHODS: We assessed participants in eight cohorts from the USA (Framingham, Mayo Clinic), France (QUALYOR, STRAMBO, OFELY), Switzerland (GERICO), Canada (CaMos), and Sweden (MrOS). We used Cox proportional hazard ratios (HRs) to estimate the association between HR-pQCT bone indices (per 1 SD of deficit) and incident fracture, adjusting for age, sex, height, weight, and cohort, and then additionally for femoral neck DXA aBMD or FRAX. FINDINGS: 7254 individuals (66% women and 34% men) were assessed. Mean baseline age was 69 years (SD 9, range 40-96). Over a mean follow-up of 4·63 years (SD 2·41) years, 765 (11%) participants had incident fractures, of whom 633 (86%) had femoral neck T scores greater than -2·5. After adjustment for age, sex, cohort, height, and weight, peripheral skeleton failure load had the greatest association with risk of fracture: tibia HR 2·40 (95% CI 1·98-2·91) and radius 2·13 (1·77-2·56) per 1 SD decrease. HRs for other bone indices ranged from 1·12 (95% CI 1·03-1·23) per 1 SD increase in tibia cortical porosity to 1·58 (1·45-1·72) per 1 SD decrease in radius trabecular volumetric bone density. After further adjustment for femoral neck aBMD or FRAX score, the associations were reduced but remained significant for most bone parameters. A model including cortical volumetric bone density, trabecular number, and trabecular thickness at the distal radius and a model including these indices plus cortical area at the tibia were the best predictors of fracture. INTERPRETATION: HR-pQCT indices and failure load improved prediction of fracture beyond femoral neck aBMD or FRAX scores alone. Our findings from a large international cohort of men and women support previous reports that deficits in trabecular and cortical bone density and structure independently contribute to fracture risk. These measurements and morphological assessment of the peripheral skeleton might improve identification of people at the highest risk of fracture. FUNDING: National Institutes of Health National Institute of Arthritis Musculoskeletal and Skin Diseases.

The QUALYOR (QUalité Osseuse LYon Orléans) study: a new cohort for non invasive evaluation of bone quality in postmenopausal osteoporosis. Rationale and study design.

The diagnostic performance of densitometry is inadequate. New techniques of non-invasive evaluation of bone quality may improve fracture risk prediction. Testing the value of these techniques is the goal of the QUALYOR cohort. INTRODUCTION: The bone mineral density (BMD) of postmenopausal women who sustain osteoporotic fracture is generally above the World Health Organization definition for osteoporosis. Therefore, new approaches to improve the detection of women at high risk for fracture are warranted. METHODS: We have designed and recruited a new cohort to assess the predictive value of several techniques to assess bone quality, including high-resolution peripheral quantitative computerized tomography (HRpQCT), hip QCT, calcaneus texture analysis, and biochemical markers. We have enrolled 1575 postmenopausal women, aged at least 50, with an areal BMD femoral neck or lumbar spine T-score between - 1.0 and - 3.0. Clinical risk factors for fracture have been collected along with serum and blood samples. RESULTS: We describe the design of the QUALYOR study. Among these 1575 women, 80% were aged at least 60. The mean femoral neck T-score was - 1.6 and the mean lumbar spine T-score was -1.2. This cohort is currently being followed up. CONCLUSIONS: QUALYOR will provide important information on the relationship between bone quality variables and fracture risk in women with moderately decreased BMD.

Periostin and transforming growth factor ß-induced protein (TGFßIp) are both expressed by osteoblasts and osteoclasts.

Periostin (Postn) and transforming growth factor ß-induced protein (TGFßIp) are two closely related extracellular matrix (ECM) proteins predominantly distributed in collagen-rich connective tissues submitted to mechanical strain, including bone and more specifically the periosteum. We have investigated the expression of Postn and TGFßIp mRNA by primary osteoblasts isolated from mouse periosteum and calvaria, or by the osteoblast-like MC3T3-E1 cell line, and by osteoclasts from mouse long bones differentiated in vitro. Secretion of Postn was measured with a specific ELISA. Postn and TGFßIp mRNA were concomitantly expressed in all three osteoblast models all along the differentiation process in a time-dependent manner. Both Postn and TGFßIp transcripts appeared early in osteoblast differentiation, and their expression increased 3-10 times in mature osteoblast cells. Expression decreased after differentiation was achieved and when the cultures mineralised. ELISA for secreted Postn showed a similar pattern. When MC3T3-E1 cells were treated with TGF-ß, Postn and TGFßIp mRNA expression and secretion were stimulated, whereas 1.25(OH)(2)D(3) had no detectable effect. Osteoclasts also expressed both Postn and TGFßIp during in vitro differentiation. Expression of both Postn and TGFßIp peaks in the early phases of osteoblast differentiation, and decreases later at the start of mineralisation. A novel finding is that Postn and TGFßIp are expressed by osteoclasts in vitro. Therefore Postn and TGFßIp proteins are potential biomarkers of early osteoblast differentiation and new bone formation.

Non-enzymatic glycation of bone collagen modifies osteoclastic activity and differentiation.

Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.

NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis.

We previously identified functional N-methyl-D-aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and Rank ligand (RankL). Using RT-PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi-quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose-dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western-blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF-kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF-kappa B pathway.