Osteoporosis can cause chronic pain, but the mechanisms are unclear. This study investigates pain behaviours in mouse models of osteoporosis and fracture together with nociceptive markers expression in bone and dorsal root ganglia (DRGs). It also quantifies nerve markers in serum of patients with or without osteoporotic fractures and pain. Ovariectomy (OVX) or Sham surgery (Sham-OVX) of C57/Bl6 mice was performed (n = 10/group) and evoked and spontaneous pain behaviours assessed. In another experiment, OVX or Sham-OVX mice underwent a femoral osteotomy or sham osteotomy (n = 8/group) and pain behaviours measured. Gene expression of pain markers in bone and DRGs was quantified by RT-PCR. Nerve markers were quantified in serum of osteoporotic patients with or without fractures and pain using specific ELISAs. OVX did not cause changes in pain behaviours nor alter nociceptive gene expression in bone and DRGs. Osteotomy and Sham osteotomy both affected pain behaviours in mice compared to non-operated controls but did not significantly change nociceptive gene expression in bone and DRGs. OVX before osteotomy worsens weight-bearing compared to Sham-OVX. Fracture and pain did not affect nerve markers expression levels in serum of osteoporotic patients. This study demonstrates that OVX and subsequent bone loss in mice are insufficient to induce pain behaviours but may intensify pain after fracture. Our clinical analysis does not show a correlation between circulating nerve markers and fracture pain reported by the patients but suggests possible sex differences in pain markers that need to be further investigated.

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. Cite this article: Bone Joint Res 2023;12(10):657–666.

This study is the first comprehensive characterisation of the pain phenotype after fracture using both evoked and naturalistic behaviours in adult male and ovariectomised female mice. It also shows that an anti-nerve growth factor (NGF) therapy could be considered to reduce pain after fracture surgery. Bone fractures are common due to the ageing population and very painful even after healing. The phenotype of this pain is still poorly understood. We aimed to characterise it in a femoral fracture model in mice. We employed both adult male, and female ovariectomised (OVX) mice to mimic osteoporotic fractures. Mice underwent a unilateral femoral fracture maintained by an external fixator or a sham surgery. Pain behaviours, including mechanical and thermal sensitivity, weight bearing and LABORAS, were measured from baseline to 6 weeks after fracture. The effect on pain of an antibody against nerve growth factor (anti-NGF) was assessed. Changes in nerve density at the fracture callus were analysed by immunohistochemistry. Following surgery, all groups exhibited high levels of invoked nociception. Mechanical and thermal hyperalgesia were observed from 1 week after surgery, with nociceptive sensitization in the fracture group maintained for the 6 weeks, whereas it resolved in the sham group after 3 weeks. OVX induced reduction in pain thresholds, which was maintained after fracture. The frequency of naturalistic behaviours did not change between groups. Anti-NGF administered before and weekly after surgery alleviated fracture-induced mechanical nociception. The density of nerve fibres in the fracture callus was similar in all groups 6 weeks after surgery. Fractures in rodent models are highly painful in both sexes. This pain-like phenotype is prolonged and should be routinely considered in fracture healing studies as it can affect the study outcome. The anti-NGF alleviates fracture-induced mechanical pain.

