Horizontal fissuring at the osteochondral interface: a novel and unique pathological feature in patients with obesity-related osteoarthritis.

OBJECTIVES: Obesity is a well-recognised risk factor for osteoarthritis (OA). Our aim is to characterise body mass index (BMI)-associated pathological changes in the osteochondral unit and determine if obesity is the major causal antecedent of early joint replacement in patients with OA. METHODS: We analysed the correlation between BMI and the age at which patients undergo total knee replacement (TKR) in 41 023 patients from the Australian Orthopaedic Association National Joint Replacement Registry. We then investigated the effect of BMI on pathological changes of the tibia plateau of knee joint in a representative subset of the registry. RESULTS: 57.58% of patients in Australia who had TKR were obese. Patients with overweight, obese class I & II or obese class III received a TKR 1.89, 4.48 and 8.08 years earlier than patients with normal weight, respectively. Microscopic examination revealed that horizontal fissuring at the osteochondral interface was the major pathological feature of obesity-related OA. The frequency of horizontal fissure was strongly associated with increased BMI in the predominant compartment. An increase in one unit of BMI (1 kg/m2) increased the odds of horizontal fissures by 14.7%. 84.4% of the horizontal fissures were attributable to obesity. Reduced cartilage degradation and alteration of subchondral bone microstructure were also associated with increased BMI. CONCLUSIONS: The key pathological feature in OA patients with obesity is horizontal fissuring at the osteochondral unit interface. Obesity is strongly associated with a younger age of first TKR, which may be a result of horizontal fissures.

Mechanical overload-induced release of extracellular mitochondrial particles from tendon cells leads to inflammation in tendinopathy.

Tendinopathy is one of the most common musculoskeletal diseases, and mechanical overload is considered its primary cause. However, the underlying mechanism through which mechanical overload induces tendinopathy has not been determined. In this study, we identified for the first time that tendon cells can release extracellular mitochondria (ExtraMito) particles, a subtype of medium extracellular particles (mEPs), into the environment through a process regulated by mechanical loading. RNA sequencing systematically revealed that oxygen-related reactions, extracellular particles, and inflammation were present in diseased human tendons, suggesting that these factors play a role in the pathogenesis of tendinopathy. We simulated the disease condition by imposing a 9% strain overload on three-dimensional mouse tendon constructs in our cyclic uniaxial stretching bioreactor. The three-dimensional mouse tendon constructs under normal loading with 6% strain exhibited an extended mitochondrial network, as observed through live-cell confocal laser scanning microscopy. In contrast, mechanical overload led to a fragmented mitochondrial network. Our microscopic and immunoblot results demonstrated that mechanical loading induced tendon cells to release ExtraMito particles. Furthermore, we showed that mEPs released from tendon cells overloaded with a 9% strain (mEP9%) induced macrophage chemotaxis and increased the production of proinflammatory cytokines, including IL-6, CXCL1, and IL-18, from macrophages compared to mEP0%, mEP3%, and mEP6%. Partial depletion of the ExtraMito particles from mEP9% by magnetic-activated cell sorting significantly reduced macrophage chemotaxis. N-acetyl-L-cysteine treatment preserved the mitochondrial network in overloaded tendon cells, diminishing overload-induced macrophage chemotaxis toward mEP9%. These findings revealed a novel mechanism of tendinopathy; in an overloaded environment, ExtraMito particles convey mechanical response signals from tendon cells to the immune microenvironment, culminating in tendinopathy.

Impact of age and donor sites on bioactivities of tendon cells in autologous tenocyte implantation (OrthoATI™) for treatment of chronic tendinopathy.

