Expression of semaphorin-3A in the joint and role in osteoarthritis.

Osteoarthritis (OA) is characterised by the deterioration of cartilage in the joints and pain. We hypothesise that semaphorin-3A (sema-3A), a chemorepellent for sensory nerves, plays a role in joint degradation and pain. We used the mechanical joint loading (MJL) model of OA to investigate sema-3A expression in the joint and examine its association with the development of OA and pain. We also analyse its effect on chondrocyte differentiation using the ATDC5 cell line. We demonstrate that sema-3A is present in most tissues in the healthy joint and its expression increases in highly innervated tissues, such as cruciate ligaments, synovial lining and subchondral bone, in loaded compared to nonloaded control joints. In contrast, sema-3A expression in cartilage was decreased in the severe OA induced by the application of high loads. There was a significant increase in circulating sema-3A, 6 weeks after MJL compared to the nonloaded mice. mRNA for sema-3A and its receptor Plexin A1 were upregulated in the dorsal root ganglia of mice submitted to MJL. These increases were supressed by zoledronate, an inhibitor of bone pain. Sema-3A was expressed at all stages of Chondrocyte maturation and, when added exogenously, stimulated expression of markers of chondrocyte differentiation. This indicates that sema-3A could affect joint tissues distinctively during the development of OA. In highly innervated joint tissues, sema-3A could control innervation and/or induce pain-associated neuronal changes. In cartilage, sema-3A could favour its degeneration by modifying chondrocyte differentiation.

Unbiased gene expression analysis of the delayed fracture healing observed in Zucker diabetic fatty rats.

Aims: Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods: Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results: Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion: These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients.

Bone marrow lesions: plugging the holes in our knowledge using animal models.

Bone marrow lesions (BMLs), which are early signs of osteoarthritis (OA) that are associated with the presence, onset and severity of pain, represent an emerging imaging biomarker and clinical target. Little is known, however, regarding their early spatial and temporal development, structural relationships or aetiopathogenesis, because of the sparsity of human early OA imaging and paucity of relevant tissue samples. The use of animal models is a logical approach to fill the gaps in our knowledge, and it can be informed by appraising models in which BMLs and closely related subchondral cysts have already been reported, including in spontaneous OA and pain models. The utility of these models in OA research, their relevance to clinical BMLs and practical considerations for their optimal deployment can also inform medical and veterinary clinicians and researchers alike.

A new method for segmentation and analysis of bone callus in rodent fracture models using micro-CT.

Fracture burden has created a need to better understand bone repair processes under different pathophysiological states. Evaluation of structural and material properties of the mineralized callus, which is integral to restoring biomechanical stability is, therefore, vital. Microcomputed tomography (micro-CT) can facilitate noninvasive imaging of fracture repair, however, current methods for callus segmentation are only semiautomated, restricted to defined regions, time/labor intensive, and prone to user variation. Herein, we share a new automatic method for segmenting callus in micro-CT tomograms that will allow for objective, quantitative analysis of the bone fracture microarchitecture. Fractured and nonfractured mouse femurs were scanned and processed by both manual and automated segmentation of fracture callus from cortical bone after which microarchitectural parameters were analyzed. All segmentation and analysis steps were performed using CTAn (Bruker) with automatic segmentation performed using the software's image-processing plugins. Results showed automatic segmentation reliably and consistently segmented callus from cortical bone, demonstrating good agreement with manual methods with low bias: tissue volume (TV): -0.320 mm3 , bone volume (BV): 0.0358 mm3 , and bone volume/tissue volume (BV/TV): -3.52%, and was faster and eliminated user-bias and variation. Method scalability and translatability across rodent models were verified in scans of fractured rat femora showing good agreement with manual methods with low bias: TV: -3.654 mm3 , BV: 0.830 mm3 , and BV/TV: 7.81%. Together, these data validate a new automated method for segmentation of callus and cortical bone in micro-CT tomograms that we share as a fast, reliable, and less user-dependent tool for application to study bone callus in fracture, and potentially elsewhere.

What Did We Learn About Fracture Pain from Animal Models?