Objective: In osteoarthritis (OA), the pain-structure relationship remains complex and poorly understood. Here, we used the mechanical joint loading (MJL) model of OA to investigate both knee pathology and nociceptive behaviour. Design: MJL was used to induce OA in the right knees of 12-week-old male C57BL/6 mice (40 cycles, 9N, 3x/week for 2 weeks). Mechanical sensitivity thresholds and weight-bearing ratios were measured before loading and at weeks one, three and six post-loading. At these time points, separate groups of loaded and non-loaded mice (n = 12/group) were sacrificed, joints collected, and fur corticosterone levels measured. mu CT analyses of subchondral bone integrity was performed before joint sections were prepared for nerve quantification, cartilage or synovium grading (scoring system from 0 to 6). Results: Loaded mice showed increased mechanical hypersensitivity paired with altered weight-bearing. Initial ipsilateral cartilage lesions 1-week post-loading (1.8 +/- 0.4) had worsened at weeks three (3.0 +/- 0.6, CI = -1.8-0.6) and six (2.8 +/- 0.4, CI = -1.6-0.4). This increase in lesion severity correlated with mechanical hypersensitivity development (correlation; 0.729, P = 0.0071). Loaded mice displayed increased synovitis (3.6 +/- 0.5) compared to non-loaded mice (1.5 +/- 0.5, CI = -2.2-0.3) 1-week post-loading which returned to normal by weeks three and six. Similarly, corticosterone levels were only increased at week one post-loading (0.21 +/- 0.04 ng/mg) compared to non-loaded controls (0.14 +/- 0.01 ng/mg, CI = -1.8 -0.1). Subchondral bone integrity and nerve volume remained unchanged. Conclusions: Our data indicates that although the loading induces an initial stress reaction and local inflammation, these processes are not directly responsible for the nociceptive phenotype observed. Instead, MJL-induced allodynia is mainly associated with OA-like progression of cartilage lesions. Crown Copyright (C) 2020 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. All rights reserved.

Purpose: Osteoarthritis (OA) is a common skeletal disease affecting nearly 3.3% of the world population. The main symptoms are chronic joint pain and stiffness caused by progressive changes in the joint tissues. Abnormal nerve growth into joints was observed in OA contributing to pain. Semaphorin-3A (sema-3A) is a secreted axonal chemo-repellent which inhibits axonal growth in a concentration-dependent manner. The purpose of this study was to determine whether sema-3A plays a role in the development of joint pain and degeneration. Methods: Two mouse models of OA, the mechanical joint loading (MJL) model and STR/ort mice that spontaneously develop OA were used in this study. For the MJL model, the right knees of C57BL/6 mice (male, 12-week-old) were loaded (40 cycles of loading regimen at 9N or 11N for two weeks, 3 times/week). The knee joints and serum were collected at week 6 post loading. Male STR/ort mice were maintained for 30-40 weeks for severe OA phenotype to develop and joints collected. Joint sections were used for immunocytochemistry using sema-3A antibody (Abcam). Sema-3A levels in serum were detected by ELISA (CUSABIO). QPCR was used to analyse sema-3A mRNA expression during mouse chondrogenic cell line ATDC5 differentiation and in rat articular cartilage and dorsal root ganglia (DRGs). We used lentivirus (10ul, 5x108 TU/ml) as a tool to overexpress sema-3A-GFP in mouse knee joints by intra-articular injection using the MJL model of OA. Knee joints were collected at week 1 after loading, and then processed for cryostat sections. Results: Sema-3A is expressed in cartilage, cruciate ligaments, synovial lining and subchondral bone in healthy mouse knee joints. The overall expression of sema-3A increased in these tissues and osteophytes with the development of OA, both in the MJL and STR/ort mouse models. The expression of sema-3A in cartilage doesn’t change with OA. There was a significant increase in sema-3A expression (+25%) in mouse serum 6 weeks after MJL compared to the non-loaded mice. Sema-3A is expressed at all stages of ATDC5 chondrocyte differentiation, with a peak of expression during chondrocyte maturation. Sema-3A was also found in rat DRGs but its expression was more than 10 times higher in rat articular cartilage compared to DRGs. Intra-articular injection of lentivirus in mouse knee joints successfully expressed sema-3A-GFP in articular cartilage, meniscus, patella and tibia and fibular junction. Conclusions: Our results demonstrate that sema-3A is upregulated in the highly innervated joint tissues, suggesting that it may control joint innervation during the development of OA. Intra-articular injection of lentivirus overexpressing sema-3A is efficient and further studies will use this tool to study sema-3A function during OA development.

