Bone Research Society 2021 Abstracts

Physiological mineralization occurs naturally and is responsible for the formation and regeneration of hard tissues. On the other hand, mineralization of soft tissues, such as mineralization of the vasculature, has been associated to several diseases, including cardiac diseases, cancer and Alzheimer’s disease. In this work we introduce the mineralomics (briefly, the characterization and study of minerals present in a biological context) and discuss how it may help us unveil key biological andmedical information about cardiovascular diseases, breast cancer and Alzheimer’s disease. On cardiovascular diseases, if not the direct cause of death, calcification contributes considerably to complications that can lead to heart failure or heart attack. The origins and mechanisms of formation of vascular calcification are still strongly debated, with competing theories present in the literature. Almost a decade ago, we showed that nano-sized and micron-sized calcified spherical particles formed by a single crystal of magnesiumcontaining calcium phosphate were the first calcified structure that could be detected in vascular tissue. Starting with the characterization of the mineral present on the vascular tissue and moving to the relationship between this mineral and the cells and proteins in the vascular system, we were able to propose a new mechanism for the origins of the first calcified structures in the vascular tissue. Using the same principles of characterization of minerals, we also examined minerals found in breast tumors. Our results demonstrated the presence of nanosized and micron-sized This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received in original form September 2, 2021; accepted September 5, 2021. JBMR Plus (WOA), Vol. 5, No. S5, November 2021, e10552. DOI: 10.1002/jbm4.10552 © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.


Overview of the BRS 2021 Online Annual Meeting
The Bone Research Society (BRS), formerly the Bone and Tooth Society, was founded in 1950. The BRS is one of the largest national scientific societies in Europe dedicated to clinical and basic research into mineralised tissues and is the oldest such society in the world. Meetings are held annually, attracting a wide audience from throughout the UK and beyond. The presentations are traditionally balanced between clinical and laboratory studies. The participation of young scientists and clinicians is actively encouraged. Due to the unprecedented times which we had all experienced over the last year and like many other meetings, the BRS Annual Meeting 2021 was held online rather than in person, but that didn't stop our community continuing to produce high quality science and connecting with each other to exchange ideas and knowledge. BRS 2021 was the platform for this, where the society ran a full programme of a webinars online over three days, with invited speakers, oral communications, virtual posters and satellite symposia from our Industry partners. In addition, three workshops were held in the eveningsthe Rare Bone Disease workshop, Muscle

Abstract
Physiological mineralization occurs naturally and is responsible for the formation and regeneration of hard tissues. On the other hand, mineralization of soft tissues, such as mineralization of the vasculature, has been associated to several diseases, including cardiac diseases, cancer and Alzheimer's disease. In this work we introduce the mineralomics (briefly, the characterization and study of minerals present in a biological context) and discuss how it may help us unveil key biological and medical information about cardiovascular diseases, breast cancer and Alzheimer's disease. On cardiovascular diseases, if not the direct cause of death, calcification contributes considerably to complications that can lead to heart failure or heart attack. The origins and mechanisms of formation of vascular calcification are still strongly debated, with competing theories present in the literature. Almost a decade ago, we showed that nano-sized and micron-sized calcified spherical particles formed by a single crystal of magnesiumcontaining calcium phosphate were the first calcified structure that could be detected in vascular tissue. Starting with the characterization of the mineral present on the vascular tissue and moving to the relationship between this mineral and the cells and proteins in the vascular system, we were able to propose a new mechanism for the origins of the first calcified structures in the vascular tissue. Using the same principles of characterization of minerals, we also examined minerals found in breast tumors. Our results demonstrated the presence of nanosized and micron-sized spherical particles made of highly crystalline whitlockite, which are exclusively found in the arterial wall of malignant invasive tumors. More recently, we have also been working in the characterization of minerals present in the brains of Alzheimer's patients. Although mechanisms for the initial formation of minerals in the brain are not yet fully understood, the abnormalities mentioned in literature are directly related to a number of diseases, such as Fahr's disease, hypoparathyroidism, Alzheimer's disease, among others. Our characterization study shows the unique morphology, composition, crystallinity, and location of minerals in brain tissue samples from aged donors, Alzheimer's patients, and young donors. Moreover, we discovered that these minerals are related to the Tau protein, and are primarily located on nuclei of brain cells. The causes of this phenomenon are still elusive and are being investigated by my group, since they could have an important role on charting brain aging and further implications for the study of mental disorders such as Alzheimer's. Seen as a whole, our work demonstrates the potential of the mineralomics field and shows that mineralomics enables the study of pathological minerals, which have long held and undervalued power to predict disease onset and provide valuable information about diseases and their mechanisms. We believe that mineralomics has the potential to provide critical insights into human (patho)biology and to contribute to breakthroughs in medicine, especially when integrated into a multiomics research approach. Furthermore, the pronounced female preponderance in fracture incidence observed in white populations is not seen among blacks or Asians in whom age-adjusted female to male incidence ratios approximate unity. Life expectancy is increasing around the globe and the number of elderly individuals is rising in every geographic region. Assuming constant age-specific incidence rates for fracture, the number of hip fractures occurring worldwide among people aged 65 years and over will rise from 1.66 million in 1990 to 6.26 million in 2050. Studies performed in the United States, Scandinavia, and the United Kingdom, between 1930 and the late 1980s, consistently reported increases in the age-adjusted incidence of hip fractures among men and women. This increase appears to have leveled off, in the northern regions of the United States, as well as more recently in Europe. Rates in Asian populations continue to show substantial rises between the 1960s and the present time.
In the most recent data available from the United States, the incidence of first ever hip fracture declined by 1.37% per year among women and 0.06% per year among men. The cumulative incidence of a second hip fracture after 10 years was 11% among women and 6% among men, when death was treated as a competing risk. The reduction in hip fracture occurrence was even greater than that expected from the declining incidence of hip fractures more generally. Age-period-cohort models have suggested influences of all three contributors to these secular trends. Among current risk factors for low bone density and trauma (low body mass index, cigarette smoking, alcohol consumption, physical inactivity, and dietary calcium intake) the trends are best explained by physical inactivity. Developmental contributors to peak bone and muscle mass, for example maternal nutrition and lifestyle, also appear capable of contributing to cohort effects. Finally, debate continues on the role of more aggressive osteoporosis risk assessment and therapeutic strategies in contributing to the secular decline in hip fracture rates generally. Although pharmacologic intervention might be efficacious, only a minority of hip fracture patients remain so treated, and the scope for even greater reductions in incidence remains an enticing prospect.
GWAS findings can be leveraged in Mendelian RANDOMIZATION (MR) studies intended to examine causal relationships with risk factors. For example, in a MR study of fracture risk, although grip strength was found to have a causal, protective effect on fracture risk, no effect was seen for vitamin D levels. MR has also been applied to interrogate adverse effects of drug therapies. For example, whereas concerns have been raised over possible increased cardiovascular risk following romosozumab, SNPs associated with reduced SOST expression were recently found to be unrelated to cardiovascular endpoints. MR studies are subject to a number of assumptions; however, newer analysis techniques enable these to be tested and controlled for. GWAS findings can also be used to generate polygenic risk scores, which can be used to predict disease occurrence. However, to date, the additional predictive value of polygenic risk scores for fracture risk is somewhat low when combined with conventional factors, particularly BMD. High-dimensional imaging data may also enable improved accuracy of disease prediction, as illustrated by recent findings that hip shape modes, derived from principle components analysis of hip DXA scans, are predictive of total hip replacement in UK Biobank.

Abstract
Disuse osteoporosis occurs after extended periods of bed rest or nerve damage leading to increased risk of fracture. It remains to be established, however, whether the trajectory of bone loss is equivalent in bone's cortical and trabecular compartments following long-term disuse. We aim to use μCT to evaluate whether trabecular and cortical bone compartments behave similarly in response to defined periods of disuse. The right hind-limb of seventeen 12-week-old female mice was subjected to sciatic neurectomy (SN, left limb served as control) and euthanized in four groups (n = 3-5/group) at 5, 35, 65, and 95 days. Trabecular bone was examined at the proximal metaphysis and cortical bone mass and geometry were evaluated along almost the entire tibia length. We found trabecular bone volume/total volume, number, and mineral density rapidly decreased within the first 5 days and exhibited a trajectory of loss that plateaued only after 65 days post-SN. In contrast, decreased cortical crosssectional area (CSA, and others measures of mass/geometry) along the tibia length reached levels of significance (p < 0.001) only much later, at 35 days, and surprisingly no further deterioration was observed thereafter. These data indicate that disuserelated cortical bone loss exhibits delayed onset yet stabilizes more rapidly than trabecular bone (Fig. 1). These data suggest that trabecular and cortical bone compartments behave as distinct modules in response to disuse.
Do 3D epiphyseal bone architectural changes in ageing STR/ Ort and healthy mice reveal early imaging biomarkers of osteoarthritis?

Abstract
Novel imaging biomarkers are required to advance research into, and treatment of, knee joint osteoarthritis (OA). Currently, early-stage OA is undetectable in humans, and treatment effectiveness cannot be reliably monitored. STR/Ort mice are an age-related model of progressive OA, in which predisposition (at 10 weeks of age), early-stage onset (at 20 weeks), and late-stage OA (at 40 weeks) are well-defined. Using STR/Ort (OA) and CBA (healthy parental control) mice at these age intervals, knee joints were non-invasively μCT-imaged with effective pixel size 5 μm. Tibial epiphyses were semi-automatically segmented from joints and separated into their constituent anatomical components: cortical bone, trabecular bone, and marrow space volume (Fig. 1A-D). Crucially, these bony features can be detected in human knees using clinical in vivo scanners (XtremeCT II HR-pQCT, Scanco Medical) making translation of our research realistically attainable in the near-future. 3D analyses of tibial epiphyses followed by two-way ANOVA confirmed significant age-and/or strain-related differences in epiphyseal cortical bone volume (p ≤ 0.001), trabecular bone volume (p ≤ 0.001), mean trabecular and cortical bone thicknesses (both p values ≤ 0.010), trabecular volume relative to cortical volume (p ≤ 0.001), and degree of anisotropy, a descriptive measurement of trabecular orientation (p = 0.001, Fig. 1E). The two mouse strains had different epiphyseal growth patterns throughout life with respect to total epiphyseal volume (p = 0.023), marrow space volume (p = 0.002), and trabecular volume relative to epiphyseal interior volume (p = 0.024), as well as changes in trabecular anisotropy indicating divergent and JBMR Plus (WOA) n 6 of 51 potentially exciting age-related interactions in these OA-prone and healthy mouse strains. Our findings disclose new imaging biomarkers of pre-OA, OA onset, and OA progression in the STR/Ort mouse, an established animal model of spontaneous age-related OA. Due to our exploitation of only gross-anatomical features in the tibial epiphysis, which can also be segmented from clinical CT scans, we anticipate future translation of this promising research into human clinical practice.
The novel high bone mass-associated Smad9 transcription factor is predominantly expressed in osteochondral progenitor populations of zebrafish skeletal elements Georgina McDonald, Mengdi Wang, Chrissy Hammond, Dylan Bergen University of Bristol, Bristol, UK

Abstract
Background: Osteoporosis is a prevalent age-related disease characterized by low bone mineral density and fragility fractures. In the clinic there is a demand for new osteoanabolic treatments to strengthen bones. A powerful way to identify new osteoanabolic targets is by understanding the genetics of high bone mass (HBM). Recently, a damaging p.Leu22Pro mutation in SMAD9 was identified in a HBM pedigree, which was independently confirmed in two unrelated HBM cases. SMAD9 is an inhibiting transcription factor in the bone morphogenetic protein (BMP) signaling pathway, whereas SMAD1/5 activate transcription.
Zebrafish are a suitable model as they have similar skeletal physiology to humans and can be easily pharmacologically manipulated and imaged. Methods: We used zebrafish transgenic reporters to visualize osteoblasts (sp7:GFP) and the BMP-pathway (BMPre:GFP). Protein expression was visualized with α-Smad9, α-GFP and α-collagen2a1 antibodies. Three day-old larvae were treated with 0.01% DMSO, 4μM dorsomorphin (DM), 1μM retinoic acid (RA), or 25μM prednisolone (PD) for 2 days. Six-hour nifurpirinol (5μM) treatment was used to ablate osteoblasts in the sp7:mCherry-NTR transgenic. Adult caudal fins were amputated or fractured and assessed 7 days later. Experiments were ethically reviewed and performed under UK Home Office license (30/3801).
Results: This research shows that Smad9 is expressed by a group of osteochondral precursor cells in the developing skeleton, which do not express Smad1/5. Specifically, Smad9 is expressed within cartilage-bone interfaces and joints during early craniofacial development. Smad9 + cells are proliferative and expand during osteoblast regeneration after chemically induced ablation. DM, RA, and PD are drugs known to manipulate skeletal cell fate. RA caused a loss of Smad9 expression and inhibited osteoblast regeneration. PD reduced Smad9 expression at bone and cartilage sites whereas DM had no effect. Smad9 expression changes when joint morphology is altered in mutants and by immobilization. In caudal dermal bone fin regeneration and fracture repair, we observed elevated Smad9 expression in osteoblasts that make newly formed bone.
Conclusions: We show that Smad9 is a marker for an undifferentiated osteochondral precursor population in both larval and adult skeletal tissues. This population can be pharmacologically manipulated and responded to osteoblast ablation in larvae. In adult dermal bone of the caudal fin Smad9 expression is unregulated in response to fracture repair and bone regeneration. Because BMP-inhibitor DM did not show an effect, we showed that Smad9 is likely a repressor R-Smad in vivo. These findings make Smad9 an attractive candidate for an osteoanabolic drug target.
Repairing myeloma bone disease; can the combination of bone anabolic and antiresorptive therapy improve bone quality and repair?