Progress in bone fracture repair research has been made possible due to the development of reproducible models of fracture in rodents with more clinically relevant fracture fixation, where there is considerably better assessment of the factors that affect fracture healing and/or novel therapeutics. However, chronic or persistent pain is one of the worst, longest-lasting and most difficult symptoms to manage after fracture repair, and an ongoing challenge remains for animal welfare as limited information exists regarding pain scoring and management in these rodent fracture models. This failure of adequate pre-clinical pain assessment following osteotomy in the rodent population may not only subject the animal to severe pain states but may also affect the outcome of the bone healing study. Animal models to study pain were also mainly developed in rodents, and there is increasing validation of fracture and pain models to quantitatively evaluate fracture pain and to study the factors that generate and maintain fracture pain and develop new therapies for treating fracture pain. This review aims to discuss the different animal models for fracture pain research and characterize what can be learned from using animal models of fracture regarding behavioral pain states and new molecular targets for future management of these behaviors.

Applied mechanical loading to mouse hindlimb acutely increases skeletal perfusion and chronically enhanced vascular porosity.

Blood supply is essential for osteogenesis, yet its relationship to load-related increases in bone mass is poorly defined. Herein, we aim to investigate the link between load-induced osteogenesis and the blood supply (bone perfusion and vascular porosity) using an established osteogenic noninvasive model of axial loading. Accordingly, 12 N mechanical loads were applied to the right tibiae of six male C57BL6 mice at 10-12 wk of age, 3 times/wk for 2 wk. Skeletal perfusion was measured acutely (postloading) and chronically in loaded and contralateral, nonloaded hindlimbs by laser-Doppler imaging. Vascular and lacunar porosity of the cortical bone and tibia load-related changes in trabecular and cortical bone was measured by nanoCT and micro-CT, respectively. We found that the mean skeletal perfusion (loaded: nonloaded limb ratio) increased by 56% immediately following the first loading episode (vs. baseline, P < 0.01), and a similar increase was observed after all loading episodes, demonstrating that these acute responses were conserved for 2 wk of loading. Loading failed, however, to engender any significant chronic changes in mean perfusion between the beginning and the end of the experiment. In contrast, 2 wk of loading engendered an increased vascular canal number in the tibial cortical compartment (midshaft) and, as expected, also increased trabecular and cortical bone volumes and modified tibial architecture in the loaded limb. Our results indicate that each episode of loading both generates acute enhancement in skeletal blood perfusion and also stimulates chronic vascular architectural changes in the bone cortices, which coincide with load-induced increases in bone mass.NEW & NOTEWORTHY This study investigated modifications to the blood supply (bone perfusion and intracortical vascular canals) in mechanoadaptive responses in C57BL6 mice. Each episode of mechanical loading acutely increases skeletal perfusion. Two weeks of mechanical loading increased bone mass and cortical vascular canal number, while there was no chronic increase in hindlimb perfusion. Our findings suggest that the blood supply may participate in the processes that govern load-induced bone formation.

Noninvasive Mechanical Joint Loading as an Alternative Model for Osteoarthritic Pain.

OBJECTIVE: Mechanisms responsible for osteoarthritic (OA) pain remain poorly understood, and current analgesic therapies are often insufficient. This study was undertaken to characterize and pharmacologically test the pain phenotype of a noninvasive mechanical joint loading model of OA, thus providing an alternative murine model for OA pain. METHODS: The right knees of 12-week-old male C57BL/6 mice were loaded at 9N or 11N (40 cycles, 3 times per week for 2 weeks). Behavioral measurements of limb disuse and mechanical and thermal hypersensitivity were acquired before mechanical joint loading and monitored for 6 weeks postloading. The severity of articular cartilage lesions was determined postmortem with the Osteoarthritis Research Society International scoring system. To assess efficacy of various treatments for pain, 9N-loaded mice were treated for 4 weeks with diclofenac (10 mg/kg), gabapentin (100 mg/kg), or anti-nerve growth factor (anti-NGF) (3 mg/kg). RESULTS: Mechanical hypersensitivity and weight bearing worsened significantly in 9N-loaded mice (n = 8) and 11N-loaded mice (n = 8) 2 weeks postloading, compared to baseline values and nonloaded controls. Maximum OA scores of ipsilateral knees confirmed increased cartilage lesions in 9N-loaded mice (mean ± SEM 2.8 ± 0.2; P < 0.001) and 11N-loaded mice (5.3 ± 0.3; P < 0.001), compared to nonloaded controls (1.0 ± 0.0). Gabapentin and diclofenac restored pain behaviors to baseline values after 2 weeks of daily treatment, and gabapentin was more effective than diclofenac. A single injection of anti-NGF alleviated nociception 2 days after treatment and remained effective for 2 weeks, with a second dose inducing stronger and more prolonged analgesia. CONCLUSION: Our findings show that mechanical joint loading induces OA lesions in mice and a robust pain phenotype that can be reversed using analgesics known to alleviate OA pain in patients. This establishes the use of mechanical joint loading as an alternative model for the study of OA pain.