Purpose: Osteoarthritis (OA) is a common degenerative joint disease associated with chronic and debilitating pain in the affected joints which significantly reduces mobility and quality of life in patients. The mechanisms for osteoarthritic pain are poorly understood and current therapies to reduce pain are often insufficient. The lack of murine models of osteoarthritic pain, that show both histological OA cartilage lesions and a robust reproducible pain phenotype, has deterred the understanding of the disease. In this study we used the recently established, adjustable and non-invasive murine joint loading model of OA to characterize the development of a pain phenotype. Methods: The right knee of male mice (12 week-old, C57BL/6) were loaded at 2N, 9N and 11N three times per week for two weeks under anaesthesia. Behavioural measurements were taken at baseline before loading, and monitored on a weekly basis for six weeks post loading. Mechanical hyperalgesia was measured using manual von Frey in both ipsilateral and contralateral hind paws; thermal hyperalgesia was measured using the hot and cold plates whilst the rotarod and weight bearing tests were used to measure disuse of affected limb. Development of pain was compared to control mice which did not undergo a loading regime but were subjected to isoflurane anaesthesia for the same duration as loaded mice. Severity of OA lesions in the articular cartilage was determined post-mortem on joint sections using the grading scheme from the OARSI histopathology initiative. Results: Following two weeks of loading 9N- and 11N-loaded mice developed a pain phenotype which was more severe than that of the 2N-loaded mice. Both the 11N- and 9N-loaded mice developed ipsilateral mechanical hypersensitivity two weeks post loading whilst contralateral hypersensitivity developed at a later stage. The mechanical hypersensitivity was accompanied by reduced motor activity and altered weight bearing. The non-loaded control group did not show changes over time in any of the pain measurements. There was no thermal hypersensitivity phenotype in any of the mice. Histological analysis revealed that loading at 11N induces OA-like lesions of significantly higher severity than in 9N- and 2N-loaded mice. Mice loaded at 2N showed signs of low OA in the ipsilateral knee which suggests that the static load of 2N is sufficient to induce mild damage in the knee. The contralateral knee of 2N-loaded mice showed no lesions. This correlates with the weak ipsilateral mechanical hypersensitivity but no contralateral pain seen in the 2N-loaded mice. The contralateral knee of both 9N and 11N-loaded mice showed mild OA lesions suggesting that in these mice the ipsilateral knee is damaged to such an extent that they compensate with the contralateral leg. This is in agreement with the contralateral development of mechanical hypersensitivity seen in these mice. Conclusions: We have characterized for the first time the pain phenotype in a novel non-invasive murine joint loading model. Our results show that this model of OA produces a robust and reproducible pain phenotype as well as showing OA lesions, both of which vary in severity depending on the load magnitude. This establishes the use of the non-invasive murine joint loading model as an appropriate model to measure osteoarthritic pain.

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 (GLP-1r) agonists, 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. Lay Abstract Bone weakening is an important complication in individuals with type 2 diabetes (T2DM). This review summarises the effects on skeletal health of drugs that are similar to the hormone Glucagon-like peptide-1 (GLP-1), which are now used increasingly for the treatment of T2DM and could lead to a stronger skeleton.

Objective: Short-term neurectomy-induced disuse (SN) has been shown to restore load responses in aged mice. We examined whether this restoration was further enhanced in both cortical and trabecular bone by simply extending the SN. Methods: Following load: strain calibration, tibiae in female C57BL/J6 mice at 8, 14 and 20 weeks and 18 months (n=8/group) were loaded and bone changes measured. Effects of long-term SN examined in twenty-six 18 months-old mice, neurectomised for 5 or 100 days with/without subsequent loading. Cortical and trabecular responses were measured histomorphometrically or by micro-computed tomography. Results: Loading increased new cortical bone formation, elevating cross-sectional area in 8, 14 and 20 week-old (p <0.05), but not 18 month-old aged mice. Histomorphometry showed that short-term SN reinstated load-responses in aged mice, with significant 33% and 117% increases in bone accrual at 47% and 37%, but not 27% of tibia length. Cortical responses to loading was heightened and widespread, now evident at all locations, following prolonged SN (108, 167 and 98% at 47, 37 and 27% of tibial length, respectively). In contrast, loading failed to modify trabecular bone mass or architecture. Conclusions: Mechanoadaptation become deficient with ageing and prolonging disuse amplifies this response in cortical but not trabecular bone.

Abstract Aims Chemotherapy-induced peripheral neuropathy (CIPN) is a dose limiting side effect in the use of the platinum-based antineoplastic drug oxaliplatin as a treatment for colorectal cancer. Currently there is no treatment available to reverse the neurotoxicity which presents as pain, sensory loss and cold allodynia in up to 80% of patients. The aim of this study is to investigate if pregabalin can reverse the allodynia caused by oxaliplatin in CIPN. Methods CIPN was induced in 10 male C57BL/6 mice (6 weeks-old) with a single intraperitoneal injection of oxaliplatin (15 mg/kg i.p.). Signs of thermal and mechanical allodynia were assessed from baseline to 20 days after injection by Cold/Hot plate (Bioseb, France) at 20 °C and hand-held von Frey (vF) hairs of gradually increasing weights. Pregabalin (3 mg/kg and 10 mg/kg p.o.) was administered to treat CIPN. Results Mechanical and thermal allodynia were established 3 days post-oxaliplatin injection and remained stable for 14 days. At day 15, pregabalin (3 mg/kg p.o.) reversed mechanical allodynia to baseline scores at 2 h (H) post-dosing and thermal allodynia at 1 and 2H post-dosing. Following a 2-day wash out where scores returned to neuropathic baseline, pregabalin (10 mg/kg p.o.) reverted scores for mechanical and thermal allodynia to baseline scores at both 1 and 2H. Thermal testing was performed either immediately after vF or alone and our results were similar, showing no iatrogenic effects of vF on thermal sensitivity. Correlation analysis of the responses to thermal and mechanical stimuli showed no significant trend, indicating that oxaliplatin-induced peripheral neuropathy affects the mechanical and thermal modalities in different ways. Conclusion Oxaliplatin-induced peripheral neuropathy as measured by thermal and mechanical allodynia is reversible by a single dose of pregabalin. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 642720. RM is funded by a Marie Sklodowska-Curie grant agreement No. 642720 doing a joint PhD with the Royal Veterinary Collage and Transpharmation Ltd. AF and NU are employees of Transpharmation Ltd.

In contrast to previously reported elevations in serum sclerostin levels in diabetic patients, the present study shows that the impaired bone microarchitecture and cellular turnover associated with type 2 diabetes mellitus (T2DM)-like conditions in ZDF rats are not correlated with changes in serum and bone sclerostin expression.T2DM is associated with impaired skeletal structure and a higher prevalence of bone fractures. Sclerostin, a negative regulator of bone formation, is elevated in serum of diabetic patients. We aimed to relate changes in bone architecture and cellular activities to sclerostin production in the Zucker diabetic fatty (ZDF) rat.Bone density and architecture were measured by micro-CT and bone remodelling by histomorphometry in tibiae and femurs of 14-week-old male ZDF rats and lean Zucker controls (n = 6/group).ZDF rats showed lower trabecular bone mineral density and bone mass compared to controls, due to decreases in bone volume and thickness, along with impaired bone connectivity and cortical bone geometry. Bone remodelling was impaired in diabetic rats, demonstrated by decreased bone formation rate and increased percentage of tartrate-resistant acid phosphatase-positive osteoclastic surfaces. Serum sclerostin levels (ELISA) were higher in ZDF compared to lean rats at 9 weeks (+40 %, p < 0.01), but this difference disappeared as their glucose control deteriorated and by week 14, ZDF rats had lower sclerostin levels than control rats (-44 %, p < 0.0001). Bone sclerostin mRNA (qPCR) and protein (immunohistochemistry) were similar in ZDF, and lean rats at 14 weeks and genotype did not affect the number of empty osteocytic lacunae in cortical and trabecular bone.T2DM results in impaired skeletal architecture through altered remodelling pathways, but despite altered serum levels, it does not appear that sclerostin contributes to the deleterious effect of T2DM in rat bone.