Abstract
Background: Approximately 90% of patients diagnosed with multiple myeloma (MM) will suffer from myeloma-induced bone disease (MBD). MBD causes increased fracture risk, poor mobility and chronic pain for patients. Antiresorptives (eg, zoledronic acid [ZA]) are standard of care, but there is increasing interest in the clinical use of bone anabolics to attempt to repair bone. Inhibitors of TGF-β have shown bone anabolic effects and MBD repair in preclinical studies, but these have not been compared to, or combined with, antiresorptive treatments in a chemotherapy-treated model. Aims: We hypothesized that combination of antiresorptive and anabolic therapies would repair MBD more effectively than either monotherapy. Methods: NSG mice (n = 8/group) were inoculated via tail vein injection with 1 Â 10 6 U266-GFP-Luc human myeloma cells. unit, and therefore this regenerative capacity offers a potential route to identify new potential osteoanabolic factors as they both have mineralized matrix resorbing and building cells. However, it is long thought that zebrafish scales were more odontogenic rather than osteogenic in their formation processes. Results: To address this, we defined the transcriptomic profiles of ontogenetic and regenerating scales of zebrafish. We identified 604 differentially expressed genes (DEGs) that were enriched for extracellular matrix, ossification, and cell adhesion pathways but not in enamel or tooth formation processes. To further determine whether DEGs resembled genes involved in bone formation, we deployed gene set enrichment analyses of DEG human orthologous on monogenic and polygenic association studies. Indeed, we found that human orthologues of DEGs were 2.8 times more likely to cause human monogenic skeletal diseases (p < 8 Â 10 À11 ), and they showed enrichment for human orthologues associated with polygenetic disease traits (UK-Biobank) including stature, bone density and osteoarthritis (p < 0.005). To determine whether zebrafish scales DEGs and their association profiles were evolutionary conserved, zebrafish mutants of two human orthologues that were robustly associated with height and osteoarthritis (COL11A2) or estimated bone mineral density only (SPP1) were phenotypically assessed for both exoskeletal and endoskeletal abnormalities. Consistent with our genetic association studies, col11a2 Y228X/Y228X mutants showed exoskeletal and endoskeletal features consistent with abnormal growth and osteoarthritis. The spp1 P160X/P160X mutants showed mineralization defects in regenerating scales whereas endoskeletal elements depicted elevated eBMD (p < 0.05).
Conclusions: We show that scales have a stronger osteogenic expression profile than previously thought, Despite the fact that there are many differences between scale and endoskeletal developmental processes, our data highlight a relation with bone diseases. Understanding scale regeneration processes beyond their physiological context could therefore reveal novel evolutionarily conserved osteoanabolic processes. Thus, regenerating scales offer an attractive alternative of identifying new genes and pathways involved in bone formation that resemble a sub-population of genes that are relevant to human skeletal disease.
Investigating skeletal architecture in the mdx mouse

Mark Hopkinson, Dominic Wells, Andrew Pitsillides
Royal Veterinary College, London, UK

Abstract
Duchenne muscular dystrophy (DMD), caused by dystrophin gene mutations, elicits poor bone health; DMD patients have short stature, low BMD, osteomalacia, osteopenia, and increased fracture risk. These skeletal characteristics have historically been attributed solely to muscle weakness and correspondingly reduced bone loading. There are, however, studies indicating that dystrophin may have direct roles in skeletal tissues. Most have reported skeletal defects, after the onset of muscle damage, in either mdx mice or human DMD patients. Few, importantly, have focused on whether bone changes may occur sooner, before the onset of muscle damage, and shown similar reduction in cortical and trabecular bone volume and impaired collagen organization culminating in weaker bones in mdx mice. Interrogation of these data reveals that their histological and μCT based phenotyping was however limited only to selected bone regions, highlighting that microarchitectural bone changes in entire mdx bones remained unresolved. My studies address these shortcomings by undertaking an analysis of entire tibia and femur of 3-week-old male mdx (versus wild-type [WT], n = 6). by μCT. Analyses revealed that mdx mice exhibit elevated tibial cortical bone volume (p < 0.05) and a divergent shape (versus WT); rounder cross-sectional profile close to the tibiofibular junction (p < 0.01). Greatest differences were however evident in tibial trabecular regions, where mdx mice exhibited a less mature organization with more numerous, thinner, less separated, highly connected trabeculae (p < 0.001). This was somewhat at odds with the interesting observation that mdx mouse tibiae were also longer (versus WT) (p < 0.05). Analysis of the femur reinforced the findings of greater cortical bone area, which was significantly thicker at the midshaft and distally (p < 0.05). Intriguingly, mdx femoral shape divergence was also aligned with those in tibias, where specific regions also comprised significantly rounder cross-sectional profiles (p < 0.01) and greater predicted indices of bending strength (between 30% and 50% of the proximodistal length) (p < 0.05). Akin to the tibia, the femoral trabecular bone in mdx mice comprised more numerous, thinner, less separated, and less connected trabeculae and, in addition, occupied a significantly smaller medullary area (p < 0.001). Tissue mineral density of cortical bone was also significantly lower in mdx mice, most notably around the knee joint in the distal femur and proximal tibia (p < 0.05).
Overall, this study revealed that mdx mice possess tibial and femoral microarchitecture differences when compared to WT mice prior to the onset of muscle damage, consistent with the hypothesis that dystrophin serves direct roles in skeletal tissues.
Are epiphyseal growth plates sensitive to short-term loadinduced mechanical stimuli?

Abstract
Endochondral ossification (EO), critical during skeletal growth, is a pivotal contributor to the massive morbidity and £billions/year burden of osteoarthritis and delayed fracture repair. EO is sensitive to local biomechanics, yet these control mechanisms remain ill-defined. To decipher these mechanisms, our studies exploit in vivo tibia loading to characterize growth plate (GP) responses to defined mechanical stimuli.
In vivo μCT scanning of 12-week-old mice approaching skeletal maturity revealed that transient applied dynamic compressive loads (six episodes, comprising 40, 2 Hz 12 N cycles over 2 weeks) initially delayed but later accelerated EO, culminating in significantly longer tibias 12 weeks thereafter. Specifically, mean tibia length change (versus starting length) was 36.5% greater (p < 0.01) 8 weeks after commencement of the loading protocol, compared to basal growth-related changes in nonloaded limbs. This acceleration was preserved 12 weeks postloading, when mean tibia length change was 58.9% (p < 0.01) greater than in non-loaded limbs. To pinpoint the specific chondrocyte stage(s) targeted, studies focused on acute load-related changes in GP chondrocyte proliferation optimized in younger 3-week-old or 6-week-old mice with inherently faster growth. BrdU-dosing with immunofluorescent-tracking of labeled nuclear DNA disclosed rapid (6 hours) dynamic load-induced triggering of proliferation in proximal tibial GPs, with markedly raised numbers of labeled nuclei (versus contralateral) predominantly in the lateral compartment, where loads are known to be greater. This indicates that mechanical stimuli influence murine GPs by controlling resident chondrocyte proliferation to accelerate EO. Additional studies analyzing three-dimensional ultra-high resolution joint scans, loaded in real-time, from differently aged mice have disclosed marked deformation within the entirely cartilaginous GP of young 8-week-old mice, which lack the GP bone "bridges" seen in skeletally-mature, 36-week-old or 60-week-old mice. Bone morphology reveals less epiphyseal trabecular bone, more porous basal plates and thinner metaphyseal trabeculae, which imply less effective metaphyseal load-transfer in younger mice. Ongoing digital volume correlation analysis is seeking to measure intra-GP strains directly in such images to define GP deformation in healthy, aging and OA mice for the first time. Current focus is on: (i) developing quantitate methods for regional assessment of BrdU-EdU labeling to define spatiotemporal relationships to GP load-engendered strains; (ii) examining responses at alternative endpoints (4-24 hours) to fully characterize acute proliferative response trajectory; and (iii) histological evaluation of changes in GP zone heights and organization. It is envisioned that merging of studies, measuring load-induced GP deformation, proliferation, changes in zonal organization, bridge formation and growth rates within our tibial loading model will together define roles for load-induced mechanical stimuli in controlling EO.
Transcriptomic analysis reveals a link between mitochondrial dysfunction and experimental chronic kidney disease-mineral bone disorder The Royal Veterinary College, London, UK. 5 The Janssen Pharmaceutical Companies of Johnson and Johnson, Pennsylvania, USA. 6 University of Brighton, Brighton, UK

Abstract
Objectives: Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a systemic disorder that presents with dysregulated mineral metabolism causing ectopic calcification and compromised bone formation. It is characterized by hyperphosphatemia, elevated parathyroid hormone (PTH), as well as fibroblast growth factor-23 (FGF23) levels. Uremic toxins, eg, indoxyl sulfate (IS), that promote oxidative stress are also associated with the development and progression of CKD in humans. To obtain an improved understanding of how this altered systemic milieu results in CKD-MBD, we used whole transcriptome sequencing (RNA-seq) to identify possible molecular mechanisms responsible for the poor bone health in CKD-MBD. Methods: To induce CKD-MBD, male C57BL/6 mice were fed a diet containing 0.6% calcium, 0.9% phosphate, and 0.2% adenine. Control mice received the same diet without adenine. Tissue was analyzed at 5-day intervals over a 35-day time course to identify temporal changes with disease progression. Serum was analyzed for BUN, creatinine, calcium, phosphorus, PTH, FGF23, and bone turnover markers. Urine specific gravity (SG) and albumin and creatinine concentration were determined. The architecture and bone mineral density of cortical and trabecular bone were determined by μCT and histomorphometry. RNA-seq analysis was completed on femurs (marrow removed) and data were analyzed by Graphia software. In vitro experiments of mitochondrial function in murine primary osteoblasts treated with IS (2mM) were completed to extend the RNA-seq results.
Results: CKD mice presented with hyperphosphatemia, hyperparathyroidism, and elevated FGF23 BUN, phosphorus, and serum creatinine concentrations together with decreased urine SG, albumin, and creatinine. Kidney fibrosis was also noted. μCT and histomorphometry revealed osteopenia with structural changes to both trabecular and cortical bone in CKD mice (p < 0.01). Trabecular bone mineralization was decreased (p < 0.01) and bone turnover markers were increased (p < 0.01). Genes with a 1.5-fold change in expression levels (FDR <0.05) from day 20 and 35 time points (CKD versus control) were selected to establish gene clusters associated with CKD. An unexpected finding was the significant downregulation of genes involved in mitophagy, mitochondrial function, and oxidative phosphorylation in CKD bones. In isolated osteoblasts, IS treatment altered mitochondrial morphology, increased production of reactive oxygen species, and reduced Parkin expression, mitochondrial membrane potential, and oxidative phosphorylation.
Conclusion: This is the first transcriptomic study of bone in an experimental model of CKD-MBD. Our findings are consistent with the hypothesis that mitochondrial dysfunction contributes to the altered bone remodeling characteristic of CKD-MBD. The full clinical implications of this conclusion are still to be determined. Abstract Osteoarthritis (OA) of the hip is a painful and debilitating condition that affects over 40 million people just in Europe. One of the two main causes for hip OA is hip dysplasia, which is an instability of the hip joint. This instability is caused by incomplete coverage of the femoral hip by the acetabulum and is commonly observed in humans, including children, as well as in veterinary patients, mainly dogs (Fig. 1A). Currently, the most common surgical methods to correct the dysplasia involve the realignment of the hip socket (osteotomy) or the insertion of a bone graft to enlarge the socket (shelf arthroplasty) (Fig. 1B). Although the osteotomy is invasive, the shelf arthroplasty requires donor tissue. In addition, both procedures only have limited success rates.

Bioengineering
Here, we developed a 3D-printed regenerative, yet stable, bone implant for the treatment of hip dysplasia that can overcome the drawbacks with current treatments. A patient-specific hip implant was designed based on CT scans of three cadaveric dogs (Fig. 1C) and 3D printed using a room temperature extrusion printing process and a biomaterial ink, composed of a magnesium phosphate (MgP) ceramic phase, modified with osteoinductive strontium (Sr 2+ ) ions (MgPSr), and a degradable polycaprolactone (PCL) polymer phase. After implant fabrication, the effect of the implant composition and structure on mechanical stability, fixation to host bone and osteoconductive properties were investigated. To anticipate the in-vivo mechanical performance of the resorbable implant, the implant was tested under physiological loading conditions using a custom-made (bio)mechanical setup. The 3D printing process and biomaterial ink, allowed the successful fabrication of a patient specific hip implant that ensured perfect fit to the hip socket of a canine dysplastic hip (Fig. 1D). Moreover, mechanical and in vitro biological characterization confirmed the flexible and bone-inducing properties of the implant. Finally, ex vivo biomechanical evaluation confirmed that the developed implant can be fixed with metallic screws and, importantly, support physiological loading conditions, even after accelerated in vitro degradation (Fig. 1E,F). This novel implant opens up great perspectives with respect to the treatment of hip dysplasia as it provides a stable, long-term, and regenerative solution that can fully integrate with the host bone tissue. Moreover, the regenerative nature offers great potential for the treatment of young (pediatric) patients.