Bidirectional regulation of bone formation by exogenous and osteosarcoma-derived Sema3A.

Semaphorin 3A (Sema3A), a secreted member of the Semaphorin family, increases osteoblast differentiation, stimulates bone formation and enhances fracture healing. Here, we report a previously unknown role of Sema3A in the regulation of ectopic bone formation and osteolysis related to osteosarcoma. Human recombinant (exogenous) Sema3A promoted the expression of osteoblastic phenotype in a panel of human osteosarcoma cell lines and inhibited the ability of these cells to migrate and enhance osteoclastogenesis in vitro. In vivo, administration of exogenous Sema3A in mice after paratibial inoculation of KHOS cells increased bone volume in non-inoculated and tumour-bearing legs. In contrast, Sema3A overexpression reduced the ability of KHOS cells to cause ectopic bone formation in mice and to increase bone nodule formation by engaging DKK1/ß-catenin signalling. Thus, Sema3A is of potential therapeutic efficacy in osteosarcoma. However, inhibition of bone formation associated with continuous exposure to Sema3A may limit its long-term usefulness as therapeutic agent.

Novel skeletal effects of glucagon-like peptide-1 (GLP-1) receptor agonists.

Type 2 diabetes mellitus (T2DM) leads to bone fragility and predisposes to increased risk of fracture, poor bone healing and other skeletal complications. In addition, some anti-diabetic therapies for T2DM can have notable detrimental skeletal effects. Thus, an appropriate therapeutic strategy for T2DM should not only be effective in re-establishing good glycaemic control but also in minimising skeletal complications. There is increasing evidence that glucagon-like peptide-1 receptor agonists (GLP-1RAs), now greatly prescribed for the treatment of T2DM, have beneficial skeletal effects although the underlying mechanisms are not completely understood. This review provides an overview of the direct and indirect effects of GLP-1RAs on bone physiology, focusing on bone quality and novel mechanisms of action on the vasculature and hormonal regulation. The overall experimental studies indicate significant positive skeletal effects of GLP-1RAs on bone quality and strength although their mechanisms of actions may differ according to various GLP-1RAs and clinical studies supporting their bone protective effects are still lacking. The possibility that GLP-1RAs could improve blood supply to bone, which is essential for skeletal health, is of major interest and suggests that GLP-1 anti-diabetic therapy could benefit the rising number of elderly T2DM patients with osteoporosis and high fracture risk.

Exenatide Improves Bone Quality in a Murine Model of Genetically Inherited Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is associated with skeletal complications, including an increased risk of fractures. Reduced blood supply and bone strength may contribute to this skeletal fragility. We hypothesized that long-term administration of Exenatide, a glucagon-like peptide-1 receptor agonist, would improve bone architecture and strength of T2DM mice by increasing blood flow to bone, thereby stimulating bone formation. In this study, we used a model of obesity and severe T2DM, the leptin receptor-deficient db/db mouse to assess alterations in bone quality and hindlimb blood flow and to examine the beneficial effects of 4 weeks administration of Exenatide. As expected, diabetic mice showed marked alterations in bone structure, remodeling and strength, and basal vascular tone compared with lean mice. Exenatide treatment improved trabecular bone mass and architecture by increasing bone formation rate, but only in diabetic mice. Although there was no effect on hindlimb perfusion at the end of this treatment, Exenatide administration acutely increased tibial blood flow. While Exenatide treatment did not restore the impaired bone strength, intrinsic properties of the matrix, such as collagen maturity, were improved. The effects of Exenatide on in vitro bone formation were further investigated in primary osteoblasts cultured under high-glucose conditions, showing that Exenatide reversed the impairment in bone formation induced by glucose. In conclusion, Exenatide improves trabecular bone mass by increasing bone formation and could protect against the development of skeletal complications associated with T2DM.