OBJECTIVES: Autologous tenocyte implantation (OrthoATI™) therapy has demonstrated efficacy in treating patients with tendinopathy at various anatomical sites. This study evaluates the effect of patient age, gender, and tendon biopsy site on morphology, growth, and gene expression of autologous tendon cells used to treat chronic tendinopathy. METHODS: Patients undergoing OrthoATI™ for tendinopathies between 2020 and 2022 were initially treated by biopsies taken from patella tendon (PT) or palmaris longus tendon (PL). The biopsies were sent to a Good Manufacturing Practice (GMP) cell laboratory where tendon cells were isolated, cultured, and expanded for four to six weeks. Cell morphology was assessed using phase contrast microscopy. Droplet digital PCR (ddPCR) was utilized for gene expression analysis. Dichotomous results were compared between groups using x2 or Fisher's exact tests with no adjustment for multiple comparisons. The nonparametric Mann-Whitney U and Kruskal-Wallis tests were utilized for the sex and age (<35y, 35-44y, 45-54y, >55y) analyses, respectively. All analyses were performed using IBM SPSS v27, and a two-tailed P-value of <0.05 was considered statistically significant. RESULTS: 149 patients were included in the analysis. The PT was biopsied in 63 patients, and PL in 86 patients. There were no observer effects for age and gender between the PT and PL groups. There was no statistical significance between the PT and PL tendons for cell morphology, average cell population doubling time (PDT) (PT 83.9 vs PL 82.7 â€‹h, p â€‹= â€‹0.482), cellular yield (PT 16.2 vs PL 15.2 â€‹× â€‹106, p â€‹= â€‹0.099), and cell viability (PT 98.7 vs PL 99.0%, p â€‹= â€‹0.277). Additionally, ddPCR analyses showed no statistical significance found in tenogenic gene expression, including collagen type I (COL1, p â€‹= â€‹0.86), tenomodulin (TNMD, p â€‹= â€‹0.837) and scleraxis (SCX, p â€‹= â€‹0.331) between PT- and PL-derived tendon cells. An age stratification analysis found no effect on growth and gene expression. COL1 was found to be higher in males when compared to females (P â€‹< â€‹0.001), but otherwise no difference was seen in growth and gene expression in the gender analysis. No postbiopsy clinical complications were reported for either group. CONCLUSION: This study has shown that the growth and bioactivities of tendon cells from tendon biopsies for OrthoATI™ are not affected by tendon donor site and age. LEVEL OF EVIDENCE: IV.

Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system.

Identification of functional programmable mechanical stimulation (PMS) on tendon not only provides the insight of the tendon homeostasis under physical/pathological condition, but also guides a better engineering strategy for tendon regeneration. The aims of the study are to design a bioreactor system with PMS to mimic the in vivo loading conditions, and to define the impact of different cyclic tensile strain on tendon. Rabbit Achilles tendons were loaded in the bioreactor with/without cyclic tensile loading (0.25 Hz for 8 h/day, 0-9% for 6 days). Tendons without loading lost its structure integrity as evidenced by disorientated collagen fiber, increased type III collagen expression, and increased cell apoptosis. Tendons with 3% of cyclic tensile loading had moderate matrix deterioration and elevated expression levels of MMP-1, 3, and 12, whilst exceeded loading regime of 9% caused massive rupture of collagen bundle. However, 6% of cyclic tensile strain was able to maintain the structural integrity and cellular function. Our data indicated that an optimal PMS is required to maintain the tendon homeostasis and there is only a narrow range of tensile strain that can induce the anabolic action. The clinical impact of this study is that optimized eccentric training program is needed to achieve maximum beneficial effects on chronic tendinopathy management.

Systematic evaluation of three porcine-derived collagen membranes for guided bone regeneration.

Guided bone regeneration is one of the most common surgical treatment modalities performed when an additional alveolar bone is required to stabilize dental implants in partially and fully edentulous patients. The addition of a barrier membrane prevents non-osteogenic tissue invasion into the bone cavity, which is key to the success of guided bone regeneration. Barrier membranes can be broadly classified as non-resorbable or resorbable. In contrast to non-resorbable membranes, resorbable barrier membranes do not require a second surgical procedure for membrane removal. Commercially available resorbable barrier membranes are either synthetically manufactured or derived from xenogeneic collagen. Although collagen barrier membranes have become increasingly popular amongst clinicians, largely due to their superior handling qualities compared to other commercially available barrier membranes, there have been no studies to date that have compared commercially available porcine-derived collagen membranes with respect to surface topography, collagen fibril structure, physical barrier property, and immunogenic composition. This study evaluated three commercially available non-crosslinked porcine-derived collagen membranes (Striate+TM, Bio-Gide® and CreosTM Xenoprotect). Scanning electron microscopy revealed similar collagen fibril distribution on both the rough and smooth sides of the membranes as well as the similar diameters of collagen fibrils. However, D-periodicity of the fibrillar collagen is significantly different among the membranes, with Striate+TM membrane having the closest D-periodicity to native collagen I. This suggests that there is less deformation of collagen during manufacturing process. All collagen membranes showed superior barrier property evidenced by blocking 0.2-16.4 µm beads passing through the membranes. To examine the immunogenic agents in these membranes, we examined the membranes for the presence of DNA and alpha-gal by immunohistochemistry. No alpha-gal or DNA was detected in any membranes. However, using a more sensitive detection method (real-time polymerase chain reaction), a relatively strong DNA signal was detected in Bio-Gide® membrane, but not Striate+TM and CreosTM Xenoprotect membranes. Our study concluded that these membranes are similar but not identical, probably due to the different ages and sources of porcine tissues, as well as different manufacturing processes. We recommend further studies to understand the clinical implications of these findings.

Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts.

Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast's 'resorptive apparatus'. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast's unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

Small molecule inhibitors of IkappaB kinase signaling inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo.

The NFκB pathway plays a critical role in the regulation of osteoclast activity, and activation of the pathway is dependent on IκB kinase (IKK), which phosphorylates IκB, targeting it for proteasomal degradation. Pharmacological inhibitors of IKK exhibit anti-inflammatory properties and prevent bone erosions in models of inflammatory arthritis. However, the effects of these agents on osteoblast function and ovariectomy-induced bone loss remain unknown. Here we examined the effects of the IKK inhibitors celastrol, BMS-345541, and parthenolide on bone cell function in vitro and ovariectomy-induced bone loss in vivo. All three compounds inhibited RANKL-induced signaling in osteoclasts, caused osteoclast apoptosis, and inhibited osteoclast formation. Although parthenolide and BMS-345541 had no inhibitory effects on osteoblast function, celastrol prevented IL1ß-induced TAK1 activation and inhibited osteoblast growth, differentiation, and bone nodule formation. The selective IKK inhibitors parthenolide and BMS-345541 prevented ovariectomy-induced bone loss by inhibiting osteoclastic bone resorption. We conclude that pharmacological inhibitors of IKK inhibit several critical signaling pathways in osteoclasts necessary for cell survival, formation, and activity in vitro and bone loss in vivo. Accordingly, IKK inhibitors may be of value in the prevention and treatment of bone diseases characterized by increased bone loss such as postmenopausal osteoporosis.

Intramuscular injection of Botox causes tendon atrophy by induction of senescence of tendon-derived stem cells.

BACKGROUND: Botulinum toxin (Botox) injection is in widespread clinical use for the treatment of muscle spasms and tendinopathy but the mechanism of action is poorly understood. HYPOTHESIS: We hypothesised that the reduction of patellar-tendon mechanical-loading following intra-muscular injection of Botox results in tendon atrophy that is at least in part mediated by the induction of senescence of tendon-derived stem cells (TDSCs). STUDY DESIGN: Controlled laboratory study METHODS: A total of 36 mice were randomly divided into 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into the right vastus lateralis of quadriceps muscles either with Botox (to induce mechanical stress deprivation of the patellar tendon) or with normal saline as a control. At 2 weeks post-injection, animals were euthanized prior to tissues being harvested for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity or the presence of senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, the PTEN/AKT-mediated cell senescence pathway was evaluated in TDSCs from both groups. RESULTS: Two weeks after Botox injection, patellar tendons displayed several atrophic features including tissue volume reduction, collagen fibre misalignment and increased degradation. A colony formation assay revealed a significantly reduced number of colony forming units of TDSCs in the Botox-injected group compared to controls. Multipotent differentiation capacities of TDSCs were also diminished after Botox injection. To examine if mechanically deprived TDSC are capable of forming tendon tissue, we used an isolated bioreactor system to culture tendon constructs using TDSC. These results showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of the signalling pathway revealed that injection of Botox into quadriceps muscles causes PTEN/AKT-mediated cell senescence of TDSCs. CONCLUSION: Intramuscular injection of Botox interferes with tendon homeostasis by inducing tendon atrophy and senescence of TDSCs. Botox injection may have long-term adverse consequences for the treatment of tendinopathy. CLINICAL RELEVANCE: Intramuscular Botox injection for tendinopathy or tendon injury could result in adverse effects in human tendons and evaluation of its long-term efficacy is warranted.

A bio-inductive collagen scaffold that supports human primary tendon-derived cell growth for rotator cuff repair.