Abstract
Bone biomineralization is a fundamental process critical to the development of the skeleton and is orchestrated by nanoscopic extracellular vesicles known as matrix vesicles (MVs). MVs are embedded in the mineralizing extracellular matrix and are hypothesized to accumulate amorphous calcium and inorganic phosphate to induce hydroxyapatite mineral nucleation. The phosphatase orphan phosphatase 1 (PHOSPHO1) has been shown to be critical to this process and young PHOSPHO1-null (Phospho1 À/À ) mice exhibit severe bone hypomineralization. The biochemical mechanism through which PHOSPHO1 substrates are generated within MVs, however, is still unknown. Skeletal development in Phospho1 À/À and wild-type animals was characterized in mouse embryos at 17 days of development (E17) using mesoscopic optical projection tomography (OPT) while the ultrastructure was characterized using focused ion beam-scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM). To understand the biogenesis and biochemistry of MVs vesicles were isolated by differential ultracentrifugation from primary osteoblast cultures under mineralizing conditions and subjected to mass spectrometry. OPT revealed a marked loss of mineralized bone throughout the skeleton in Phospho1 À/À animals compared to controls (p < 0.001) affecting both endochondral and intramembranousderived bones. The ultrastructure of wild-type embryonic bone investigated by FIB-SEM exhibited a distinct core of mineralized material associated with a forming surface characterized by mineralization foci propagating through a fibrous matrix (Fig. 1). In contrast, Phospho1 À/À bone was entirely fibrous in places with numerous electron dense particles embedded throughout a collagenous network (Fig. 1). Correlated TEM revealed these to be unmineralized MVs containing osmophilic material but no mineral crystals. Lipidomics performed on vesicle isolates showed a significant enrichment of some lysophospholipid species in Phospho1 À/À MVs (p < 0.05), supporting the hypothesis that the breakdown of vesicle membrane phospholipids contributes to intravesicular generation of PHOSPHO1 substrates. Characterization of the vesicle proteome by mass spectrometry was also performed to gain insights into potential mechanisms of vesicle biogenesis. Protein interaction network analysis revealed a potential role for RhoA/Rac1 GTPase signaling in integrating protein trafficking to determine vesicle cargo and cytoskeletal dynamics during vesicle budding. PHOSPHO1 is critical for embryonic bone development and its ablation prevents mineral nucleation from MVs. The generation of PHOSPHO1 substrates within vesicles likely occurs via breakdown of the vesicle membrane, however further detailed experiments are required to fully elucidate the detailed biochemistry occurring within MVs.

Abstract
Background: Feline tooth resorption (TR) is a painful and progressive disease commonly diagnosed in small animal practice and characterized by loss of the mineralized tissues of the tooth. Surveys of the feline population reports incidences of up to 29% in the mixed breeds but this increases to 75% in some purebred populations. Odontoclasts are the osteoclasts of the dental microenvironment responsible for the resorption of milk teeth to allow eruption of the permanent dentition. Unlike bone, teeth do not remodel so odontoclasts have a very limited role later in life. In TR however the odontoclasts become reactivated and start to attack the adult dentition ( Figure). Methods and Results: We performed RNAseq of the dental transcriptome of TR-ve and TR+ve teeth and identified 1732 differentially expressed genes. Pathway analysis highlighted a number of genes of interest involved in osteoclast differentiation and calcium signaling; three of these have been investigated further: matrix metalloproteinase 9 (MMP9), cathepsin K (CTSK), and purinergic receptor P2X4 (P2X4R). Single nucleotide polymorphism (SNP) analysis of the RNAseq data revealed high-impact SNPs associated with osteoclast differentiation and activity exclusively in TR+ve (coenzyme Q4, myosin heavy chain 13, mitochondria encoded cytochrome B) and TR-ve teeth (MMP14). Immunohistochemistry co-localized the expression of MMP9, CTSK, and P2X4R to TRAP positive, multinucleated cells located in resorption pits on the mineralized surfaces of TR+ve teeth. The effect of inhibition of MMP9 during odontoclastogenesis was investigated using a semi-selective MMP9 inhibitor and siRNAs. Preliminary investigations into the effect of CTSK and P2X4R siRNAs were performed. Drug inhibition of MMP9 caused a dose-dependent reduction in odontoclast numbers and resorption activity. Targeting of MMP9 using siRNA resulted in decreased odontoclast numbers but had no impact on the numbers of resorption pits formed on dentine when compared to scrambled controls. When repeated using osteoplates, a decrease in resorption area was observed between MMP9 and scrambled controls. Significantly less TRAP positive cells formed on plastic following electroporation with siRNAs against CTSK and P2X4R when compared to scrambled controls. Conclusions: By mapping the pattern of expression of these genes and their gene products during odontoclast differentiation and investigating the effect on odontoclast activity when blocking them, we can elucidate their role in disease initiation and progression. This could form the basis for the development of genetic tests to help breed down the incidence in the purebred cat population, as well as identify potential future drug targets. Caloric restriction (CR) is a dietary intervention that involves reduction of total calories below ad libitum (AL) intake without nutritional insufficiency or malnutrition. Over the past 20 years, the potential for CR to delay the onset of age-related chronic diseases and offer direct cardioprotective effects has become increasingly well recognized. However, the effect of CR on VC has yet to be examined. VSMCs from the aortas of CR mice were isolated and cultured under calcifying conditions (3mM Pi). VSMCs derived from CR mice showed reduced calcification compared to VSMCs from AL mice (p < 0.0001). Calcified VSMCs from CR mice show reduced protein expression of runt-related transcription factor (RUNX2) (two fold, p < 0.05). Comparable results were obtained following treatment with the CR mimetic metformin (1μM) and in vitro starvation in cell culture. Investigation of the latter treatment by stable isotope labeling using amino acids in cell culture (SILAC) based quantitative mass spectrometry further identified upregulation of ubiquitin proteasome and autophagy pathway proteins.
To investigate the regulation of RUNX2 by CR, we first blocked its degradation via the ubiquitin proteasome pathway (UPS) with MG132 (0.5μM). Next, we employed Bafilomycin-A (10nM), a selective V-ATPase inhibitor to block autophagic trafficking. Blocking both trafficking pathways increased RUNX2 expression in calcifying VSMCs derived from CR mice (two fold, p < 0.05).
Silencing key genes underpinning UPS (Cullin2 and UBE2H) and autophagy (ATG3 and LAMP1) pathways increased VSMC calcification (p < 0.05). Furthermore, calcified VSMCs treated with metformin show translocation of RUNX2 from the nucleus to the cytoplasm where RUNX2 is present with in the autophagosomes. Silencing Atg3 blocks the translocation of nuclear RUNX2 to cytoplasm suggesting a novel mechanism of RUNX2 degradation via autophagosomes. In summary, our data strongly suggests that CR and the CR mimetic metformin inhibit VC by promoting autophagy and UPS-mediated degradation of RUNX2. This novel finding could lead to the application of CR-mimetics to combat vascular calcification. ethical concerns about the use of animals in research warrants the development of in vitro models that can recapitulate the bone remodeling cycle. Advances in our understanding of the bone remodeling cycle have demonstrated that the osteocyte is central to its control, by regulating the differentiation and activity of the major bone cells involved, the osteoclasts and the osteoblasts. At present, in vitro bone models lack osteocytes, due to the challenging and demanding conditions required for maintaining them in long-term culture. We have developed a Self-Structuring Bone Model (SSBM) that can support osteoblasts and osteocytes long-term and provide a platform for bone remodeling studies in vitro, by building on a successful methodology developed at the University of Birmingham. An immortalized human osteoblast cell line, hFOB 1.19, was seeded and cultured on fibrin hydrogels that were prepared and formed between two calcium phosphate cement anchors. Over a subsequent 12-week culture, the cells cause contraction of the hydrogel scaffold around the anchors, forming a dense fibrin scaffold. The matrix of this scaffold is gradually replaced with a progressively mineralizing, collagen-rich, extracellular matrix, confirmed using imaging techniques including XRF, confocal microscopy, and μCT. The progress of construct formation and maturation was compared against similar constructs seeded with primary rat periosteal cells (as used in the original SSBM development study: Iordachescu et al. 2019) and our initial findings show that this methodology is transferable and repeatable using the hFOB 1.19 cell line. Further work is ongoing to fully characterize the degree of osteocyte differentiation, as well as the development of the constructs' structural composition over the experimental timeframe using qPCR, ELISA, and scanning electron microscopy analysis. The need for a simple, reliable and reproducible in vitro bone model is apparent. Here we describe the first 3D human-derived self-structuring construct of its kind to differentiate and culture osteoblasts and osteocytes long-term. Our ambition now is to use this as a platform to enable complete recapitulation of the bone remodeling cycle in vitro.
Proteomic identification of the targets of the pro-metastatic transcription factor TWIST1 within breast cancer bone metastasis Steven Wood, Ana Lopez, Phillippe Clezardin, Janet Brown University of Sheffield, Sheffield, UK Abstract Rationale and hypothesis: Considerable progress has been made in breast cancer treatment; however, despite this, metastatic spread is currently a major cause of patient morbidity and reduced quality of life. Bone is one of the major sites of metastatic spread within breast cancer, with spread to the skeleton being detectable in over 70% of advanced breast cancer patients. The transcription factor TWIST1 and its downstream target micro-RNA (miR-10b) have been identified as key regulators in the early stages of metastasis of cancers. Mechanistic understanding of the genes and proteins regulated by TWIST1 -miR-10b signaling will yield novel drug targets (and potential biomarkers) relevant to the early stages of cancer spread.
Objectives: The complete set of downstream targets of miR-10b within bone-metastatic breast cancer cells cannot be predicted by in silico analysis alone. Thus, state-of-the-art proteomic methods were used to identify the proteins which alter in expression in response to miR-10b. Methodology: A bone metastatic variant of the triple negative breast cancer cell line MDA-MB-231 (termed BOM2) was transfected with either miR-10b synthetic oligonucleotides or control oligonucleotides. Quantitative proteomics using pulsed SILAC (pSI-LAC) was employed to identify the proteins which alter in expression with the intracellular compartment of these cells in response to miR-10b. In a complimentary study, bio-orthogonal noncanonical amino acid tagging (BONCAT) was used, to identify the proteins differentially secreted by these cells in response to miR-10b. Results: Over 6000 proteins were identified and quantified within the BOM2-cell lysates, with 407 proteins displaying a statistically significant difference in expression (p < 0.05), in response to miR-10b (using four biological replicates) (see Fig. 1). Extensive bioinformatic analysis has resulted a panel of 10 proteins, taken forward for validation. In a parallel series of experiments, the BONCAT method identified over 40 proteins differentially secreted by BOM2 cells upon expression of miR-10b (see Fig. 1). These differentially secreted proteins include key components of extra-cellular matrix remodeling and cell-signaling.
Conclusion: Proteomic analysis of the bone homing BOM2 cell line has identified a panel of both intracellular and secreted proteins, which alter in response to the pro-metastatic micro-RNA miR-10b. Identification of these proteins has provided insights into the mechanism of action of miR-10b within the early stages metastasis. In addition, they are being further pursued as potential biomarkers within the clinical management of breast cancer.

Clinical Cases
Vanishing bones and persistent pain resistant to treatment n 14 of 51 Case: The 23-year-old woman first presented at the age of 3 years with a right distal radius fracture and radiographic evidence of osteolysis affecting her right carpal bones. Genetic testing confirmed heterozygosity for the p.Ser69Leu mutation. Her father presented as an inflammatory type of arthritis at the age of 5 years and was known to have de novo mutation. Due to ongoing pain and progressive osteolysis, she received a number of treatments including IV Pamidronate monthly for 20 months (weight-adjusted dose $37 mg monthly). Due to concerns over long-term use of bisphosphonates at her age, and her ongoing pain, Pamidronate was discontinued and calcitonin (200 IU intranasally followed by subcutaneous injections of 50 IU/3x week) initiated. Osteolysis progressed affecting both her carpal and tarsal bones and she was walking to limited extent by the age of 5 years. Following a drug holiday of 2 years, clinical progression, and ongoing pain, she was treated with IV zoledronic acid (first dose of 0.0125 mg/kg followed by 0.025 mg/kg) every 6 months, and had received four doses in total when she presented with a fracture of her medial column of distal humerus and was admitted with urinary sepsis. In 2011, 40 mg Denosumab injections 6-monthly (four doses) were initiated followed by 2-monthly 60-mg injections (July 2013 to November 2013). Denosumab dose increased to monthly injections from January 2014 until June 2014 when she presented with an atypical subtrochanteric right femur fracture that was managed with an intramedullary nail and 18 months of teriparatide. Unfortunately, her condition has progressed radiologically showing complete osteolysis of the carpal bones, right elbow, humeral trochlear is essentially dissolved, and erosions in both feet. She has a low-level proteinuria managed with an ACEI (father had primary focal segmental glomerulosclerosis). Club-shaped metaphyses and Pamidronate-induced osteopetrosis noted on X-rays. Serial DXA scans show normal bone density and HRpQCT (2011-2017) are unchanged. Conclusion: We present a case of MCTO that has been very challenging to manage. Imaging and genetic testing pays a crucial role in establishing the diagnosis but MCTO management often raises important and difficult treatment questions. Currently, targeted therapies are lacking.
Experiences of a virtual multidisciplinary team meeting process to review unsolved musculoskeletal families from the 100 K Genomes Project