Low dose of propranolol does not affect rat osteotomy healing and callus strength.

Experimental studies suggest that the ß-blocker propranolol stimulates bone formation but little work has investigated its effect on fracture healing. In this study, we examined if a low dose of propranolol, previously shown to be preventive against bone loss in rats, improves bone repair. Female Wistar rats were injected with saline or propranolol (0.1 mg/kg/day) (n = 20/group), 5 days a week for 8 weeks. Three weeks after the beginning of treatment, all rats underwent a mid-diaphyseal transverse osteotomy in the left femur. Radiographic analysis of ostetomy healing was performed 2 and 5 weeks after osteotomy. Rats were sacrificed at 5 weeks and femora collected for measurements of fracture strength by torsional testing, callus volume, and mineral content by micro-CT analysis and histology of fracture callus. Eighty nine percent of osteotomies achieved apparent radiological union by 5 weeks in both groups. Propranolol treatment did not significantly alter the torsional strength of the fractured femur compared with controls. The volume and mineralization of fracture callus at 5 weeks were not significantly different in both groups. Histology showed that endochondral ossification was not affected by propranolol. Altogether, our results demonstrate that propranolol using the regimen described does not significantly improve or inhibit rat osteotomy healing and mechanical strength.

Mammalian Sulf1 RNA alternative splicing and its significance to tumour growth regulation.

SULF1/SULF2 enzymes regulate the activities of several growth factors by selective hydrolysis of 6-O-sulphates of heparan sulphate proteoglycan co-receptors, the sulfation of which is essential for signal transduction of some ligand/receptor interactions but not others. This study demonstrates the existence of SULF1 variants with a wide spectrum of splicing patterns in mammalian tumours. The levels and relative proportions of SULF1/SULF2 splice variants markedly vary in different tumours with a potential to regulate cell growth differentially. Although mammalian Sulf1 compared with Sulf2 gene generates a much larger number of splice variants, both enzymes follow generally similar distribution and signalling association trends in hepatocellular carcinomas.

Neuropeptide Y expression and function during osteoblast differentiation--insights from transthyretin knockout mice.

To better understand the role of neuropeptide Y (NPY) in bone homeostasis, as its function in the regulation of bone mass is unclear, we assessed its expression in this tissue. By immunohistochemistry, we demonstrated, both at embryonic stages and in the adult, that NPY is synthesized by osteoblasts, osteocytes, and chondrocytes. Moreover, peptidylglycine alpha-amidating monooxygenase, the enzyme responsible for NPY activation by amidation, was also expressed in these cell types. Using transthyretin (TTR) KO mice as a model of augmented NPY levels, we showed that this strain has increased NPY content in the bone, further validating the expression of this neuropeptide by bone cells. Moreover, the higher amidated neuropeptide levels in TTR KO mice were related to increased bone mineral density and trabecular volume. Additionally, RT-PCR analysis established that NPY is not only expressed in MC3T3-E1 osteoblastic cells and bone marrow stromal cells (BMSCs), but is also detectable by RIA in BMSCs undergoing osteoblastic differentiation. In agreement with our in vivo observations, in vitro, TTR KO BMSCs differentiated in osteoblasts had increased NPY levels and exhibited enhanced competence in undergoing osteoblastic differentiation. In summary, this work contributes to a better understanding of the role of NPY in the regulation of bone formation by showing that this neuropeptide is expressed in bone cells and that increased amidated neuropeptide content is related to increased bone mass.