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection ( P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively ( P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.

SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

Patients with acromegaly have a higher prevalence of vertebral fractures despite normal bone mineral density (BMD), suggesting that GH overexpression has adverse effects on skeletal architecture and strength. We used giant bovine GH (bGH) transgenic mice to analyze the effects of high serum GH levels on BMD, architecture, and mechanical strength. Five-month-old hemizygous male bGH mice were compared with age- and sex-matched nontransgenic littermates controls (NT; n=16/group). Bone architecture and BMD were analyzed in tibia and lumbar vertebrae using microcomputed tomography. Femora were tested to failure using three-point bending and bone cellular activity determined by bone histomorphometry. bGH transgenic mice displayed significant increases in body weight and bone lengths. bGH tibia showed decreases in trabecular bone volume fraction, thickness, and number compared with NT ones, whereas trabecular pattern factor and structure model index were significantly increased, indicating deterioration in bone structure. Although cortical tissue perimeter was increased in transgenic mice, cortical thickness was reduced. bGH mice showed similar trabecular BMD but reduced trabecular thickness in lumbar vertebra relative to controls. Cortical BMD and thickness were significantly reduced in bGH lumbar vertebra. Mechanical testing of femora confirmed that bGH femora have decreased intrinsic mechanical properties compared with NT ones. Bone turnover is increased in favor of bone resorption in bGH tibia and vertebra compared with controls, and serum PTH levels is also enhanced in bGH mice. These data collectively suggest that high serum GH levels negatively affect bone architecture and quality at multiple skeletal sites.

Some anti-diabetic therapies can have adverse effects on bone health and increase fracture risk. In this study, we tested the skeletal effects of chronic administration of two Glucagon-like peptide-1 receptor agonists (GLP-1RA), increasingly used for type 2 diabetes treatment, in a model of osteoporosis associated bone loss and examined the expression and activation of GLP-1R in bone cells. Mice were ovariectomised (OVX) to induce bone loss and four weeks later they were treated with Liraglutide (LIR) 0.3mg/kg/day, Exenatide (Ex-4) 10μg/kg/day or saline for four weeks. Mice were injected with calcein and alizarin red prior to euthanasia, to label bone-mineralising surfaces. Tibial micro-architecture was determined by micro-CT and bone formation and resorption parameters measured by histomorphometric analysis. Serum was collected to measure calcitonin and sclerostin levels, inhibitors of bone resorption and formation, respectively. GLP-1R mRNA and protein expression were evaluated in the bone, bone marrow and bone cells using RT-PCR and immunohistochemistry. Primary osteoclasts and osteoblasts were cultured to evaluate the effect of GLP-1RA on bone resorption and formation in vitro. GLP-1RA significantly increased trabecular bone mass, connectivity and structure parameters but had no effect on cortical bone. There was no effect of GLP-1RA on bone formation in vivo but an increase in osteoclast number and osteoclast surfaces was observed with Ex-4. GLP-1R was expressed in bone marrow cells, primary osteoclasts and osteoblasts and in late osteocytic cell line. Both Ex-4 and LIR stimulated osteoclastic differentiation in vitro but slightly reduced the area resorbed per osteoclast. They had no effect on bone nodule formation in vitro. Serum calcitonin levels were increased and sclerostin levels decreased by Ex-4 but not by LIR. Thus, GLP-1RA can have beneficial effects on bone and the expression of GLP-1R in bone cells may imply that these effects are exerted directly on the tissue.