BACKGROUND: Rotator Cuff (RC) tendon tearing is a common clinical problem and there is a high incidence of revision surgery due to re-tearing. In an effort to improve patient outcome and reduce surgical revision, scaffolds have been widely used for augmentation of RC repairs. However, little is known about how scaffolds support tendon stem cell growth or facilitate tendon regeneration. The purpose of this study is to evaluate the structural and biological properties of a bioactive collagen scaffold (BCS) with the potential to promote tendon repair. Additionally, we conducted a pilot clinical study to assess the safety and feasibility of using the BCS for repair of RC tears. METHODS: A series of physical, ultrastructural, molecular and in vitro tests determined the biocompatibility and teno-inductive properties of this BCS. In addition, a prospective case study of 18 patients with RC tendon tears (>20 â€‹mm in diameter) was performed in an open-label, single-arm study, involving either mini-open or arthroscopic surgical RC repair with the BCS. Clinical assessment of RC repair status was undertaken by MRI-imaging at baseline, 6 and 12 months and patient evaluated questionnaires were taken at baseline as well as 3, 6 & 12 months. RESULTS: The BCS consists of highly purified type-I collagen, in bundles of varying diameter, arranged in a higher order tri-laminar structure. BCS have minimal immunogenicity, being cell and essentially DNA-free as well as uniformly negative for the porcine α-Gal protein. BCS seeded with human primary tendon-derived cells and exposed to 6% uniaxial loading conditions in vitro, supported increased levels of growth and proliferation as well as up-regulating expression of tenocyte differentiation marker genes including TNMD, Ten-C, Mohawk and Collagen-1α1. To test the safety and feasibility of using the BCS for augmentation of RC repairs, we followed the IDEAL framework and conducted a first, open-label single arm prospective case series study of 18 patients. One patient was withdrawn from the study at 3 months due to wound infection unrelated to the BCS. The remaining 17 cases showed that the BCS is safe to be implanted. The patients reported encouraging improvements in functional outcomes (ASES, OSS and Constant-Murley scores), as well as quality of life assessments (AQoL) and a reduction in VAS pain scores. MRI assessment at 12 months revealed complete healing in 64.8% patients (11/17), 3 partial thickness re-tears (17.6%) and 3 full thickness re-tears (17.6%). CONCLUSION: The BCS is composed of type-I collagen that is free of immunogenic proteins and supports tendon-derived cell growth under mechanical loading in vitro. This pilot study shows that it is safe and feasible to use BCS for RC argumentation and further controlled prospective studies are required to demonstrate its efficacy. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The results of this study indicate that this bioactive collagen scaffold has unique properties for supporting tendon growth and that it is non-immunogenic. The clinical study further confirms that the scaffold is a promising biological device for augment of human rotator cuff repairs.

Collagen Membrane for Guided Bone Regeneration in Dental and Orthopedic Applications.

Treatment of cortical bone defects is a clinical challenge. Guided bone regeneration (GBR), commonly used in oral and maxillofacial dental surgery, may show promise for orthopedic applications in repair of cortical bone defects. However, a limitation in the use of GBR for cortical bone defects is the lack of an ideal scaffold that provides sufficient mechanical support to bridge the cortical bone with minimal interference in the repair process. We have developed a new collagen membrane, CelGro™, for use in GBR. We report the material characterization of CelGro and evaluate the performance of CelGro in translational preclinical and clinical studies. The results show CelGro has a bilayer structure of different fiber alignment and is composed almost exclusively of type I collagen. CelGro was found to be completely acellular and free from xenoantigen, α-gal (galactose-alpha-1,3-galactose). In the preclinical study of a rabbit cortical bone defect model, CelGro demonstrated enhanced bone-remodeling activity and cortical bone healing. Microcomputed tomography evaluation showed early bony bridging over the defect area 30 days postoperatively, and nearly complete restoration of mature cortical bone at the bone defect site 60 days postoperatively. Histological analysis 60 days after surgery further confirmed that CelGro enables bridging of the cortical bone defect by induction of newly formed cortical bone. Compared to a commercially available collagen membrane, Bio-Gide®, CelGro showed much better cortical alignment and reduced porosity at the defect interface. As selection of orthopedic patients with cortical bone defects is complex, we conducted a clinical study evaluating the performance of CelGro in guided bone regeneration around dental implants. CelGro was used in GBR procedures in a total of 16 implants placed in 10 participants. Cone-beam computed tomography images show significantly increased bone formation both horizontally and vertically, which provides sufficient support to stabilize implants within 4 months. Together, the findings of our study demonstrate that CelGro is an ideal membrane for GBR not only in oral and maxillofacial reconstructive surgery but also in orthopedic applications (Clinical Trial ID ACTRN12615000027516).

Beta2-adrenoreceptor ligands regulate osteoclast differentiation in vitro by direct and indirect mechanisms.