Blockade of beta-adrenergic signaling does not influence the bone mechano-adaptive response in mice.

The involvement of the sympathetic nervous system (SNS) in the modulation of bone adaptation to its load-bearing demand remains controversial. This study tested the involvement of SNS in the adaptive response of trabecular and cortical bone to either external loading or disuse. External loading consisted of cyclic strain (40 cycles, peak 1500 microstrain) applied for 7 min, 3 days/week, while disuse was induced by unilateral sciatic neurectomy (SN). C57Bl/J6 mice, female, 9 weeks old, were subjected to loading or disuse for 2 weeks. Half of the loaded and SN mice were injected with the beta-adrenergic antagonist, propranolol (PRO, 20 mug/g) 1 week before the start of loading or disuse and during all the duration of the experiment. MicroCT analysis of the tibiae showed that the applied load induced significant changes on both trabecular architecture and cortical geometry compared to the contralateral controls, indicating increased bone mass. In contrast, disuse markedly reduced trabecular and cortical indexes. However, these adaptive responses were not altered by PRO treatment. We further tested whether the lack of protective effect of PRO against disuse-induced bone loss was due to the very short duration of treatment by blocking SNS signaling for 8 weeks with either PRO (0.5 mg/ml in drinking water) or guanethidine sulfate (GS, 40 mug/g, injected). At the end of fourth week of treatment, mice underwent SN surgery so that disuse was induced for the remaining 4 weeks. Again, neither PRO nor GS treatments altered the disuse-induced bone loss in the neurectomized tibia. In addition, blockade of SNS signaling for either 3 or 8 weeks did not affect the basal trabecular bone architecture in control tibiae and in L4 vertebrae. This study shows that the mechano-adaptive response occurring in trabecular and cortical bone upon loading or disuse is not altered by inactivation of beta-adrenergic signaling. Furthermore, sympathectomy had no effect on trabecular bone at different skeletal sites. This suggests that the osteo-regulatory action of beta-adrenergic signaling is not involved in the bone mechano-adaptive response and must therefore affect other bone regulatory pathways.

Absence of mechanical loading in utero influences bone mass and architecture but not innervation in Myod-Myf5-deficient mice.

Although the responses of bone to increased loading or exercise have been studied in detail, our understanding of the effects of decreased usage of the skeleton has been limited by the scarcity of suitable models. Such models should ideally not affect bone innervation, which appears to be a mediator of physiological responses of bone to unloading. MyoD-/-/Myf5-/- (dd/ff) mice lack skeletal muscle, so the fetuses develop without any active movement in utero and die soon after birth. We used micro-compter tomography and histology to analyse their bone development and structure during endochondral ossification in parallel with the establishment of bone innervation. Long bones from mutant mice were found to be profoundly different from controls, with shorter mineralized zones and less mineralization. They lacked many characteristics of adult bones - curvatures, changes in shaft diameter and traction epiphyses where muscles originate or insert - that were evident in the controls. Histologically, dd/ff mice showed the same degree of endochondral development as wild-type animals, but presented many more osteoclasts in the newly layed bone. Innervation and the expression pattern of semaphorin-3A signalling molecules were not disturbed in the mutants. Overall, we have found no evidence for a major defect of development in dd/ff mice, and specifically no alteration or delay in endochondral ossification and bone innervation. The altered morphological features of dd/ff mice and the increased bone resorption show the role of muscle activity in bone shaping and the consequences of bone unloading.

Sympathetic nervous system does not mediate the load-induced cortical new bone formation.

UNLABELLED: The contribution of the SNS to bone's response to mechanical loading is unclear. Using a noninvasive model of axial loading of the murine tibia, we found that sciatic neurectomy enhances load-induced new cortical bone formation and that pharmacological blockade of the SNS does not affect such responses, indicating that the SNS does not mediate the osteogenic effects of loading in cortical bone. INTRODUCTION: There is increasing evidence that the sympathetic nervous system (SNS) contributes to the regulation of bone mass and may influence remodeling by modulating bones' response to mechanical load-bearing. The aim of this study was to examine the effect of sciatic neurectomy (SN) on the changes in cortical bone formation induced in response to mechanical loading and to investigate whether the SNS is directly involved in such load-induced responses. MATERIALS AND METHODS: Accordingly, load-induced responses were compared in tibias of growing and adult control C57Bl/J6 mice and in mice submitted to unilateral SN; noninvasive axial loading that induced 2,000 microstrain on the tibia lateral midshaft cortex was applied cyclically, 5 or 100 days after surgery, for 7 minutes, 3 days/week for 2 weeks, and mice received calcein on the third and last days of loading. Tibias were processed for histomorphometry, and transverse confocal images from diaphyseal sites were analyzed to quantify new cortical bone formation. Chemical SNS inactivation was achieved by prolonged daily treatment with guanethidine sulfate (GS) or by the introduction of propranolol in drinking water. RESULTS: Our results show that new cortical bone formation is enhanced by loading in all tibial sites examined and that load-induced periosteal and endosteal new bone formation was greater in the SN groups compared with sham-operated controls. This SN-related enhancement in load-induced cortical bone formation in tibias was more pronounced 100 days after neurectomy than after 5 days, suggesting that longer periods of immobilization promote a greater sensitivity to loading. In contrast, the increases in new bone formation induced in response to mechanical loading were similar in mice treated with either GS or propranolol compared with controls, indicating that inactivation of the SNS has no effect on load-induced cortical new bone formation. CONCLUSIONS: This study shows that SN, or the absence of loading function it entails, enhances loading-related new cortical bone formation in the tibia independently of the SNS.

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.

Osteoclast spreading kinetics are correlated with an oscillatory activation of a calcium-dependent potassium current.

Cell movement and spreading involve calcium-dependent processes and ionic channel activation. During bone resorption, osteoclasts alternate between spread, motile and resorptive phases. We investigated whether the electrical membrane properties of osteoclasts were linked to their membrane morphological changes. Rabbit osteoclasts were recorded by time-lapse videomicroscopy performed simultaneously with patch-clamp whole cell and single channel recordings. Original image analysis methods were developed and used to demonstrate for the first time an oscillatory activation of a spontaneous membrane current in osteoclasts, which is directly correlated to the membrane movement rate. This current was identified as a calcium-dependent potassium current (IK(Ca)) that is sensitive to both charybdotoxin and apamin and was generated by a channel with unitary conductance of approximately 25+/-2 pS. Blockade of this current also decreased osteoclast spreading and inhibited bone resorption in vitro, demonstrating a physiological role for this current in osteoclast activity. These results establish for the first time a temporal correlation between lamellipodia formation kinetics and spontaneous peaks of IK(Ca), which are both involved in the control of osteoclast spreading and bone resorption.

Glutamatergic innervation in bone.

Bone is highly innervated, and evidence for a regulation of bone metabolism by nerve fibers has been suggested by many clinical and experimental studies. However, the nature of the neuromediators involved in these processes has not been well documented. Glutamate (Glu), a major neuromediator of the central nervous system (CNS), was recently identified in nerve fibers running in bone marrow in close contact with bone cells, suggesting that Glu may also act as a neuromediator in this tissue. During the last few years, all the machinery required for glutamate signalling in the CNS was demonstrated in bone. Osteoblasts and osteoclasts express ionotropic Glu receptors (iGluR) (NMDA, AMPA, and Kainate) and metabotropic Glu receptors (mGluR) as well as Glu transporters. Electrophysiological studies have demonstrated that NMDA receptors (NMDAR) and mGluR are functional on bone cells. NMDAR are involved in osteoclast formation and bone resorption and preliminary studies suggest that they may also participate in mechanisms underlying osteoblast proliferation or differentiation, providing evidence for a direct action of Glu on bone cells. The bone loss induced in a model of sciatic neurectomy in growing rats is associated with a decrease of glutamatergic innervation, suggesting that Glu released by nerve fibers may contribute to the regulation of bone remodeling. The manipulation of Glu action in bone may, therefore, represent a new therapeutic target for pathologies associated with modifications of bone remodeling.