SUMMARY:The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed. INTRODUCTION:In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration. METHODS:We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively. RESULTS:In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups. CONCLUSIONS:Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents.

AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. We previously demonstrated that AMPK activation in osteoblasts increases in vitro bone formation while deletion of the Ampkα1 (Prkaa1) subunit, the dominant catalytic subunit expressed in bone, leads to decreased bone mass in vivo. To investigate the cause of low bone mass in the Ampkα1−/− mice, we analysed bone formation and resorption in the tibia of these mice by dynamic histomorphometry and determined whether bone turnover can be stimulated in the absence of the Ampkα1 subunit. We subjected 12-week-old Ampkα1+/+ and Ampkα1−/− mice to ovariectomy (OVX), intermittent PTH (iPTH) administration (80 μg/kg per day, 5 days/week) or both OVX and iPTH hormonal challenges. Tibiae were harvested from these mice and bone micro-architecture was determined by micro-computed tomography. We show for the first time that Ampkα1−/− mice have a high bone turnover at the basal level in favour of bone resorption. While both Ampkα1+/+ and Ampkα1−/− mice lost bone mass after OVX, the bone loss in Ampkα1−/− mice was lower compared with controls. iPTH increased trabecular and cortical bone indexes in both ovariectomised Ampkα1+/+ and Ampkα1−/− mice. However, ovariectomised Ampkα1−/− mice showed a smaller increase in bone parameters in response to iPTH compared with Ampkα1+/+ mice. By contrast, non-ovariectomised Ampkα1−/− mice responded better to iPTH treatment than non-ovariectomised Ampkα1+/+ mice. Overall, these data demonstrate that Ampkα1−/− mice are less affected by changes in bone turnover induced by OVX but respond better to the anabolic challenge induced by iPTH. These results suggest that AMPKα1 activation may play a role in the hormonal regulation of bone remodelling.

There is increasing evidence that osteoporosis, similarly to obesity and diabetes, could be another disorder of energy metabolism. AMP-activated protein kinase (AMPK) has emerged over the last decade as a key sensing mechanism in the regulation of cellular energy homeostasis and is an essential mediator of the central and peripheral effects of many hormones on the metabolism of appetite, fat and glucose. Novel work demonstrates that the AMPK signaling pathway also plays a role in bone physiology. Activation of AMPK promotes bone formation in vitro and the deletion of α or β subunit of AMPK decreases bone mass in mice. Furthermore, AMPK activity in bone cells is regulated by the same hormones that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues. AMPK is also activated by antidiabetic drugs such as metformin and thiazolidinediones (TZDs), which also impact on skeletal metabolism. Interestingly, TZDs have detrimental skeletal side effects, causing bone loss and increasing the risk of fractures, although the role of AMPK mediation is still unclear. These data are presented in this review that also discusses the potential roles of AMPK in bone as well as the possibility for AMPK to be a future therapeutic target for intervention in osteoporosis.

Epidemiological and modelling studies suggest that elimination of Onchocerca volvulus transmission (EoT) throughout Africa may not be achievable with annual mass drug administration (MDA) of ivermectin alone, particularly in areas of high endemicity and vector density.Single-dose Phase II and III clinical trials demonstrated moxidectin's superiority over ivermectin for prolonged clearance of O. volvulus microfilariae.We used the stochastic, individual-based EPIONCHO-IBM model to compare the probabilities of reaching EoT between ivermectin and moxidectin MDA for a range of endemicity levels (30 to 70% baseline microfilarial prevalence), treatment frequencies (annual and biannual) and therapeutic coverage/adherence values (65 and 80% of total population, with, respectively, 5 and 1% of systematic non-adherence).EPIONCHO-IBM's projections indicate that biannual (six-monthly) moxidectin MDA can reduce by half the number of years necessary to achieve EoT in mesoendemic areas and might be the only strategy that can achieve EoT in hyperendemic areas.Data needed to improve modelling projections include (i) the effect of repeated annual