Pharmacological modulators of beta-adrenoceptors can influence bone mineral density and fracture risk in humans. Studies reported that beta-adrenoceptor ligands stimulate bone resorption by enhancing the expression of RANK-L, whereas the mechanisms by which beta-adrenoreceptors regulate bone formation are poorly understood. Here we show that beta2-adrenoceptor is predominantly expressed by bone cells, although low levels of beta1- and beta3-adrenoceptors were detectable. Noradrenaline and the selective beta2-adrenoceptor agonists isoprenaline and salmeterol stimulated osteoclast formation and bone resorption in BM osteoblast co-cultures and increased expression of RANK-L by osteoblasts. All three ligands enhanced RANK-L induced osteoclast formation and increased osteoclast multinuclearity. There was no significant effect of noradrenaline or isoprenaline on osteoblast growth, differentiation or function. These findings confirm the importance of the sympathetic nervous system in the regulation of bone mass, and demonstrate that pharmacological agonists of beta2-adrenoceptors directly and indirectly stimulate osteoclast formation, but have no direct effect on osteoblast growth, differentiation or function.

Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor.

The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2-/-) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2-/- mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2-/- mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2-/- mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.

Fabrication of a silver nanoparticle-coated collagen membrane with anti-bacterial and anti-inflammatory activities for guided bone regeneration.

Alveolar bone loss is a common problem that affects dental implant placement. A barrier between the bone substitute and gingiva that can prevent fibro-tissue ingrowth, bacterial infection and induce bone formation is a key factor in improving the success of alveolar ridge reconstruction. This study aims to develop a bioactive collagen barrier material for guided bone regeneration, that is coupled with anti-bacterial and anti-inflammatory properties. We have evaluated two silver coating methods and found controllable and precise coating achieved by sonication compared with sputtering. The optimized AgNP-coated collagen membrane exhibited excellent anti-bacterial effects against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) with limited cellular toxicity. It also displayed effective anti-inflammatory effects by reducing the expression and release of inflammatory cytokines including IL-6 and TNF-alpha. Additionally, AgNP-coated collagen membranes were able to induce osteogenic differentiation of mesenchymal stem cells that guide bone regeneration. These findings demonstrate the potential application of AgNP-coated collagen membranes to prevent infection after bone graft introduction in alveolar ridge reconstruction.

Open source software for semi-automated histomorphometry of bone resorption and formation parameters.

Micro-CT analysis has become the standard method for assessing bone volume and architecture in small animals. However, micro-CT does not allow the assessment of bone turnover parameters such as bone formation rate and osteoclast (OC) number and surface. For these crucial variables histomorphometric analysis is still an essential technique. Histomorphometry however, is time consuming and, especially in mouse bones, OCs can be difficult to detect. The main purpose of this study was to develop and validate a relatively easy and rapid method to measure static and dynamic bone histomorphometry parameters. Here we present the adaptation of established staining protocols and three novel open source image analysis packages: TrapHisto, OsteoidHisto and CalceinHisto that allow rapid, semi-automated analysis of histomorphometric bone resorption, osteoid, and calcein double labelling parameters respectively. These three programs are based on ImageJ, but use a relatively simple user interface that hides the underlying complexity of the image analysis.

Thonzonium bromide inhibits RANKL-induced osteoclast formation and bone resorption in vitro and prevents LPS-induced bone loss in vivo.

Osteoclasts (OCs) play a pivotal role in a variety of lytic bone diseases including osteoporosis, arthritis, bone tumors, Paget's disease and the aseptic loosening of orthopedic implants. The primary focus for the development of bone-protective therapies in these diseases has centered on the suppression of OC formation and function. In this study we report that thonzonium bromide (TB), a monocationic surface-active agent, inhibited RANKL-induced OC formation, the appearance of OC-specific marker genes and bone-resorbing activity in vitro. Mechanistically, TB blocked the RANKL-induced activation of NF-κB, ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation. TB disrupted F-actin ring formation resulting in disturbances in cytoskeletal structure in mature OCs during bone resorption. Furthermore, TB exhibited protective effects in an in vivo murine model of LPS-induced calvarial osteolysis. Collectively, these data suggest that TB might be a useful alternative therapy in preventing or treating osteolytic diseases.

Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth.

The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that sorting nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development.

Influence of age and gender on microarchitecture and bone remodeling in subchondral bone of the osteoarthritic femoral head.