Abstract
To shorten the diagnostic odyssey for rare disease patients, the 100,000 Genomes Project (100KGP) undertook genome sequencing (GS) for rare disease and cancer patients. A subsidiary aim of the project was to establish the utility of GS in the NHS. The musculoskeletal domain recruited 750 families in whom a clinical diagnosis of osteogenesis imperfecta, unexplained skeletal dysplasia, multisuture/syndromic craniosynostosis, or multiple epiphyseal dysplasia had been made (Table). GS data were assessed using standard clinical bioinformatic pipelines focusing on curated gene-panels. Initial diagnostic yields across these categories was 12%-31%. Here we present preliminary findings arising from closer scrutiny of specific families of interest that remained unsolved. Recruiting clinicians were invited to attend a virtual multidisciplinary team (MDT) meeting to present families and assess the results of further bioinformatic analyses. Additional analyses included searching for homozygous loss-of-function variants and manual review of read-alignments for genes implicated based on a strong clinical suspicion for a specific diagnosis. Clinicians were asked to circulate background information prior to meeting, including the suspected diagnosis and sets of genes/ pathways potentially involved. Multiple disciplines attended the meeting, including clinical geneticists, adult and pediatric endocrinologists/rheumatologists, musculoskeletal radiologists and data analysts. Of seven families presented over two sessions, four are now considered solved. This included a three-generational family with suspected Greig syndrome where targeted GLI3 sequencing performed in 2015 had been negative. We identified a 1.2 Mb chromosome 7 inversion disrupting GLI3 ( Figure). A separate scenario involves an affected father-daughter with hypophosphatemic rickets where a 112-kb duplication from 11p15.5 had been inserted into intron 9 of PHEX. A recommendation was made that RNA analysis should be performed to confirm the effects of this rearrangement at the transcript level. Other individuals were identified with homozygous frameshifts in PRKG2 and PKDCC, helping to confirm recently proposed skeletal dysplasia disease genes. For the remaining families, recommendations were made for additional genes to analyze (n = 2), further radiological investigations (n = 1) and sampling of the parents to complete the trio (n = 1).The experience from running our first MDT meetings highlighted the need for close collaboration with the clinical community when analyzing GS data. Furthermore, the process supports a strategy to select high priority genes and pathways based on characteristic phenotypic features. n 16 of 51 examined the relationship between rHOA, including its individual components, and hip pain using a novel dual-energy X-ray absorptiometry (DXA)-based method.
Methods: Left hip DXAs were obtained from UK Biobank. An automated method was developed to obtain minimum joint space width (mJSW) in millimeters (mm) from points placed around the femoral head and acetabulum with a Random Forest machine learning-based approach. Osteophyte areas measured in square millimeters (mm 2 ) at the lateral acetabulum, superior and inferior femoral head were derived manually. Semiquantitative measures of osteophytes and joint space narrowing (JSN) were combined to provide a measure of rHOA. Logistic regression was used to examine the relationships between these variables and hip pain, obtained via questionnaires. Separately, acetabular dysplasia (AD) is thought to cause OA via joint instability due to lack of acetabular coverage of the femoral head. We aimed to examine whether the aforementioned hip morphologies are associated with radiographic hip osteoarthritis (rHOA) and hip pain, and, if so, whether they are associated with a distribution of osteophytes in keeping with FAI or more global joint degeneration.
Methods: Left hip dual-energy X-ray absorptiometry (DXA) scans taken on UK Biobank participants had alpha angle (AA), lateral center-edge angle (LCEA), and joint space narrowing (JSN) derived using automatically placed points around the hip. Cam and pincer morphologies, and AD were defined using AA and LCEA ( Fig. 1). Osteophytes were measured manually and the points were moved so as not to encompass them. rHOA grades were based on the presence of JSN and an osteophyte.
Results: A total of 6807 individuals were selected (mean age: 62.7 years; 3382/3425 males/females). Cam morphology was more prevalent in males than females (15.4% and 1.8%,

Abstract
Background: Musculoskeletal conditions are a leading contributor to disability across sub-Saharan Africa, with prevalence increasing with age. Understanding people's beliefs about health and illness in midlife can improve prevention and ensure healthcare delivery is appropriately designed, enabling healthcare to reach the people who need it. This study aimed to characterize the understandings and beliefs of midlife women towards musculoskeletal health in older age in Zimbabwe.
Methods: Twenty semi-structured interviews were conducted with women aged 40-60 years in Harare, Zimbabwe. Purposive sampling identified women, taking into account age, comorbidities, HIV status and socio-economic background. Data were audio-recorded, transcribed and analyzed thematically.
Results: Participants provided their understandings about what happened to women's bones as they aged. These most commonly included joint, back, and leg pain. Most women described this as "eating," "burning in the bones," "biting," and "aching." Some talked about bones "losing strength," making it more difficult to participate in everyday life. There was a belief that bones no longer healed after fracture or dislocation and that they could become brittle, "loose," and fall out of place. Several contrasted these changes to the bones of children that they described as "healthy" and "active." Many women were unsure what caused these changes and saw them as an inevitable part of aging. Several provided biological explanations about arthritis, with participants explaining that loss of "gel" (synovial fluid) caused friction and pain. Being overweight was linked to musculoskeletal problems. Some thought excess weight made bones "ache" because of increased pressure, another thought that being overweight made them more likely to break bones if they fell. Participants also said that the bones in older people moved out of place due to lack of bone marrow. Inadequate nutrition was mentioned as a cause of musculoskeletal problems, both in terms of the types of foods older people ate and the quantity. Participants also talked about the impact of women's lifestyle that they contrasted with men's. Several thought that working hard, "carrying the weight of a man" during intercourse, pregnancy, and childbearing made their bones weaker. One woman attributed her joint pain to supernatural causes or "witchcraft." Conclusions: This study provides novel findings about understandings of musculoskeletal health and aging in midlife women in Zimbabwe. Information can be used to develop contextually appropriate educational and treatment interventions and enhance communication with healthcare professionals, improving musculoskeletal health in midlife women before they enter older age.

Abstract
Osteoarthritis is the most prevalent systemic musculoskeletal disorder characterized by articular cartilage degeneration and subchondral bone sclerosis. Suppressor of cytokine signaling 2 (SOCS2) is an intracellular negative regulator of growth hormone signaling, thus mice deficient in SOCS2 (Socs2À/À) display accelerated bone growth. Here we sought to examine the contribution of accelerated growth to osteoarthritis development using this murine model of excessive longitudinal growth. We examined the vulnerability of 16 week-old male Socs2À/À mice to osteoarthritis following surgical induction of disease (destabilization of the medial meniscus [DMM]), and with aging (12-13 months old), by histology and micro-computed tomography (μCT). We observed increase in the number and density of growth plate bridges in Socs2À/À tibias in comparison to wild-type (WT), regardless of osteoarthritis intervention (Fig. 1 p < 0.05). Articular cartilage lesion severity scores (mean and maximum) were similar in WT and Socs2À/À mice with either DMM, or with aging. μCT analysis revealed decreases in subchondral bone thickness, epiphyseal trabecular number and thickness in the medial compartment of Socs2À/À knee joints, in comparison to WT (p < 0.001). DMM had no effect on subchondral bone plate thickness in comparison to sham in neither WT nor Socs2À/À knee joints. Together these data from the study of Socs2À/À mice suggest that enhanced growth hormone signaling on long bones accelerates growth plate fusion but has no effect on osteoarthritis vulnerability in this model.   Abstract Introduction: Increased fat mass may increase bone mass through greater biomechanical load on bones. Further, fat mass has an endocrine function which may influence bone metabolism both positively and negatively. The mediating role of endocrine factors in the positive relationship between fat mass and bone mass in prepubertal and early-pubertal children is unclear. The aim of this study was to examine the association between fat mass and bone mineral content (BMC), and to investigate whether this relationship is mediated by insulin, free leptin index, adiponectin, dehydroepiandrosterone sulfate, testosterone, and estradiol in girls and boys aged 9 to 11 years. Methods: We utilized cross-sectional data from the 2-year follow-up of the Physical Activity and Nutrition in Childhood study, an ongoing longitudinal study in a population sample of Finnish children (n = 396; 203 girls). Total body less head (TBLH) BMC and fat mass were assessed with dual-energy X-ray absorptiometry. Endocrine factors were assessed from fasted venous blood samples. We applied the novel four-way decomposition method to analyze associations between fat mass, endocrine factors, and BMC, adjusting for age, stature, pubertal status, lean mass, and baseline BMC. The four-way decomposition method allows the total effect of an exposure on an outcome to be decomposed into a controlled direct effect, a reference interaction, a mediated interaction, and a pure indirect effect. Results: Fat mass had a positive controlled direct effect on BMC in girls and boys (β = 0.033 to 0.68, p < 0.001). We observed a negative interaction between fat mass and adiponectin with BMC in girls (β = À0.003, p = 0.033), and a negative mediated interaction between fat mass and free leptin index with BMC in boys (β = À0.007, p = 0.007).
Conclusions: In children with greater levels of adiponectin and free leptin index, the relationship between fat mass and BMC became less positive, in girls and boys, respectively. Fat mass primarily positively influenced BMC through pathways not related to the endocrine factors we assessed, likely through mechanical loading. Because the relationship between fat mass and endocrine factors with BMC is likely moderated by weight status and pubertal stage, further research is needed to assess whether these observations extend to children and adolescents with overweight and obese weight status. respectively), such that of 187 postmenopausal women, the 31 with HIV had 0.035 (95% confidence interval [CI], 0.004-0.065) g/cm 2 lower TB-LH BMD and 0.056 (95% CI, 0.008-0.104) g/cm 2 lower total hip BMD than the 156 without HIV. Conclusion: Osteoporosis rather than sarcopenia is the common musculoskeletal disease of aging in rural South Africa; postmenopausal women with HIV may experience greater bone losses than women without HIV. Findings raise concerns over future age-related fracture risk in Southern Africa, and suggest HIV clinics in South Africa should consider routine bone health assessment, particularly in postmenopausal women.
The impact of adiposity and lean mass on bone health and metabolic profile in Asian women of reproductive age Abstract Background and Objectives: It is increasingly recognized that the risk of fractures is increased in individuals with type 2 diabetes, challenging the traditional view of a positive influence of adiposity on bone mineral density (BMD). Obesity increases fat deposition in muscle tissue and production of inflammatory cytokines. These cytokines can interfere with bone resorption and bone formation, influencing overall bone health negatively. These findings underscore the complex interrelationships between fat mass (FM), lean mass (LM), and bone health in obesity and metabolic diseases. Women tend to have higher degree of adiposity, lose bone earlier and faster compared to men. We therefore investigated the impact of LM, FM, and metabolic profile on bone health in Asian women of reproductive age.
Methods: A total of 191 Asian women of reproductive age from the Singapore Preconception Study of Long-Term Mother and Child Outcomes (S-PRESTO) were studied. BMD of femoral neck (BMD FN ), lumbar spine (BMD LS ), whole body (BMD WB ), LM, and FM were measured by dual-energy X-ray absorptiometry (DXA) scans. Metabolic markers fasting plasma glucose (FPG), 2-hour plasma glucose (2-hrPG), fasting plasma insulin (FPI), blood lipid concentrations, and high-sensitivity C-reactive protein (HS-CRP) were measured. incidence of hip fracture will increase sixfold in Africa and Asia by 2050. To date there are no longitudinal studies focusing on musculoskeletal aging in SSA. The aims were to describe and characterize changes in bone mass of older adults and to determine prevalence of osteoporosis and osteopenia in the population. Methods: The Gambian Bone and Muscle Aging Study (GamBAS; ISRCTN17900679) is a prospective observational study in women and men from rural Gambia. Participants were recruited into sexstratified, 5-year age bands. The primary outcome was change in total hip areal bone mineral density (aBMD) over a 1.5-year to 2-year period (follow-up was randomized to account for seasonality). A GE-Lunar Prodigy scanner was used to obtain proximal femur scans for assessment of aBMD. Annualized percentage change in aBMD was calculated. Total hip BMD T-scores were calculated using National Health and Nutrition Examination Survey (NHANES) data and Z-scores were calculated using manufacturer reference data as per Conclusions: These are the first longitudinal musculoskeletal DXA data in an older-adult population from SSA. The change at the hip was comparative to that in higher-income countries where fracture risk is high. Over 40% of women were osteopenic and osteoporotic. These data provide important insights into musculoskeletal health in The Gambian population, which was previously thought to be at low risk of fracture.

Abstract
Background: Osteoporosis is of concern given its association with fragility fractures. Adipose tissue, a source of inflammatory markers and adipokines, is a potentially important determinant of poorer bone health.
Methods: The MRC National Survey of Health and Development (NSHD) is a prospective birth cohort study; 766 men and 820 women included in these cross-sectional analyses had DXA (total hip, spine, whole body areal BMD [aBMD]) and pQCT (radius: 4% and 50% sites) measurements. Multiple linear regression, stratified by sex, was used to determine associations between inflammatory markers (IL-6, adiponectin, and leptin) and bone adjusted for fat mass residuals (the residual of fat mass after adjustment for lean mass and height Conclusions: Overall, these results highlight differing associations between markers of inflammation and adiposity with bone, which might influence the aging process. Although sex interactions were not tested, these results suggest that some associations may differ in men and women.
Epigenetic age acceleration associations with skeletal outcomes: Differential impacts in men and women however, data from the sub-Saharan African region are scarce. Yet, this century, the number of older adults in sub-Saharan Africa is expected to grow faster than any other region globally. We aimed to use emerging data on hip fracture incidence in South Africa to estimate future burden of hip fracture for the country over the next three decades.
Methods: Previously collected data on hip fracture patients from eight districts within the Gauteng, KwaZulu-Natal, and Western Cape regions of South Africa were re-analyzed. All patients aged ≥40 years with a radiograph-confirmed hip fracture over a 12-month period in one of 94 included hospitals were enrolled. High-velocity trauma, pathological and periprosthetic fractures were excluded. Age-, sex-and ethnicity-specific incidence rates were generated and standardized to the 2011 South African census population and to future South African population projections estimated by the United Nations (UN Chronic renal failure is the main cause of metastatic calcifications. Different factors predispose the dialysis patient to calcium salt deposits. We report two cases of tumors calcinosis in a hemodialysis patients. Case reports Case 1: A 61-year-old-patient who is followed for hypertensive nephropathy for which he is under periodic hemodialysis. He consults for mechanical pain in the upper extremity of the right leg and both shoulders. Standard radiographs revealed polycyclic, heterogeneous formations, occupying the periarticular space of the two shoulders, especially on the right one. In the right knee, there is a multiple calcifications of the superior and postero-external soft tissue. The magnetic resonance imaging (MRI) notes multiple images of macro-calcifications (Fig. 1). The biological assessment found calcemia at 103 mg/L, hyperphosphatemia at 71 mg/ L, hyperparathyroidism at 128.50 pg/mL, and uric acid at 69 mg/L. Case 2: A 53-year-old man in hemodialysis for 6 years. He consults for mechanical knee pain. Physical exam found a 6-cm swelling at the latero-extremity of the left knee. Radiograph and ultrasound revealed polycyclic and heterogenous formations retrocondylar (Fig. 2). Biological exam found hyperphosphoremia at 60 mg/L, calcium at 110 mg/L, and hyperparathyroidism at 300 pg/mL. Discussion: Different factors predispose the dialysis patient to calcium salt deposits: hyperparathyroidism, elevation of phosphocalcic product, excess intake of calcium or vitamin D, poor compliance of the patient with regular intake of chelators, insufficient dialysis, or a dialysate rich in aluminum.
The clinical context and the radiological data made the diagnosis of pseudotumoral calcinosis in our patients with chronic dialysis. The management is based on the correction of the Ca/ph product. The reduction of calcifications go through the correction of hyperparathyroidism or the increase of dialysis sessions with low-Ca dialysate, while checking the aluminum level in the dialysate. Kidney transplantation can promote rapid decrease in calcifications and is desirable in cases of severe calcinosis. Surgery with as complete a resection as possible is recommended when there is a risk of vascular damage or on joint mobility. direct links to bone mineralization and strength via sex-specific gene expression (Goring et al. (2) ). Herein, we aimed to identify whether sexual dimorphism in the cortical vasculature exists in humans during arduous military training. We have undertaken high-resolution peripheral quantitative computed tomography imaging of age-matched male (n = 5) and female (n = 6) British Army Officers during 1, 14, 28, and 44 weeks of training. These diaphyseal tibial scans with a voxel size of 61 μm were imported into ImageJ, where intracortical blood vessel canals were manually separated from trabecular pores to extract cortical porosity (Ct.Po), cortical bone tissue volume (Ct.TV), canal volume (Ca.V), canal diameter (Ca.Dm), and canal density (Ca.Dn). Significant sexual divergence in cortical parameters was detected from baseline: Ct.Po (p < 0.05), Ct.TV (p < 0.001), and Ca.Dn (p < 0.005) were higher, Ca.V was similar (p > 0.05), and Ca.Dm was lower (p < 0.001) in men than women across all time points (p > 0.05 for exercise in every parameter). Men therefore have more and narrower blood vessel canals than women, who have fewer but wider canals. Our results indicate that regulation of the cortical vasculature may be sex-specific, which could contribute to increased stress fracture risk in women compared with men. These findings can aid future development of exercise regimes targeting bone injury prevention in both sexes.

Abstract
Introduction: Osteosarcoma (OS) is the most common primary cancer of bone, typically describing highly aggressive pediatric tumors characterized by an aberrant, immature, extracellular matrix (ECM) (Shapiro (1) ; Shapiro and Eyre (2) ). The manipulated matrix of OS is believed to drive malignant progression, though mechanisms for which are yet to be concluded (Cui et al. (3) ). In recent years, novel approaches such as nonlinear microscopy have been utilized in the field of oncology for rapid, noninvasive detection of tumors-via examination of the ECM-as well as uncovering potential therapeutic targets (Nadiarnykh et al. (4) ; Provenzano et al. (5) ). Herein, we aim to investigate the potential of multimodal, nonlinear microscopy for distinction of OS from healthy bone. Method: Multimodal, nonlinear microscopy-including second harmonic generation (SHG) imaging and two-photon excitation fluorescence (TPEF)-was employed to examine decalcified and paraffin-embedded human bone and OS tissue sections. The imaging protocol was optimized on human bone biopsies (n = 3 male n = 3 female). Image analysis by ImageJ and CT-FIRE-enabled quantification of matrix-collagen parameters including fiber length, width, angle, straightness, and number.  (Fig. 1A,B) and OS tissue (Fig. 1C,D). Conclusion: Further investigations into matrix disparities between normal bone and various malignancy grades of OS will provide clearer insight into potential diagnostic signatures that may be used in clinical settings for OS diagnosis. This preliminary data highlights the promising potential of nonlinear microscopy for research and diagnosis of OS. Our continued work aims to utilize nonlinear, optical modalities for in-depth characterization of the unique OS matrix and elucidate a potential diagnostic Introduction: Triaxial accelerometers are widely used to assess physical activity; however, this is generally in terms of energy consumption; methods for classifying the output in terms of different types of activity, of relevance to effects of exercise on the skeleton, are not currently available.
Purpose: The purpose of this study was to assess the accuracy of four machine learning models (k-Nearest Neighbors (NN) Manhattan, k-NN Euclidian, Decision Tree (DT), and Support Vector Machine (SVM)) on a binary movement classification (stationary and walking) and a tertiary movement classification (stationary, walking, and running) from postmenopausal women. Methods: Eighty postmenopausal women performed a submaximal incremental shuttle test on an indoor track, of which 30 also performed the same test on an indoor treadmill, all while wearing a hip-worn triaxial accelerometer. The raw accelerometer data were preprocessed by filtering out noise and segmenting the data into 2-second segments per subject. A feature set was thengenerated that consisted of four different wavelet decompositions across three levels using the Daubechies 2 mother wavelet. To evaluate the method using leave-one-out validation, each feature set per subject was used sequentially as the test set for the four machine learning models, with the remainder of the subjects making up the training set.

Results:
The highest mean leave-one-out classification accuracy for the binary classifier, 99.57% AE 1.383%, was produced by the k-NN Manhattan classifier. The highest mean leave-one-out classification accuracy for the tertiary classifier, 92.64% AE 5.385%, was also produced by the k-NN Manhattan classifier. Sixty (60) of 110 tests for the binary k-NN Manhattan classifier produced results of 100% with two tests that produced results significantly lower than the mean (>3 standard deviations [SDs]). The tertiary k-NN Manhattan classifier's highest individual test accuracy was 99.78% and had three tests that produced results significantly lower than the mean (>3 SD).
Conclusions: The k-NN Manhattan classifier trained with triaxial accelerometer data provided accurate recognition of specific types of activity in postmenopausal women.
The mediating role of lean soft tissue in the relationship between somatic maturation and bone density in adolescent practitioners and non-practitioners of sports spine and total body less head [TBLH]) and LST. The mediation analyses were performed using the command "med4way" on Stata 15.1, with statistical significance level at p < 0.05. The theoretical mediation model is presented in Figure 1.
Results: For both sexes, LST mediated the association between somatic maturation and aBMD at all skeletal sites (mediation percentage ranging from 36.3% to 75.4%). For sport and non-sport groups, the LST also mediated the association between somatic maturation and aBMD at all skeletal sites (mediation percentage ranging from 51.6% to 85.6%). The direct effect was observed in all groups, except for lower limbs and TBLH in the non-sport group. Conclusion: The association between somatic maturation and aBMD was mediated by LST in adolescents of both sexes and regardless of involvement in organized sports. Our findings highlighted the role of improving LST to mitigate the association of somatic maturation with aBMD.

Department of Rheumatology, Ibn Rochd University Hospital
Center, Hassan II University, Faculty of Medicine and Pharmacy.

Casablanca, Casablanca, Morocco
Abstract Background: Pycnodysostosis, or Toulouse-Lautrec syndrome, is a rare autosomal recessive skeletal dysplasia, due to a defect in the gene encoding cathepsin K. Its prevalence is 1/100,000. It is characterized by short-limbed, short stature, typical facial appearance; convex nasal ridge and small jaw with obtuse mandibular angle, osteosclerosis with increased bone fragility, acroosteolysis of the distal phalanges, delayed closure of the cranial sutures, and dysplasia of the clavicle. The diagnosis is usually based on the clinic and bone X-rays, or better by the study of the gene abnormally located on the cathepsin K gene (1q21). We present two isolated and separate cases, with typical clinical-radiological criteria, who were admitted for the assessment of bone status. We describe a 4-year-old Emirati child with CSHS, whose hypophosphatemic rickets and dysplastic skeletal lesions failed to heal after 1 year of treatment with oral phosphate supplements and alfacalcidol (conventional treatment). The diagnosis of CSHN with FGF23-mediated hypophosphatemic rickets was made in our patient due to increased urinary phosphate excretion and hypophosphatemia, in the face of normal serum parathyroid hormone (PTH) levels and inappropriately elevated plasma FGF23 levels ( Table 1). The whole-exome sequence on cells from nevoid skin biopsy revealed a somatic missense variant c.182A>G p.(Gin61Arg) (chr11:533874;hg19) in the HRAS gene (OMIM *190020; chromosome 11p15.5).
Results: Recently, burosumab, a fully human immunoglobulin G1 monoclonal antibody to FGF23 was approved for the treat-ment of children with X-linked hypophosphatemia (XLH), a multisystem disorder caused by increased expression of FGF23. Treatment of our patient with burosumab, for 12 months, resulted in normalization of her serum inorganic phosphate and alkaline phosphatase levels (Graph 1), healing of rickets, and improvement in her symptoms of myopathy and quality of life. Conclusion: To the best of our knowledge, this is the first case report to describe the usage of the new treatment modality (burosumab) in a child with CSHS. Burosumab may have a role in the treatment of CSHS and dysplastic skeletal lesions.
Ipsen, Newton, MA, USA. 8 Ipsen, Slough, UK Abstract Background: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare, severely debilitating genetic disorder characterized by episodic heterotopic ossification (HO) and flare-ups. Identification of biomarkers would be useful to predict flare-ups, monitor FOP disease progression, and to identify when interventions are warranted and assess responses to these interventions.   were reported in two patients (flank pain, musculoskeletal/ connective tissue disorder, and unknown); these were not related or unlikely related to treatment. No patient discontinued because of an AE or met prespecified discontinuation criteria. Conclusion: The data suggest that AA improves mobility, pain, and HRQoL in adults with pediatric-onset HPP, with a favorable benefit/risk profile. We describe the muscle function of children with NF1 using a range of assessment tools, and have studied the relationship with other features of NF1 such as small muscle size, poor balance, perceived fatigue, and poor quality of life. Methods: Children had their dynamic muscle function measured using Leonardo Mechanography (Novotec Medical), which measures peak power and force generation on single twolegged jumping and multiple one-legged hopping, respectively, and also jumping efficiency and balance. Grip force (isometric muscle function) and 6-minute walk test (6MWT, assessing functional capacity) were also measured. Magnetic resonance imaging of the right thigh was undertaken to measure crosssectional area of knee extensor muscles. Questionnaires of perceived fatigue (PedsQL Multi-dimensional Fatigue Scale) and quality of life (Child Outcome Rating Scale) were also completed. Data was compared to age-and gender-specific reference data where available, to calculate standard deviation scores (SDS). Results: A total of 44 children were recruited (20 males, 24 females; age range 6.1-16.5 years, mean age 10.6 years). Children with NF1 had significantly reduced jumping power SDS (mean À2.1; 95% confidence interval [CI], À2.5 to À1.8), jumping efficiency SDS (mean À1.2; 95% CI, À1.5 to À0.9), hopping force SDS (mean À2.4; 95% CI, À2.8 to À1.9), grip force SDS (mean À2.4; 95% CI, À2.7 to À2.1), and 6MWT SDS (mean À2.4; 95% CI, À2.8 to À2.0) (p < 0.001 for all), regardless of age or gender. Muscle size strongly correlated with jumping power (r = 0.80, p < 0.001), jumping efficiency (r = 0.45, p = 0.003), hopping force (r = 0.54, p < 0.001), and grip force (r = 0.53, p < 0.001), even when controlling for age, gender, height, and weight. No correlations were evident with measures of muscle function for balance performance, fatigue scores, or quality of life scores. Discussion/Conclusion: Children with NF1 have very significant impairments in a variety of muscle function tests, indicating deficits in strength, endurance, and dynamic muscle function. These deficits exist throughout childhood, and regardless of gender. These deficits in muscle function correlate with muscle size, suggesting this impairment is at least partly due to issues with muscle bulk, highlighting the potential for improvement with physiotherapy exercises aimed at improving muscle bulk. These deficits do not seem to strongly impact on other features of NF1, presumably because these features are multifactorial in etiology.

IMS and SUM Medical College and Hospital, Bhubaneswar, India
Abstract Introduction: Pachydermoperiostosis (PDP) is a rare disorder characterized by digital clubbing, thickening of the skin (pachyderma), hyperhidrosis, and new periosteal bone formation. PDP can sometimes clinically mimic acromegaly due to many similar findings in both of these disorders. We describe an interesting case of a young male who was referred for evaluation of acromegaly but was subsequently diagnosed to have a complete form of PDP. Case Description: A 22-year-old male presented with complaints of enlargement of distal ends of hand and feet, coarsening of facial features and increased seborrhea for around 8 years. There was no history of chronic fever, headache, or cardiac, pulmonary, hematologic, or gastro intestinal disease in past. There was no history of similar complaints in any family members. Clinical examination revealed presence of bilateral grade IV digital clubbing in upper and lower limbs, prominent facial furrows, hyperhidrosis, and bilateral blepharoptosis. The rest of the systemic examination was clinically unremarkable. Complete blood count, hepatic function tests, renal function tests, serum electrolytes, and glycemic parameters were within normal limits. Chest X-ray and 2D echocardiogram were normal. Hormonal analysis including thyroid function tests and insulinlike growth factor 1 were normal. Two-hour post-glucose growth hormone values were suppressed. X-ray of both hands and feet showed cortical thickening, periosteal reaction, and bulbous enlargement of finger tips. Based on the above findings a diagnosis of pachydermoperiostosis was made. The patient was counseled regarding the nature of the disease and advised regarding use of nonsteroidal anti-inflammatory drugs (NSAIDs) on a short-term as and when required basis for symptomatic management. Discussion: PDP is a rare inherited form of primary hypertrophic osteoarthropathy. PDP can be diagnosed based on established major and minor criteria. The three major criteria include pachyderma, periostosis and digital clubbing. Similarly, the minor criteria include hyperhidrosis, arthralgia, gastric ulcer, cutis verticis gyrata, blepharoptosis, joint effusion, edema, seborrhea, acne, and flushing. The disease has a male preponderance. The three major forms of PDP include: complete form (involving all three major symptoms); incomplete form (presence of periostosis but without pachyderma), and forme fruste (presence of pachyderma with minimal or no skeletal anomalies). Symptomatic management with NSAIDs, analgesics, and bisphosphonates have been tried. Apart from acromegaly, other clinical mimics include psoriatic arthritis, rheumatoid arthritis, and even thyroid acropachy. Conclusion: PDP is a rare disorder that can clinically simulate acromegaly. Knowledge about this entity among physicians would be helpful for early identification and appropriate management.

Abstract
Although muscle weakness is commonly reported in adults with X-linked hypophosphatemia (XLH), there is a dearth of information on multiple components of physical function in this population. We examined upper body strength, lower body power, functional capacity, mobility, and physical activity (PA) level and explored the relationships among these variables in adults with XLH. Participants were recruited as part of a UK-based prospective cohort study, the RUDY Study. They underwent a clinical visit and physical examination, including assessment of handgrip strength, jump power (mechanography), 6-minute walk test (6MWT), short physical performance battery (SPPB), and the International Physical Activity Questionnaire (IPAQ). Performance data were analyzed using parametric and nonparametric tests, whereas correlations between physical activity and performance data were assessed by univariate analysis. Twenty-nine adults with XLH (52% males) with a mean age of 45.5 AE 15.9 years were recruited. Jump power and 6MWT distances were 47% and 39% lower, respectively, in individuals with XLH compared with normative values (p < 0.0001), whereas handgrip strength values were similar to expected values (p = 0.51). Twelve of 29 participants (41%) had an SPBB score of ≤9, indicating impaired mobility. Low and moderate PA levels were noted in 72% and 28% of the participants, and none were classified as highly active. These deficits were similar in individuals of both sexes and were not associated with age. Univariate analysis revealed only a correlation between PA and exerciseinduced feeling inventory (EFI) (r = 0.505, p = 0.023). Adults with XLH are characterized by marked deficits in lower limb muscle power and functional capacity, with a high incidence of impaired mobility and inactivity. In addition to metabolic effects of XLH, low physical activity may contribute to deficits in lower limb power. Further studies are required to understand underlying mechanisms and to develop novel treatment approaches to improve physical function and mobility.

Abstract
Introduction: This study aimed to identify novel monogenic causes of unexplained high bone mass (HBM), defined as a total hip and/or first lumbar vertebral bone mineral density (BMD) Zscore of ≥+3.2.
Methods: All participants in the UK HBM cohort (355 HBM cases, 200 unaffected relatives) underwent clinical assessment and DXA scanning. Whole-exome sequencing (WES) was undertaken in likely informative pedigrees, with data analyzed for carriage of at least one novel or rare (minor allele frequency [MAF] <0.005) nonsynonymous single-nucleotide variant (SNV) or indel in a highly conserved region of a gene, segregating with HBM within the pedigree. Data were then filtered based on functional prediction using Polyphen-2 and Sorting Intolerant From Tolerant (SIFT). WES data from the UK HBM cohort and 126 HBM cases in the Anglo-Australasian Osteoporosis Genetics Consortium (AOGC) were then interrogated to identify other individuals carrying either the same or another rare variant within the same gene. WES data from 493 low bone mass (LBM) cases in AOGC were analyzed to ensure absence of the variant. Variants were validated using Sanger sequencing. Protein homology modeling was performed using the SwissModel server with visualization in PyMOL. Results: Exome sequencing in a pedigree with unexplained and apparently autosomal dominant HBM (Fig. 1)

Abstract
Background: Despite their rigid appearance, bones are dynamic organs going through continuous remodeling cycles. Osteoclasts are responsible for bone resorption and their activation is regulated by hormones, cytokines, growth factors, and physical properties of the bone microenvironment. Changes to this microenvironment can have profound effects on both osteoclastogenesis and osteoclast resorption activity. The aim of this project was to gain a better understanding of how an altered microenvironment influences osteoclast biology using companion animal cancers as a model of disrupted microenvironment. Feline oral squamous cell carcinoma (FOSCC) and canine osteosarcoma were selected because they represent good models of their human counterparts and they have the ability to invade bone and alter osteoclast activity in vivo. Method: A novel FOSCC cell line was isolated from a bone invasive tumor and characterized. This bone invasive cell line, and a previously characterized, non-bone invasive FOSCC cell line were used. Conditioned media from the cell lines was added during osteoclast differentiation from feline bone marrow precursors in presence of colony stimulating factor 1 (CSF-1) and receptor activator of nuclear factor κB ligand (RANKL). Similarly, the effect of four different canine osteosarcoma cell lines conditioned media was tested on osteoclast differentiation from canine bone marrow cells. To determine which factors (known and unknown) might have an effect on osteoclast differentiation and activity, conditioned media from all the cell lines tested was analyzed with mass spectrophotometry. Results: FOSCC conditioned media was able to enhance osteoclast formation and resorption activity on mineralized substrates. Moreover, when cultured in hypoxic conditions (0.5% O 2 ) the presence of FOSCC conditioned media positively influenced survival of feline osteoclast precursor cells. Of the four canine osteosarcoma cell lines tested, only one showed an enhancing effect on osteoclastogenesis on plastic. A list of proteins emerged from mass spectrophotometry analysis of the conditioned media. These were searched against the literature to identify possible candidates able to mediate the effects observed experimentally.
Conclusions: The FOSCC cell lines enhancing effect observed on osteoclast differentiation and activity is likely to be mediated by factors secreted by the cell lines in their microenvironment (the conditioned media). Some of the proteins identified by mass spectrophotometry have already been recognized to modulate osteoclast biology but many others have not. It would be of interest to establish functionally which proteins might be having the effects observed because they could represent potential therapeutic targets.

Bioengineering
Regional segmentation of the murine bone cortex exposes spatial heterogeneity in lacunar: vascular organization Jacob Trend, Alisha Sharma, Patricia Goggin, Lysanne Michels, Heather Norman, Katrin Deinhardt, Phillipp Schneider, Claire Clarkin University of Southampton, Southampton, UK

Abstract
Mechanical stressors are distributed heterogeneously across the bone cortex, and variation in regional cortical microarchitecture may reflect historical loading responses. Spatial alterations in bone microarchitecture have also been reported in skeletal pathology including osteoporosis. (1) Although cortical microarchitecture comprises of allied networks including osteocyte lacunae and intracortical canals, the mechanisms driving their communication and organization remain poorly understood.
Here, we describe an automated means to regionalize the bone cortex and characterize microstructural heterogeneity within the adult murine tibias using high-resolution synchrotron radiation computed tomography (SR CT). The tibia-fibula junction (TFJ) of 13-month-old male mice (n = 3) was scanned postmortem by SR CT at 1.65 μm and an automated segmentation tool developed to isolate anterior, posterior, medial, and lateral regions, utilizing a combination of functions involving BoneJ's moment of inertia function within Fiji software. To investigate regional interactions between vascular canals and the lacunar network, 3D distance mapping tools were also developed within Dragonfly 2021.1. Consistent with literature, (2) the posterior region exhibited greater canal density (0.23 per/100 μm 10-3 AE 0.11) versus the anterior (0.07 per/100 μm 10-3 AE 0.02), lateral (0.065 per/100 μm 10-3 AE 0.002), and medial (0.072 per/100 μm 10-3 AE 0.006) regions (p = 0.02; one-way ANOVA). Osteocyte lacunae were also largest in the posterior region (652.7 μm 3 AE 47.01) versus anterior (338.6 μm 3 AE 64.03), and lateral (410.0 μm 3 AE 14.7) regions (p = 0.003; one-way ANOVA). Evidence suggests that diffusion from vascular supply should support osteocyte survival to a distance of 100 μm. (3) Computational 3D mapping of osteocyte lacunae distances from a vascular source comprising either (i) endosteal surface, (ii) periosteal surface, or (iii) intracortical canals across the TFJ showed that 6.4% AE 0.44% of lacunae were located >100 μm from a site of vascular supply. When distance mapping was performed in the absence of intracortical canals 26.8% AE 2.9% of lacunae within the posterior region were positioned >100 μm from a bone surface, suggesting that in this region a distinct subpopulation of osteocytes are "reliant" upon the vascular canals for survival. Furthermore, these "reliant" osteocyte lacunae found that they were significantly larger around the posterior intracortical canals JBMR Plus (WOA) n 36 of 51 (918.6 μm 3 AE 47.7) versus lacunae in the anterior (429.3 μm 3 AE 126.4, p < 0.05) and lateral (494.2μm 3 AE 41.8, p < 0.05) regions.
Our study demonstrates heterogeneity in the bone vascular microstructure that is aligned to osteocyte lacunae size and spatial arrangements, and stresses the importance for regional study of cortical heterogeneity for optimal assessment of bone health and fracture risk.
PubMed and Cochrane databases were searched using the keywords "cold atmospheric plasma," "diabetic foot" and "randomized clinical trial". From the 10 papers yielded, three duplicates were removed before screening titles and abstracts; three were included. The randomized clinical trials (RCTs) compared standard/ placebo treatment against CAP. The addition of CAP produced a statistically significant fractional decrease in wound size (Table 1). One RCT did not measure wound size, but showed that CAP decreases inflammatory cytokines. (4) Our study shows that none of the RCTs conducted to-date include diabetic foot osteomyelitis, highlighting a need for future RCTs. With a limited number of clinical trials, the improved patient outcomes and non-antibiotic approach have raised confidence in the potential of CAP for DFUs.

Cellular and Molecular
Osteoclast fusion, size, and activation are regulated by extracellular pH

Abstract
Extracellular pH is a known modulator of osteoclast function. Acidosis directly stimulates bone resorption with near-maximal effects at pH 7.0, whereas ≥pH 7.4 substantially limits osteoclast activity. This study investigated the mechanistic actions of low pH on osteoclast fusion and resorption. Mouse bone marrowderived osteoclasts were cultured on dentine discs at pH 7.4 or pH 6.9 for 5 days. Osteoclast formation and activity were measured by image analysis of tartrate-resistant acid phosphatase (TRAP)-stained discs. The effect of pH on gene and protein expression and nuclei number was investigated using qPCR, Western blotting, and immunofluorescence, respectively. Osteoclast number was ≤1.9-fold (p < 0.001) higher in cells cultured at pH 6.9 compared to pH 7.4 osteoclasts. Extensive resorptive activity was observed in pH 6.9-cultured osteoclasts; the level of resorption in pH 7.4 osteoclasts was 85% lower (p < 0.001). Culture at pH 7.4 was associated with the formation of larger, more nucleated osteoclasts (≤140 μm with 23 AE 15 nuclei/cell), whereas cells at pH 6.9 were ≤35 μm in size with 8 AE 3 nuclei/cell (p < 0.01). mRNA expression of osteoclast formation (eg, RANK, c-FMS, TRAF6) and resorption genes (eg, cathepsin K, carbonic anhydrase II) was decreased in pH 7.4 osteoclasts. At the protein-level, pH 7.4 osteoclasts unexpectedly expressed 1.5-fold more cathepsin K (p < 0.01); however, enzymatic activity was $70% greater in resorbing pH 6.9 osteoclasts. Protein expression of the fusion marker DC-STAMP was 1.5-fold greater in pH 7.4-cultured osteoclasts compared to pH 6.9; no differences were observed at the mRNA-level. Short-term acid exposure of pH 7.4 osteoclasts reduced osteoclast size by 30% 4 hours post-acidification. Osteoclast numbers increased 2.3-fold by 24-hours, potentially indicating fission. Resorptive activity is visible at 8 hours and is extensive 24 hours post-acidification. Acidification of osteoclasts originally cultured at pH 7.4 results in a near similar resorptive activity by 24 hours to osteoclasts continually cultured at pH 6.9 (p < 0.05). Taken together, the current work indicates that extracellular pH modulates osteoclast fusion and size and may prime cells for subsequent resorptive activity.

Activated CD4 T cells from the bone marrow participate in type 2 diabetes bone fragility Saul Ernesto Cifuentes-Mendiola, Arnulfo Martinez-Davalos, Ana Lilia Garcia-Hernandez
Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Mexico

Abstract
The mechanisms leading to the development of osteopathy in type 2 diabetes mellitus (T2D) are currently unknown. The presence of activated T cells in bone marrow may take part in diabetic osteopathy by inducing an inflammatory microenvironment in bone tissue, which leads to osteoblast and osteoclast dysfunction and conduces to increased bone fragility. Because of this, we focused on determining the participation of activated bone marrow T cells in microstructural alterations and bone fragility in a mouse model of T2D. A model of T2D was developed in C57BL/6 male mice with a hypercaloric diet and low doses of streptozotocin; we inhibited T cell activation with CTLA4-Ig. We evaluated the glycemic profile during the experimental time (4 to 24 weeks of age), serum cytokine concentration by cytometric bead array (CBA) flow cytometry, T cell activation in bone marrow was determined by CD69 surface marker; tumor necrosis factor α (TNF-α) and interleukin 17 (IL17) production by flow cytometry, TNF-α concentration in bone tissue were determinate by ELISA; bone microarchitecture was evaluated by μCT and fracture resistance by three-point assay. Our results showed that inhibition of T lymphocyte activation decreased blood glucose levels, insulin resistance, and serum pro-inflammatory cytokines; reduced TNF-α in bone and improved mineral density decreased trabecular bone porosity and increased resistance to fracture in comparison with diabetic mice without the inhibitor. We conclude that activated bone marrow CD4+ T cell participates in microstructural alterations and bone fragility in T2D, probably by the production of TNF-α in bone tissue. ). Cell number (cell confluence) and cell death (Sytox green staining) were measured following exposure to 0.1nM to 100μM BP for 3 to 7 days. Significant apoptosis was only seen with high doses (10-100μM) of nitrogen-containing BPs, regardless of the duration of exposure, that was prevented by rescue of the mevalonate pathway with 10μM geranylgeraniol (GGOH). Interestingly, low doses of all BPs (0.1nM-1μM) reduced basal levels of cell death and increased cell proliferation in some cell types. This effect of BP action was not affected by treatment with GGOH. Similar protective effects were observed in response to induced stress. All types of BP could protect against H 2 O 2 -induced cell death in some cell types, via a mevalonate pathwayindependent mechanism. Interestingly, risedronate alone was able to protect against UV-induced cell death. This data suggests that BPs exert a broad protective effect on multiple cell types, protecting against apoptosis and increasing cell proliferation in both basal conditions and in response to apoptotic stimuli. Although the individual effects are small, long-term exposure to low-dose BP (as would be expected in extraskeletal sites) could be anticipated to reduce the adverse effects of environmental insults that accumulate with age, so promoting healthy aging. Funded by UK-SPINE.
Analysis of the in vivo frequency and in vitro phenotypic stability of human skeletal stem/progenitor cells identified by podoplanin and CD146 expression Abstract Although the identity of the skeletal stem cells (SSCs) in humans is still a matter of debate, a new set of phenotypic markers was proposed to discriminate the SSCs and their progeny. SSCs would comprise the podoplanin (PDPN)+ CD146À CD164+ CD73+ subset; its downstream bone, cartilage, and stroma progenitor (BCSPs) would be identified by the simultaneous expression of PDPN and CD146, whereas the osteoprogenitors (OPs) and chondroprogenitors (CPs) would be found in the PDPNÀ CD146+ and PDPN+ CD146À subsets, respectively.
Envisioning the optimization of cell products for use in bone repair strategies, we investigated the frequency of the aforementioned populations in the human adult bone marrow, the stability of their immunophenotypic profiles in vitro, and how these profiles correlate with that of bone marrow stromal cells (BMSCs). Following approval (n 21768719.0.0000.5257), 15 surgical waste bone/marrow samples were collected from patients of both sex, older than 18 years, undergoing primary hip arthroplasty. After leukocyte and endothelial cell depletion, the frequencies of SSCs, BCSPs, OPs, and CPs were determined by fluorescence-activated cell sorting (FACS). Our analysis showed that the BCSPs were 3.12% AE 3.67% of the CD45À CD31À population; the CPs were 13.40% AE 10.14%; the OPs were 7.68% AE 11.01%; whereas SSCs accounted for 0.23% AE 0.26% of the cells. Due to the low frequency of the SSCs, we sorted the cells into two major subsets: the PDPNÀ CD146+ pool, which included the OPs; and the PDPN+ CD146À, which included the SSCs and the CPs. Both subsets were in vitro expanded and serially evaluated by FACS. BMSCs were used for comparison. As soon as the first passage, all cells in the PDPNÀ CD146+ population had acquired PDPN expression. Similarly, the cells of the PDPN+ CD146À subset started to express CD146. Thereafter, the expression of PDPN progressively decreased in both populations; and at the end of passage three, the phenotypic profiles of both populations were similar to that of BMSCs. Therefore, the in vitro modulation of PDPN and CD146 in the sorted populations and the convergence of their profiles toward that of BMSCs suggest that the use of these markers might not be useful for the manufacture of cell products with better and predicted regenerative properties. Further transcriptomic and functional analysis will be performed to determine the relationship of the populations identified by PDPN and CD146 in vivo versus in vitro and versus BMSCs. Paget's disease of bone (PDB) is a metabolic disease resembling excessive bone remodeling causing disorganized, structurally weak bone. The genetic etiology of PDB is heterogeneous, but a significant portion is attributed to mutations in the SQSTM1 gene. The SQSTM1 gene product (p62) is a 62-kD protein linked to a number of cellular processes including autophagy. KEAP1 and PARK1 are proteins shown to interact with p62 during autophagy.
The hypothesis is that mutations in SQSTM1 associated with PDB could alter KEAP1 and/or PARK2 interaction with p62. Wild-type (hFOB1.19) and pagetic (PSV10) osteoblast cell lines were treated with a combination of Vitamin D (VitD) and lipopolysaccharide (LPS) with or without simvastatin, a drug that blocks p62 activity in autophagy. The cells were then fixed and analyzed by immunofluorescence using antibodies against RELA, KEAP1, or PARK2. Activation of RELA (a known downstream target of p62 activation), KEAP1, and PARK2 were detected by changes in cellular localization of these proteins to the nucleus. Results showed activation of RELA in the PSV10 but not hFOB1.19 cells in response to LPS and VitD as measured by nuclear accumulation. Simvastatin addition appeared to eliminate this activation. There was also nuclear accumulation of KEAP1 in the PSV10 cells following LPS and VitD stimulation, which was again blocked by simvastatin addition. However for PARK2, although there was nuclear accumulation in the PSV10 cells following LPS and VitD stimulation, simvastatin addition did not eliminate the nuclear localization of PARK2. To dissect the role of the oncohistone and to investigate the mechanisms of osteoclast recruitment in the GCT, we used in vitro models of human mesenchymal stem cells and osteoblasts in which we stably expressed mutant H3.3 (H3.3 G34W) as well as an in vitro human osteoclast assays and patient samples.
Here, we demonstrate that the H3.3 G34W mutation confers no direct growth advantage on mutant osteoprogenitors. We show that H3.3 G34W induces changes in the epigenome and transcriptome in mesenchymal cells which are sufficient to increase osteoclast recruitment. Osteoclasts in GCT are in part brought about by reduced expression of a transforming growth factor β (TGFβ)-like soluble factor, SCUBE3, which has previously been implicated in bone biology. Osteoclasts in turn secrete semaphorin-4D (SEMA4D), which promotes maturation arrest and proliferation of mutated H3.3 osteoprogenitors. In contrast, malignant GCTs develop autonomous growth, often transforming into osteosarcomas, and no longer respond to osteoclastdepleting treatment. We provide here a mechanism for GCT initiation and its response to denosumab treatment. More generally, this study emphasizes the importance of the TME in tumorigenesis.

Clinical Cases
Bone health in pheochromocytoma/paraganglioma Results: Average age of subjects was 41 AE 13 years. A total of 18 of 32 (56%) subjects had either "lower BMD than expected for age" or osteoporosis. Although a statistically significant difference in aBMD-adjusted FRAX versus TBS-and aBMD-adjusted FRAX was present, clinically meaningful difference was not present as similar number of subjects qualified for anti-osteoporotic measures by both. Among the factors predicting low Z-score expected for age or osteoporosis, every kilogram less weight was associated with 12% lower Z-score (statistically significant). Higher 24-hour VMA level and presence of hyperadrenergic spells showed trends toward lower Z-score, without statistical significance. No association was noted with size of tumor or 25-hydroxy Vit-D3 level and iPTH levels. No statistically significant change in BMD or TBS at lumbar spine was noticed at a median follow-up of 4 months postoperatively. Their coexistence was considered to be distinct age-related conditions and was attributed primarily to aging and common risk factors including diabetes, dyslipidemia, and smoking.
Purpose of the Study: To assess the prevalence of densitometric bone loss in patients followed for coronary artery disease compared to control subjects.
Methods: This is a prospective, case-control, longitudinal study of patients followed for coronary artery disease and control subjects. The exclusion criteria were patients with known bone diseases. The patients included have been assessed and monitored at bone disease unit in rheumatology department of Ibn Rochd University Hospital and were divided into two groups: The first one with coronary artery disease and the second control group with normal coronary angiography. All patients underwent a complete clinical examination, blood and urinary phosphocalcic explorations, metabolic assessment, and bone densitometry (DXA).
Results: The study included 46 patients: 24 women and 22 men. The mean age was 65.7 AE 6.5 years. Regarding past medical history: 34% were diabetic type II, 60% of whom were under insulin therapy; 21% were dyslipidemic (80% of whom were under statins); 32% were hypertensive; 17% were chronic smokers. Ten cases (21.7%) had osteoporosis, 19 (41.3%) had osteopenia, and 17 (37%) had normal bone mineral density. The prevalence of osteoporosis and osteopenia was significantly higher in group I compared to the control group (with significant p value). Discussion and Conclusion: Numerous publications describe a link between bone fragility and cardiovascular disease. The mechanisms are multiple and still imperfectly understood. However, osteoprotegerin is one of the factors that may explain a pathophysiological link between bone fragility and the constitution of atheromatous plaque. It inhibits the receptor activator of nuclear factor κB-receptor activator of nuclear factor κB ligand (RANK-RANKL) system, a strong osteoclast activator, and also plays a role in protecting vascular diseases. Its decrease during osteoporosis could explain the occurrence of coronary artery disease. Our study joined the literature results, showing the high frequency of bone loss among patients followed for coronary artery disease. The exact link cannot be confirmed given the comorbidities found in our population. It seems important to request DXA to patients followed for vasculopathies and a cardiac assessment to osteoporotic subjects. Past history of fractures, walking aid, vision disturbances, rheumatoid arthritis, diabetes were the predictors of falls in our population (Table). Summary and conclusion: The fall history is an independent risk factor for fracture. This risk is particularly important in case of fragility bone densitometry.
The clinical history and fall risk factors should be taken into consideration in the assessment of fracture risk and in the antiosteoporotic treatment strategy. Thus, falls seem to be a legitimate indication for DXA and as a parameter that deserves to be integrated into the assessment of fracture risk by the FRAX score. Abstract Background: Spondyloarthritis (SpA) is associated with altered body composition due to many factors such as inflammation and immobilization. Patients often present with low muscle mass and decreased strength with high fat mass. The muscle changes may occur in the early stages and persist throughout the disease duration like the excess of fat mass.
Purpose: To assess the link between the activity of SpA and the change of the fat and lean body mass.
Method: Transversal and descriptive monocentric study conducted in 20 SpA patients followed in the rheumatology department at the University Hospital Ibn Rochd. The study include patients with active disease and control group with remission disease. Demographic characteristics were noted in all patients as well as disease activity and treatment characteristics. All patients underwent a dual-energy X-ray absorptiometry (DXA) scan with evaluation of the body mass; precisely, the lean and the fat mass. Finally, the data of the two groups were compared to assess the relationship between disease activity and disturbances in body mass.
Results: A total of 20 patients were included. The mean age was 30 AE 10.3 years, with men predominant (85%). The mean body mass index (BMI) was 21.1 AE 4.7 m/kg 2 . Mean duration of primitive SpA was 11.4 AE 6.3 years. Mean sedimentation rate was 33.7 AE 26.8 mm/h and C-reactive protein (CRP) 32.7 AE 33 mg/ L. The mean ankylosing spondylitis disease activity score with CRP (ASDAS-CRP) was 2.5 AE 1.2. Regarding the evaluation of body mass, we found the following results: total leg average fat mass 1067 AE 2012 g, total leg mean lean mass 10,757 AE 2568 g, and total leg bone mineral content (BMC) 391 AE 545 g. The whole-body average fat was 6484 AE 13,054 g, the average lean whole body was 54,605 AE 9889 g, and the whole body BMC was 2673 AE 419 g (Table). Discussion and Conclusion: The assessment of fat and lean mass is important in patients followed for chronic inflammatory rheumatism such as SpA. Inflammation induces an increase in fat mass and a decrease in lean mass with the risk of osteoporosis and fall, especially in patients with active rheumatism and obesity. Our study showed an increase in fat content in patients with active SpA and the decrease of the lean mass but without statistically significant difference with the control group. This can be explained by the low number of patients and the few associated comorbidities. The changes in body composition during SpA are linked to a cascade of metabolic abnormalities with inflammatory factors. There are multiple, complex interactions which remain largely unknown. In this study, fibular and tibial bone parameters were assessed in 13 individuals with SCI (aged 16-76 years). A set of peripheral quantitative computed tomography (pQCT) scans at 4%, 38%, and 66% distal-proximal tibia length were acquired within 5 weeks (baseline) and at 12 months postinjury. Changes in total bone mineral content (BMC), bone cross-sectional area (CSA), and bone mineral density (BMD) were assessed at the 4% site, and total BMC, total CSA, and cortical BMD and cortical CSA were analyzed at 38% and 66% in both bones using paired t tests. In addition, relationships between bone loss in the two bones at the same site were assessed using correlation and paired t tests.
At the 4% site, both total BMC and BMD declined over 12 months, but tibial BMC losses were greater than those observed in the fibula (À14.8% AE 12.4% and À6.9% AE 5.1%, respectively, p = 0.02). At the diaphyseal sites (38%, 66%), BMC reduced at both sites in the tibia, but only at the 66% site in the fibula, with that loss being larger in the tibia compared to the fibula (p = 0.03). The cortical BMD reduced by $2.5% in the tibia, but there was no change at either fibula site (both p > 0.4). Changes in bone CSA were similar between the two bones. No evidence of a correlation was found between individual changes in BMC between the two bones (all p > 0.25). These results support cross-sectional evidence of smaller disuserelated bone changes in the distal fibula compared to the tibia, although in contrast to previous findings bone loss in the fibula shaft was observed. The lack of association between losses in the two bones, suggests that different mechanisms; eg, local mechanical or systemic metabolic changes, following injury might be responsible.

Abstract
Osteoarthritis has traditionally been considered a "non-inflammatory" form of arthritis. However, there is increasing evidence to suggest that the characteristic articular cartilage degradation follows biochemical changes to the joint, and furthermore, there is clear evidence of immune cell invasion in the damaged joint. Regulatory T cells (Tregs) (CD4+Foxp3+) are a subpopulation of T helper cells thought to play a role in osteoclastic bone resorption; however, their role in osteoarthritis is unknown. In order to determine the aging changes on Treg presence in peripheral blood, CD4+Foxp3+ Treg quantification was undertaken in peripheral blood obtained from healthy young and healthy old participants using flow cytometric analysis, as well as quantification of bone homing markers CXCR4 and CCR4 to determine the possibility of Treg migration toward bone. Moreover, the effects of Tregs on osteoclast activity was further analyzed. Primary human osteoclast cultures were established and co-cultured with Tregs for up to 21 days. Osteoclast number, size, and resorption were quantified via tartrate-resistant acid phosphatase (TRAP) staining. Additionally, localization of Tregs in a surgical murine model of osteoarthritis (destabilization of the medial meniscus) was analyzed by immunohistochemistry for the presence of Foxp3+. Quantification of Tregs (CD4+Foxp3+) in peripheral blood by flow cytometry showed no significant difference in numbers when comparing healthy young with healthy old participants. However, the percentage of bone homing markers (CCR4 and CXCR4) expressed by CD4+Foxp3+ Tregs decreased with aging (62% in healthy young versus 40% in healthy old for CXCR4 and 42% in healthy young versus 28% in healthy old for CCR4 and, p < 0.05). Although preliminary data utilizing osteoclast cocultures showed co-culture of osteoclasts with Tregs had no effect on the number of osteoclasts formed, a decrease in osteoclast activity was observed when these cells were cultured in the presence of Tregs, in comparison to osteoclasts alone. This data suggests that Tregs play a role in osteoclast function. Together these data provide further evidence for Tregs to play a role in osteoclastic bone resorption, and this process to be disrupted by aging. Ongoing studies will determine the effects of osteoarthritis on these processes.   (Fig. 1a,b). In both boys and girls, GP measured consistently lower than TW3 (girls À0.36[95% CI, À0.54 to À0.18] years; CA-TW3 -0.33 [95% CI, À0.49 to À0.17] years). Conclusion: Zimbabwean boys were less skeletally mature than girls of the same age. The TW3 method is more applicable to the Zimbabwean population because TW3 BA was in closer agreement with CA and showed no systematic bias with CA. The TW3 and GP methods do not agree and therefore cannot be used interchangeably. Assessment of algorithmic training revealed that osteoclast numbers and the total area resorbed strongly correlated between manual-and automatically-quantified values (r = 0.75 and 0.83, respectively). Osteoclasts were faithfully segmented when visually compared to the original images. The accuracy of automated osteoclast number quantification was validated using zoledronate, a bisphosphonate with well-characterized inhibitory effects on osteoclasts, and ticagrelor, a P2Y 12 receptor antagonist with less well-known effects on osteoclast biology. A 70% reduction (p < 0.01) in osteoclast number was detected, irrespective of quantification method, when cultured with 10nM zoledronate. Both methods also detected a dosedependent decrease in osteoclast number when treated with 1 to 10μM ticagrelor (p < 0.05). Development of the ilastik algorithm reduces user variability by ≤100% (p < 0.001). Resorption pits were occasionally inaccurately identified using the ilastik algorithm; therefore, μCT-based protocols quantifying bone resorption were also developed. Dentine discs with adherent osteoclasts were scanned at 2 μm; resorption area was visualized and quantified using CTvox and CtAn software, respectively. Three-dimensional reconstructions reveal significant reductions in osteoclast resorptive activity following zoledronate treatment; μCT and manual quantification values were comparable.

Epidemiology
Overall, an automated image segmentation and analysis workflow was developed and validated to consistently and sensitively identify osteoclasts, but not resorption pits. μCT analysis could provide an alternative solution to automate bone resorption analysis. This pipeline significantly reduces user variability of endpoint measurements and analysis time by 75%.
A new method for segmentation and analysis of bone callus in rodent fracture models using μCT

Abstract
Increased 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 in indirect fracture repair, is, therefore, vital. μCT can facilitate noninvasive imaging of fracture repair within experimental settings; however, current methods for callus segmentation from cortical bone are only semiautomated, restricted to defined regions, time/labor intensive, and prone to user variation. Herein, we share a newly developed automatic method for segmenting callus from cortical bone in μCT tomograms that will allow for objective, quantitative analysis of the bone fracture microarchitecture. Fractured and nonfractured mouse femurs were μCT scanned and then processed by both manual and automated segmentation of fracture callus from cortical bone after which the microarchitectural parameters were analyzed. Results showed that the automatic segmentation method could reliably and consistently segment callus from cortical bone with Pearson's correlation coefficient showing a strong linear relationship for bone and tissue volume measurements (r = 0.99, p = 0.0002, and p = 0.0004, respectively). When compared to manual segmentation, the newly developed method was faster (30 minutes manually versus 1 minute automatic) and eliminated user-bias and variation (tissue volume % CV = 15.6%). Scalability and translatability of the method within rodent models was demonstrated using μCT scans of fractured rat femurs with varying gap sizes, which demonstrated advantages in extending evaluation to the entire fracture callus volume in our method. Together, these data serve to validate a new automated method for segmentation of callus and cortical bone in μCT tomograms that we have developed, which we share as a fast, reliable, and less user-dependent tool for application to the study of bone callus in fracture, and potentially elsewhere.

Other
Bone recovery after treatment: a feasibility study exploring changes in myeloma bone disease in patients receiving first line cancer treatment-is there a role for bone anabolics? The majority of patients with a diagnosis of multiple myeloma (MM) experience myeloma bone disease (MBD), resulting in life-changing symptoms such as chronic pain and poor mobility. Current bone-targeted therapies available for the treatment of MBD are limited to bisphosphonates or denosumab, which aim to prevent further bone destruction, but do not repair the damage. We propose that use of bone anabolic therapies in MBD could significantly improve patient outcomes.
Objectives: The primary aim of this study is to evaluate the feasibility of assessing bone recovery in MM patients undergoing first-line cancer treatment. The study will provide valuable information to design a larger-scale interventional trial assessing bone anabolic treatments for MBD. Secondary exploratory objectives include comprehensive assessment of changes in bone architecture (macroscopic and microscopic), bone turnover, and alterations to the surrounding microenvironment indicative of bone recovery.
Methods: This is a prospective, observational, feasibility study, recruiting patients at the Royal Hallamshire Hospital (Sheffield, UK) with a new diagnosis of MM, for longitudinal assessment of bone health throughout (and following) their first-line treatment.
Monitoring includes bone marrow biopsies, fasting serum samples for bone turnover markers, whole-body low-dose CT imaging, and quality of life (QOL) questionnaires. Major follow-up time points are planned at 3 months and 1 year post completion of treatment (+/À autologous stem cell transplant) and compared to baseline. Results: By March 2020 there were 20 active participants (the original recruitment target). Unfortunately, the study was closed to data collection from March 2020 as a result of the coronavirus disease 2019 (COVID-19) pandemic, and therefore multiple study time points have been lost. The study was recently approved to re-open and awaits ethical approval to incorporate later followup data collection points. Preliminary results demonstrate dramatic bone recovery for some study participants, evidenced on follow-up CT imaging and bone marrow trephine microarchitecture, potentially due to the use of bortezomib chemotherapy in treatment regimes.
Conclusions: This study will evaluate the feasibility of a full-scale early-phase interventional trial assessing efficacy of bone anabolic agents in MBD, with particular focus on acceptability, implementation, and practicality of extensive bone monitoring alongside first-line cancer-targeting treatment. In addition, the study will provide exploratory outcomes to assess changes in the bone microenvironment during treatment.

Abstract
Purpose: Accelerometer-derived moderate (MPA) and vigorous (VPA) intensities of physical activity (PA) have been shown to have positive associations with bone outcomes in children and adolescents. However, it is unclear whether a particular intensity is more beneficial. This systematic review aimed to determine whether the magnitude of association between PA and bone outcomes was consistently stronger for a particular intensity of activity (MPA, MVPA, or VPA) and to summarize the accelerometry methods used to obtain PA data in these studies. Methods: A systematic electronic search was conducted in MED-LINE, EMBASE, Web of Science, SPORTDiscus, and the Cochrane Central Register of Controlled Trials to identify observational studies that had assessed associations between accelerometerderived habitual MPA and/or MVPA and VPA with bone outcomes in children and adolescents (≤18 years). Thirty articles were included for review. Chi-square tests were used to determine which PA intensity (MPA, MVPA, or VPA) had the greatest proportion of "statistically significant associations" with bone outcomes and which intensity had the greatest proportion of "strongest within-study associations." Results: There was considerable heterogeneity in the accelerometry methods used. Studies varied in terms of the monitor make and model, wear criteria, epoch length, and cut-point definitions of activity intensities. Regardless of the accelerometry methods employed, results were still indicative of a greater benefit of VPA over MPA/MVPA to bone outcomes. Of the 570 association analyses, 186 were significant (p < 0.05). Of these significant associations, chi-square tests demonstrated that the proportion of strongest within-study associations differed by PA intensity (3 Â 2 χ 2 = 86.6, p < 0.001) and was significantly higher for VPA (90/228) than for MVPA (8/151, 2 Â 2 χ 2 = 55.3, p < 0.001) and MPA (18/191, 2 Â 2 χ 2 = 49.1, p < 0.001). Conclusion: Findings from this systematic review indicate that accelerometer-derived VPA is more beneficial to bone outcomes compared to MPA or MVPA in children and adolescents aged ≤18 years. However, the widely varying accelerometry methods used to obtain PA data prevent the precise, beneficial amount of VPA from being identified. The frequent use of long epoch lengths (most commonly 60 seconds and 15 seconds) and numerous different intensity cut-point definitions make it likely that bone-relevant PA has been misrepresented in this population. The use of shorter epoch lengths (eg, 1 second) and development of bone-specific activity intensity thresholds should be explored in the future. Introduction: (i) We previously showed that intraosseous pressure (IOP) at rest is not a constant but is variable and proportional to local perfusion pressures at the needle tip. With activity there are significant pressure fluctuations of several atmospheres above arterial pressure with ordinary weight bearing. (ii) We previously described hypodense marks on upper tibial magnetic resonance imaging (MRI) scans which appear to be vascular, and which are reduced in number in early osteoarthritis.
(iii) We note that bone fat is essentially liquid at body temperature. It flows from fractures and sprays from orthopedic saws or drills. No previous literature describes or explains this. (iv) Load appears to be transferred partly by hydraulic pressure acting through soft lipid and vascular tissues to transferring pressure on to the trabeculae and so to the cortical shaft. In this preliminary qualitative study, we sought histological evidence for those subchondral vascular marks on MRI and other subchondral structures that might allow and support hydraulic pressure load transfer during weight bearing. Method: We examined axial or transverse plane histology for vessel morphology that might confirm that the MRI marks were vascular. We also looked for features that might represent a n 48 of 51 mechanism for controlling flow in and out of the subchondral region when pressurized. Normal human upper tibial bone was used (n = 6). Results: Radiating vessels running parallel to the articular surface in the subchondral plane were found similar to those seen on MRI scans. The vessels penetrate the cortex near the joint margin. There are complex distortions at that point. Conclusion: We identified previously undescribed vessels running in the subchondral plane consistent with marks seen on MRI scans. As they reach the cortical margin, complex distortions exist which may be choke valves that shut off to prevent highpressure flow out from the cancellous interior. Such a mechanism would prevent turbulent flow in fine capillaries and around delicate adipocytes. Arthritic bone had none of the longitudinal subchondral vessels or the subcortical choke valve morphological features. We suggest that there is a possible vasculomechanical basis for osteoarthritis.
Disorders, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 9 Ipsen, Newton, MA, USA. 10 Ipsen, Slough, UK Abstract Background: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by heterotopic ossification (HO) of soft and connective tissues (often preceded by flareups), cumulative disability, and early mortality. Palovarotene (PVO) is a selective retinoic acid receptor gamma agonist under investigation for the treatment of FOP. Conducting trials in FOP has multiple challenges, including the low number of confirmed cases worldwide, a limited understanding of disease progression, and the need to identify disease-specific biomarkers and optimize assessment of HO progression.
Objective: To develop a methodological approach addressing the challenges of conducting clinical trials in FOP.
Methods: Here, we describe the methodology of: a non-interventional, prospective, protocol-specified, longitudinal natural history study (NHS; NCT02322255); a multicenter, randomized, double-blind, placebo-controlled phase II trial (NCT02190747); and an ongoing open-label extension (OLE) to the phase II trial (NCT02279095) ( Table). Studies were designed adaptively. PVO doses were administered episodically (high dose for 2 or 4 weeks, followed by low dose for 4 or ≥8 weeks, from flare-up onset) or chronically (daily). In the double-blind period of the phase II trial, participants were randomized in two cohorts: 0:3:1 (aged ≥15 years) or 3:3:2 (≥6 years) to PVO 5/2.5 mg, PVO 10/5 mg or placebo, episodically for 2 weeks (high dose) then 4 weeks (low dose). HO incidence and volume are assessed annually by standardized low dose whole-body computed tomography and/or at 12 weeks during the course of flare-ups (defined as ≥2 [≥1 in OLE Part C] of pain, swelling, stiffness, decreased range of motion, redness, or warmth). Other clinical, functional, and patient-reported outcomes are assessed using FOP-specific measures of physical function, including the Cumulative Analogue Joint Involvement Scale (CAJIS) and FOP Physical Function Questionnaire (FOP-PFQ). Studies were approved by independent ethics committees. A total of 151 unique participants were enrolled. Learnings informed the design of the phase III MOVE trial (NCT03312634). Conclusions: Novel methodological approaches are needed to develop disease-modifying treatments for serious, ultra-rare diseases such as FOP. HO is the main cause of disability in patients with FOP. This program could be used as an example to inform the development of new treatments in rare bone diseases. Femoral anteversion (FNA), and other lower limb geometry parameters in individuals with X-linked hypophosphatemia (XLH) Matteo Scorcelletti 1 , Serhan Kara 2 , Med. Lothar Seefried 3 , Jochen Zange 2 , Jörn Rittweger 2 , Alex Ireland 1