INTRODUCTION: Age and gender have been reported to have a remarkable impact on bone homeostasis. However, subchondral bone, which plays a pivotal role in the initiation and progression of OA, has been poorly investigated. This study was to investigate age- and gender-related changes of microarchitecture and bone remodeling in subchondral bone in OA. METHODS: Subchondral trabecular bone (STB) and deeper trabecular bone (DTB) specimens were extracted in the load-bearing region of femoral heads from 110 patients with OA. Micro-CT and histomorphometry were performed to analyze microarchitectural and bone remodeling changes of all specimens. RESULTS: Compared to DTB, STB showed more sclerotic microarchitecture, more active bone remodeling and higher frequency of bone cysts. There were no gender differences for both microarchitecture and bone remodeling in STB. However, gender differences were found in DTB, with thinner Tb.Th, higher Tb.N, higher OS/BV and ES/BV in males. In both STB and DTB, no correlation between microarchitecture and age was found in both genders. However, bone remodeling of STB increased significantly with age in males, while bone remodeling of DTB increased significantly with age in females. No age or gender preference was found in subchondral bone cyst (SBC) frequency. The cyst volume fraction was correlated with neither age nor gender. CONCLUSIONS: There were differences in microarchitecture and bone remodeling between STB and DTB, which may be due to the distinct biomechanical and biochemical functions of these two bone structures in maintaining joint homeostasis. OA changed the normal age- and gender-dependence of bone homeostasis in joints, in a site-specific manner.

The type 2 cannabinoid receptor regulates bone mass and ovariectomy-induced bone loss by affecting osteoblast differentiation and bone formation.

The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass and bone turnover but the mechanisms responsible are incompletely understood. In this study we investigated the role that the CB2 pathway plays in bone metabolism using a combination of genetic and pharmacological approaches. Bone mass and turnover were normal in young mice with targeted inactivation of CB2 receptor (CB2(-/-)), but by 12 months of age, they had developed high-turnover osteoporosis with relative uncoupling of bone resorption from bone formation. Primary osteoblasts from CB2(-/-) mice had a reduced capacity to form bone nodules in vitro when compared with cells from wild-type littermates and also had impaired PTH-induced alkaline phosphatase (ALP) activity. The CB2-selective agonist HU308 stimulated bone nodule formation in wild-type osteoblasts but had no effect in CB2(-/-) osteoblasts. Further studies in MC3T3-E1 osteoblast like cells showed that HU308 promoted cell migration and activated ERK phosphorylation, and these effects were blocked by the CB2 selective inverse agonist AM630. Finally, HU308 partially protected against ovariectomy induced bone loss in wild-type mice in vivo, primarily by stimulating bone formation, whereas no protective effects were observed in ovariectomized CB2(-/-) mice. These studies indicate that the CB2 regulates osteoblast differentiation in vitro and bone formation in vivo.

The TRPV1 ion channel antagonist capsazepine inhibits osteoclast and osteoblast differentiation in vitro and ovariectomy induced bone loss in vivo.

The vanilloid type 1 ion channel (TRPV1) is known to play an important role in the regulation of pain and inflammation. Pharmacological ligands of TRPV1 regulate human osteoclast formation in vitro, but the effects of these agents on osteoblast function have not been studied and their effects on bone loss in vivo are unknown. Here we examined the effects of the TRPV1 ion channel antagonist capsazepine on mouse osteoclast and osteoblast differentiation in vitro and ovariectomy induced bone loss in vivo. Capsazepine inhibited osteoclast formation and bone resorption in a dose dependent manner in bone marrow-osteoblast co-cultures and RANKL generated osteoclast cultures, whereas the TRPV1 agonist capsaicin enhanced RANKL and M-CSF stimulated osteoclast formation. Capsazepine also suppressed RANKL induced IkappaB and ERK1/2 phosphorylation and caused apoptosis of mature osteoclasts and also inhibited alkaline phosphatase activity and bone nodule formation in calvarial osteoblast cultures. Studies in vivo showed that capsazepine (1mg/kg/day) inhibited ovariectomy induced bone loss in mice and histomorphometric analysis showed inhibitory effects on indices of bone resorption and bone formation. We conclude that pharmacological blockade of TRPV1 ion channels by capsazepine inhibits osteoclastic bone resorption and protects against ovariectomy induced bone loss in mice, but also inhibits osteoblast activity and bone formation.

Pharmacologic inhibitors of IkappaB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo.

The NF-kappaB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IkappaB kinase (IKK), a key component of NF-kappaB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1beta and tumor necrosis factor alpha-induced IkappaB phosphorylation and prevented nuclear translocation of NF-kappaB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKalpha and IKKbeta, and celastrol inhibited IKKalpha/beta activation by preventing the phosphorylation of TAK1, a key receptor-associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer.