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Tartrate-resistant acid phosphatase (TRAP) serum levels reflect osteoclast number, bone remodeling activity, and fracture risk. Deletion or loss of function of TRAP results in short stature in mice and man. Yet, the impact and mechanisms of TRAP for the site- and sex-specific development of bone and cartilage is not well understood. Here, we use a global TRAP knockout (TRAPKO) and wildtype littermate control (WT) mice of both sexes to investigate TRAP as a possible sex- and site-specific regulator of bone and growth plate development. TRAPKO mice of both sexes weighed less and had shorter tibial length than their WT, features that were more accentuated in male than female TRAPKO mice. These changes were not associated with a general reduction in growth as not all organs displayed a proportionally lower mass, and serum IGF-1 was unchanged. Using μCT and site-specificity analysis of the cortical bone revealed wider proximal tibia, a higher trabecular thickness, and lower trabecular separation in male TRAPKO compared to WT mice, an effect not seen in female mice. Histomorphometric analysis revealed that the growth plate height as well as height of terminal hypertrophic chondrocytes were markedly increased, and the number of columns was decreased in TRAPKO mice of both sexes. These effects were more accentuated in female mice. Proliferation and differentiation of bone marrow derived macrophages into osteoclasts, as well as C-terminal cross links were normal in TRAPKO mice of both sexes. Collectively, our results show that TRAP regulates bone and cartilage development in a sex-and site-specific manner in mice. [Display omitted] • Reduction in bone length is more pronounced in male than female TRAPKO mice. • Unlike females, male TRAPKO mice have wider cortex and altered trabecular bone. • Increases in growth plate height are more pronounced in female than male TRAPKO mice. • Growth plate bone bridges were reduced in male, but not female, TRAPKO mice. • TRAP influences bone and cartilage development differently in males and females. [ABSTRACT FROM AUTHOR]

It is well understood that the balance of bone formation and resorption is dependent on both mechanical and biochemical factors. In addition to cell-secreted cytokines and growth factors, sex hormones like estrogen are critical to maintaining bone health. Although the direct osteoprotective function of estrogen and estrogen receptors (ERs) has been reported extensively, evidence that estrogen signaling also has a role in mediating the effects of mechanical loading on maintenance of bone mass and healing of bone injuries has more recently emerged. Recent studies have underscored the role of estrogen and ERs in many pathways of bone mechanosensation and mechanotransduction. Estrogen and ERs have been shown to augment integrin-based mechanotransduction as well as canonical Wnt/b-catenin, RhoA/ROCK, and YAP/TAZ pathways. Estrogen and ERs also influence the mechanosensitivity of not only osteocytes but also osteoblasts, osteoclasts, and marrow stromal cells. The current review will highlight these roles of estrogen and ERs in cellular mechanisms underlying bone mechanobiology and discuss their implications for management of osteoporosis and bone fractures. A greater understanding of the mechanisms behind interactions between estrogen and mechanical loading may be crucial to addressing the shortcomings of current hormonal and pharmaceutical therapies. A combined therapy approach including high-impact exercise therapy may mitigate adverse side effects and allow an effective long-term solution for the prevention, treatment, and management of bone fragility in at-risk populations. Furthermore, future implications to novel local delivery mechanisms of hormonal therapy for osteoporosis treatment, as well as the effects on bone health of applications of sex hormone therapy outside of bone disease, will be discussed. • Estrogen has positive effects on the balance of bone formation activity of bone cells. • Estrogen and estrogen receptors couple with mechanotransduction signaling pathways. • Combining estrogen and exercise to treat postmenopausal bone loss merits more study. • Estrogen improves fracture healing in animals, suggesting prospects for clinical use. [ABSTRACT FROM AUTHOR]

Hypophosphatasia (HPP) is a rare genetic disorder characterized by defective bone mineralization, leading to skeletal abnormalities and systemic complications. Asfotase alfa, a recombinant human tissue-nonspecific alkaline phosphatase (TNSALP) enzyme replacement therapy, has emerged as a promising treatment for HPP. However, a comprehensive evaluation of its efficacy and safety is warranted to guide clinical practice effectively. The study followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was registered in Prospective Register of Systematic Reviews (PROSPERO). A search strategy across databases found studies on asfotase alfa for HPP. Two researchers independently extracted and assessed data. This systematic review examined how the drug impacted clinical outcomes such as survival rates, musculoskeletal symptoms, respiratory function, growth measurements, dental health, quality of life, and laboratory results. This systematic review included 15 articles with a total of 455 HPP patients. Asfotase alfa was predominantly administered at a dose of 6 mg per kg per week among the reviewed studies. Notable findings included enhanced survival rates, relief from musculoskeletal pain, improvements in respiratory outcomes, growth parameters, dental health, and quality of life. Changes in laboratory variables indicated positive responses to treatment, including changes such as increase in alkaline phosphatase (ALP), decline in pyridoxal 5′-phosphate (PLP) and inorganic pyrophosphate (PPi) levels. Asfotase alfa demonstrates efficacy in improving clinical outcomes and safety in patients with HPP. Its therapeutic benefits extend across various domains. However, Larger, age-stratified comparative studies are needed to further investigate the drug's effects in HPP patients. • Asfotase alfa enhances survival rates in HPP patients significantly. • Treatment with asfotase alfa alleviates musculoskeletal pain in most patients. • Improved muscle strength and mobility observed with asfotase alfa therapy. • Radiographic changes indicate healing of skeletal manifestations in HPP. • Treatment enhances respiratory function, growth parameters, and dental health. [ABSTRACT FROM AUTHOR]

Children with type 1 diabetes (T1D) experience an increased risk of fracture, which may be related to altered bone development. We aimed to assess differences in bone, muscle and physical activity (PA), and explore if better muscle and PA measures would mitigate bone differences between children and adolescents with T1D and typically developing peers (TDP). We matched 56 children and adolescents with T1D (mean age 11.9 yrs) and 56 TDP (11.5 yrs) by sex and maturity from 171 participants with T1D and 66 TDP (6-17 yrs). We assessed the distal radius and tibia with high-resolution peripheral quantitative computed tomography (HR-pQCT), and the radius and tibia shaft bone and muscle with pQCT. We also measured muscle function from force-related measures in neuromuscular performance tests (push-up, grip test, countermovement and long jump). We compared PA based on questionnaire scores and accelerometers between groups. Bone, muscle, and neuromuscular performance measures were compared using MANOVA. We used mediation to explore the role of PA and muscle in bone differences. Children and adolescents with T1D had 6–10 % lower trabecular density, bone volume fraction, thickness and number at both distal radius and tibia, and 11 % higher trabecular separation at the distal radius than TDP. They also had 3–16 % higher cortical and tissue mineral density, and cortical thickness at the distal radius, 5–7 % higher cortical density and 1–3 % higher muscle density at both shaft sites compared to TDP. PA mediated the between-group difference in trabecular number (indirect effect −0.04) at the distal radius. Children and adolescents with T1D had lower trabecular bone density and deficits in trabecular micro-architecture, but higher cortical bone density and thickness at the radius and tibia compared to TDP. They engaged in less PA but had comparable muscle measures to those of TDP. PA participation may assist in mitigating deficit in trabecular number observed in children and adolescents with T1D. • Children with T1D had lower trabecular density, micro-architecture at distal sites. • Higher cortical density and thickness at distal radius and shaft sites in T1D • More physical activity may mitigate trabecular bone difference in T1D. • Prospective, advanced imaging data of bone development in children with T1D needed. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GCs) are the leading cause of secondary osteoporosis. The emerging perspective, derived primarily from 2D histological study of trabecular bone, is that GC-induced bone loss arises through the uncoupling of bone formation and resorption at the level of the basic multicellular unit (BMU), which carries out bone remodeling. Here we explore the impact of GCs on cortical bone remodeling in the rabbit model. Based upon the rapid reduction of bone formation and initial elevation of resorption caused by GCs, we hypothesized that the rate of advance (longitudinal erosion rate; LER) of cortical BMUs would be increased. To test this hypothesis we divided 20 female New Zealand White rabbits into four experimental groups: ovariohysterectomy (OVH), glucocorticoid (GC), OVH + GC and SHAM controls (n = 5 animals each). Ten weeks post-surgery (OVH or sham), and two weeks after the initiation of dosing (daily subcutaneous injections of 1.5 mg/kg of methylprednisolone sodium succinate in the GC-treated groups and 1 ml of saline for the others), the right tibiae were scanned in vivo using Synchrotron Radiation (SR) in-line phase contrast micro-CT at the Canadian Light Source. After an additional 2 weeks of dosing, the rabbits were euthanized and ex vivo images were collected using desktop micro-CT. The datasets were co-registered in 3D and LER was calculated as the distance traversed by BMU cutting-cones in the 14-day interval between scans. Counter to our hypothesis, LER was greatly reduced in GC-treated rabbits. Mean LER was lower in GC (4.27 μm/d; p < 0.001) and OVH + GC (4.19 μm/d; p < 0.001), while similar in OVH (40.13 μm/d; p = 0.990), compared to SHAM (40.44 μm/d). This approximately 90 % reduction in LER with GCs was also associated with an overall disruption of BMU progression, with radial expansion of the remodeling space occurring in all directions. This unexpected outcome suggests that GCs do not simply uncouple formation and resorption within cortical BMUs and highlights the value of the time-lapsed 4D approach employed. [Display omitted] • In vivo imaging of rabbits was used to directly evaluate the longitudinal erosion rate of basic multicellular units. • Basic multicellular units in sham and OVH-operated rabbits advance at similar rates. • Glucocorticoid treatment reduces the longitudinal erosion rate by ~90 %. • Remodeling spaces in glucocorticoid-treated rabbits expand radially but not longitudinally. [ABSTRACT FROM AUTHOR]

Gaucher disease is one of the most common lysosomal storage disorders. Osteonecrosis is a principal clinical manifestation of Gaucher disease and often leads to joint collapse and fractures. T1-weighted (T1w) modality in MRI is widely used to monitor bone involvement in Gaucher disease and to diagnose osteonecrosis. However, objective and quantitative methods for characterizing osteonecrosis are still limited. In this work, we present a deep learning-based quantification approach for the segmentation of osteonecrosis and the extraction of characteristic parameters. We first constructed two independent U-net models to segment the osteonecrosis and bone marrow unaffected by osteonecrosis (UBM) in spine and femur respectively, based on T1w images from patients in the UK national Gaucherite study database. We manually delineated parcellation maps including osteonecrosis and UBM from 364 T1w images (176 for spine, 188 for femur) as the training datasets, and the trained models were subsequently applied to all the 917 T1w images in the database. To quantify the segmentation, we calculated morphological parameters including the volume of osteonecrosis, the volume of UBM, and the fraction of total marrow occupied by osteonecrosis. Then, we examined the correlation between calculated features and the bone marrow burden score for marrow infiltration of the corresponding image, and no strong correlation was found. In addition, we analyzed the influence of splenectomy and the interval between the age at first symptom and the age of onset of treatment on the quantitative measurements of osteonecrosis. The results are consistent with previous studies, showing that prior splenectomy is closely associated with the fractional volume of osteonecrosis, and there is a positive relationship between the duration of untreated disease and the quantifications of osteonecrosis. We propose this technique as an efficient and reliable tool for assessing the extent of osteonecrosis in MR images of patients and improving prediction of clinically important adverse events. • U-net models achieve automatic segmentation of osteonecrosis in spine and femur T1w images of Gaucher disease patients • Prior splenectomy is closely associated with the fractional volume of osteonecrosis • The interval between the age at first symptom and the age at first specific treatment is correlated with osteonecrosis [ABSTRACT FROM AUTHOR]

The influence of iron on collagen synthesis and vitamin D metabolism has implications for bone health. This cross-sectional observational study investigated associations between markers of iron status and tibial structure, vitamin D metabolites, and circulating biochemical markers of bone metabolism in young healthy men. A total of 343 male British Army recruits participated (age 22 ± 3 y, height 1.77 ± 0.06 m, body mass 75.5 ± 10.1 kg). Circulating biochemical markers of iron status, vitamin D metabolites, and bone metabolism, and tibial structure and density by high-resolution peripheral quantitative computed tomography scans (HRpQCT) were measured in participants during week 1 of basic military training. Associations between markers of iron status and HRpQCT outcomes, bone metabolism, and vitamin D metabolites were tested, controlling for age, height, lean body mass, and childhood exercise volume. Higher ferritin was associated with higher total, trabecular, and cortical volumetric bone mineral density, trabecular volume, cortical area and thickness, stiffness, and failure load (all p ≤ 0.037). Higher soluble transferrin receptor (sTfR) was associated with lower trabecular number, and higher trabecular thickness and separation, cortical thickness, and cortical pore diameter (all p ≤ 0.033). Higher haemoglobin was associated with higher cortical thickness (p = 0.043). Higher ferritin was associated with lower βCTX, PINP, total 25(OH)D, and total 24,25(OH) 2 D, and higher 1,25(OH) 2 D:24,25(OH) 2 D ratio (all p ≤ 0.029). Higher sTfR was associated with higher PINP, total 25(OH)D, and total 24,25(OH) 2 D (all p ≤ 0.025). The greater density, size, and strength of the tibia, and lower circulating concentrations of markers of bone resorption and formation with better iron stores (higher ferritin) are likely as a result of the direct role of iron in collagen synthesis. • We explored the relationship between iron status and bone structure and metabolism. • Higher ferritin was associated with higher density and size of tibial bone. • Higher ferritin was associated with lower bone resorption and formation. • Higher ferritine was associated with lower total 25(OH)D. [ABSTRACT FROM AUTHOR]

This study sought to further develop and validate a previously proposed physics-based model that maps denaturation kinetics from differential scanning calorimetry (DSC) to the isometric tension generated during hydrothermal isometric tension (HIT) testing of collagenous tissues. The primary objectives of this study were to verify and validate two physics-based model parameters: α, which indicates the amount of instantaneous isometric tension developed per unit of collagen denaturation, and β, which captures the proportionality between temperature and the generated isometric tension post denaturation initiation. These parameters were used as measures of bone collagen quality, employing data from HIT and DSC testing of human bone collagen from two previous studies. Additionally, given the physical basis of the model, the study aimed to further validate Max.Slope, the rate of change in isometric tensile stress with change in temperature, as an independent measure of collagen network connectivity. Max.Slope has previously been positively correlated with measures of cortical bone fracture resistance. Towards this verification and validation, the hypotheses were a) that α would correlate strongly with HIT denaturation temperature, T d , and the enthalpy of melting (ΔH) from DSC, and b) that β would correlate positively and strongly with Max.Slope. The model was employed in the analysis of HIT-DSC data from the testing of demineralized bone collagen isolated from cadaveric human femurs in two prior studies. In one study, data were collected from HIT-DSC testing of cortical bone collagen from 74 donors. Among them, 38 had a history of type 2 diabetes +/− chronic kidney disease, while the remaining 36 had no history of T2D again with or without CKD. Cortical bone specimens were extracted from the lateral mid-shaft. The second study involved 15 donor femora, with four cortical bone specimens extracted from each. Of these four, two specimens underwent a 4-week incubation in 0.1 M ribose at 37 °C to induce non-enzymatic ribation and advanced glycation endproducts, while the other two served as non-ribated controls. The examination involved investigating correlations between the model parameters α and β and various measures, such as Max.Slope, Td, ΔH, age, and duration of type 2 diabetes. The results revealed positive correlations between the model parameter β and Max.Slope (r = 0.55–0.58). The parameter α was found to be associated with T d , but also sensitive to the shape of the HIT curve around T d resulting in difficulties with variability and interpretation. As a result, while both hypotheses are confirmed, Max.Slope and β are better indicators of bone collagen quality because they are measures of the connectivity or, more generally, the integrity of the bone collagen network. • Hydrothermal isometric tension (HIT) testing for evaluation of bone collagen quality • Improved physics-based model of hydrothermal isometric tension (HIT) testing is developed. • Model used to interpret and validate bone collagen quality measure Max.Slope is derived from HIT. • Correlations between model parameters α, β, and Max.Slope are confirmed. • Inclusion of tissue collagen density is crucial for assessment across tissue types and donors. [ABSTRACT FROM AUTHOR]

The field of osteoimmunology has primarily focused on fracture healing in isolated musculoskeletal injuries. The innate immune system is the initial response to fracture, with inflammatory macrophages, cytokines, and neutrophils arriving first at the fracture hematoma, followed by an anti-inflammatory phase to begin the process of new bone formation. This review aims to first discuss the current literature and knowledge gaps on the immune responses governing single fracture healing by encompassing the individual role of macrophages, neutrophils, cytokines, mesenchymal stem cells, bone cells, and other immune cells. This paper discusses the interactive effects of these cellular responses underscoring the field of osteoimmunology. The critical role of the metabolic environment in guiding the immune system properties will be highlighted along with some effective therapeutics for fracture healing in the context of osteoimmunology. However, compared to isolated fractures, which frequently heal well, long bone fractures in over 30 % of polytrauma patients exhibit impaired healing. Clinical evidence suggests there may be distinct physiologic and inflammatory pathways altered in polytrauma resulting in nonunion. Nonunion is associated with worse patient outcomes and increased societal healthcare costs. The dysregulated immunomodulatory/inflammatory response seen in polytrauma may lead to this increased nonunion rate. This paper will investigate the differences in immune response between isolated and polytrauma fractures. Finally, future directions for fracture studies are explored with consideration of the emerging roles of newly discovered immune cell functions in fracture healing, the existing challenges and conflicting results in the field, the translational potential of these studies in clinic, and the more complex nature of polytrauma fractures that can alter cell functions in different tissues., Competing Interests: Declaration of competing interest Authors of this paper have no conflicts to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Skeletal muscle and bone interact to maintain their structure and function. Physical exercise is the most effective and easily applicable strategy to maintain their functions; however, exercise-induced interactions by soluble factors remained elusive. Our study aimed to identify exercise-induced interactions between muscle and bone by examining (1) the effects of myokine on bone and (2) the effects of osteocalcin (OCN) on skeletal muscle. To understand the effects of exercise-induced myokines on bone, we examined the effects of FNDC5 for aerobic exercise and IGF-1 for resistance exercise using a muscle-specific myokine overexpression model. To examine OCN effects on muscle, mice were intraperitoneally administered OCN-neutralizing antibody during long-term exercise. Our result showed that aerobic exercise tended to increase serum HA-tag protein attached to FNDC5 in muscle-specific overexpression groups. In addition, osteoblastic activation was increased only after aerobic exercise with HA/FNDC5 overexpression. Resistance exercise did not alter circulating HA-tag (muscle-derived IGF-1) and bone metabolism after IGF-1/HA overexpression. In the OCN study, aerobic exercise enhanced endurance capacity by restoring muscle glycogen content; however, OCN neutralization returned these to baseline. After resistance exercise, OCN suppression inhibited muscle hypertrophy and strength gains by preventing protein synthesis. Our results suggest that aerobic exercise following FNDC5 muscle overexpression promotes osteoblast activity, which may be partially caused by muscle-derived FNDC5 secretion. In addition, OCN was necessary for muscle adaptation in both aerobic and resistance exercises., Competing Interests: Declaration of competing interest All authors have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Craniofacial morphogenesis depends on complex cell fate decisions during the differentiation of post-migratory cranial neural crest cells. Molecular mechanisms of cell differentiation of mesenchymal cells to developing bones, cartilage, teeth, tongue, and other craniofacial tissues are still poorly understood. We performed single-cell transcriptomic analysis of craniofacial mesenchymal cells derived from cranial NCCs in mouse embryo. Using FACS sorting of Wnt1-Cre2 progeny, we carefully mapped the cell heterogeneity in the craniofacial region during the initial stages of cartilage and bone formation. Transcriptomic data and in vivo validations identified molecular determinants of major cell populations involved in the development of lower and upper jaw, teeth, tongue, dermis, or periocular mesenchyme. Single-cell transcriptomic analysis of Meis2-deficient mice revealed critical gene expression differences, including increased osteogenic and cell adhesion markers. This leads to affected mesenchymal cell differentiation and increased ossification, resulting in impaired bone, cartilage, and tongue formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. Defects in bone homeostasis result in common age-related diseases including osteoporosis. Serum levels of miR-324-3p positively correlate with several features of bone maintenance. In contrast here, using in vivo micro-computed tomography and histology, global miR-324-null mice demonstrated increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice had reduced lipid content while TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. Ex vivo assays showed that the high bone mass phenotype of miR-324-null mice resulted from both increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator Runx2 were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, in vitro Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in bone homeostasis by regulating aspects of both bone formation and remodelling. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial Permeability Transition Pore (MPTP) and its key positive regulator, Cyclophilin D (CypD), control activity of cell oxidative metabolism important for differentiation of stem cells of various lineages including osteogenic lineage. Our previous work (Sautchuk et al., 2022) showed that CypD gene, Ppif , is transcriptionally repressed during osteogenic differentiation by regulatory Smad transcription factors in BMP canonical pathway, a major driver of osteoblast (OB) differentiation. Such a repression favors closure of the MPTP, priming OBs to higher usage of mitochondrial oxidative metabolism. The physiological role of CypD/MPTP regulation was demonstrated by its inverse correlation with BMP signaling in aging and bone fracture healing in addition to the negative effect of CypD gain-of-function (GOF) on bone maintenance. Here we show evidence that CypD GOF also negatively affects bone development and growth as well as fracture healing in adult mice. Developing craniofacial and long bones presented with delayed ossification and decreased growth rate, respectively, whereas in fracture, bony callus volume was diminished. Given that Genome Wide Association Studies showed that PPIF locus is associated with both body height and bone mineral density, our new data provide functional evidence for the role of PPIF gene product, CypD, and thus MPTP in bone growth and repair. [Display omitted] • CypD GOF negatively affects cranial and long bone development, and fracture repair. • MPTP opening in OBs triggers an Ihh -led compensatory response in the growth plate. • GWAS showed that PPIF locus is associated with body height and bone mineral density. • Our data provide functional evidence for the role of PPIF in bone growth and repair. [ABSTRACT FROM AUTHOR]

Bone is influenced by many factors such as genetics and mechanical loading, but the short-term physiological effects of these factors on bone (re)modelling are not well characterised. This study investigated the effects of endurance trainability phenotype, sex, and interval running training (7-week intervention) on bone collagen formation in rats using a deuterium oxide stable isotope tracer method. Bone samples of the femur diaphysis, proximal tibia, mid-shaft tibia, and distal tibia were collected after necropsy from forty-six 9 ± 3-month male and female rats selectively bred for yielding low (LRT) or high (HRT) responses to endurance training. Bone collagen proteins were isolated and hydrolysed, and fractional synthetic rates (FSRs) were determined by the incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. There was a significant large main effect of phenotype at the femur site (p < 0.001; η2 g = 0.473) with HRT rats showing greater bone collagen FSRs than LRT rats. There was a significant large main effect of phenotype (p = 0.008; η2 g = 0.178) and a significant large main effect of sex (p = 0.005; η2 g = 0.196) at the proximal site of the tibia with HRT rats showing greater bone collagen FSRs than LRT rats, and male rats showing greater bone collagen FSRs compared to female rats. There was a significant large main effect of training at the mid-shaft site of the tibia (p = 0.012; η2 g = 0.159), with rats that underwent interval running training having greater bone collagen FSRs than control rats. Similarly, there was a significant large main effect of training at the distal site of the tibia (p = 0.050; η2 g = 0.156), with rats in the interval running training group having greater bone collagen FSRs compared to rats in the control group. Collectively, this evidence highlights that bone responses to physiological effects are site-specific, indicating that interval running training has positive effects on bone collagen synthesis at the tibial mid-shaft and distal sites, whilst genetic factors affect bone collagen synthesis at the femur diaphysis (phenotype) and proximal tibia (phenotype and sex) in rats. • This is the first study to report the interactive effects of endurance training heritability, sex, and running exercise on bone collagen synthesis using stable isotopes. • Bone formation is affected differently by genetic (phenotype and sex) and environmental (exercise) effects. • Interval running training has a positive effect on bone collagen synthesis at the tibial mid-shaft and distal sites. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) is associated with increased fracture risk in White adults. However, the impact of DM on fractures in Black adults is unknown. This systematic review and meta-analysis investigated the association between DM and fractures in adults of African ancestry. MEDLINE, Scopus, CINAHL and Embase databases were searched from their inception up to November 2023 for all studies in the English language investigating the epidemiology of fractures (prevalence, incidence, or risk) in Black men and women (age ≥ 18 years) with type 1 or type 2 DM. Effect sizes for prevalence of previous fractures (%) and incident fracture risk (hazard ratio [HR]) were calculated using a random-effects model on Stata (version 18.0). There were 13 eligible studies, of which 12 were conducted in Black adults from the United States, while one was conducted in adults of West African ancestry from Trinidad and Tobago. We found no fracture data in Black adults with DM living in Africa. Five studies were included in a meta-analysis of incident fracture risk, reporting data from 2926 Black and 6531 White adults with DM. There was increased risk of fractures in Black adults with DM compared to non-DM (HR = 1.65; 95 % confidence interval [CI]: 1.14, 2.39). The risk of fractures was also higher in White adults with DM compared to non-DM (HR = 1.31; 95 % CI: 1.06, 1.61) among these studies. Five studies were included in a meta-analysis of fracture prevalence, of which three also reported fracture prevalence in White adults. There were 175 previous fractures among 993 Black adults with DM and 384 previous fractures among 1467 White adults with DM, with a pooled prevalence of 17.5 % (95 % CI: 15.4, 19.6) and 25.8 % (95 % CI: 4.8, 46.8), respectively. Our results indicate a high burden of fractures in Black adults with DM. • Diabetes is associated with increased fracture risk in Black adults. • There is a high prevalence of previous fractures in Black adults with diabetes. • Fracture risk may be higher in Black adults compared to White adults with diabetes. • More cost-effective and sensitive diagnostic fracture tools are required in Africa. [ABSTRACT FROM AUTHOR]

The traditional understanding of bone mechanosensation implicates osteocytes, canaliculi, and the lacunocanalicular network in biomechanical adaptation. However, recent findings challenge this notion, as shown in advanced teleost fish where anosteocytic bone lacking osteocytes are nevertheless responsive to mechanical load. To investigate specific molecular mechanisms involved in bone mechanoadaptation in osteocytic and anosteocytic fish bone, we conducted a 5-min single swim-training experiment with zebrafish and ricefish, respectively. Through RNASeq analysis of fish spines, analyzed at various time points following swim training, we uncovered distinct gene expression patterns in osteocytic and anosteocytic fish bones. Notably, osteocytic fish bone exhibited an early response to mechanical load, contrasting to a delayed response observed in anosteocytic fish bones, both within 8 h following stimulation. We identified an increase in osteoblast differentiation in anosteocytic bone following training, while chordoblast activity was delayed. This temporal response suggests a time-dependent adaptation in anosteocytic bone, indicating the presence of intricate feedback networks within bone that lacks osteocytes. • Osteocytic and anosteocytic fish performed a single 5-minute swimming bout. • Osteocytic and anosteocytic fish bone has distinct gene expression patterns after swim training. • RNASeq reveals rapid increase in bone-related DEGs in osteocytic fish bone. • RNASeq reveals delayed expression of bone-related DEGs in anosteocytic fish bone. • The notion osteocytes are sole cells responsible for mechanoadaptation may require revision. [ABSTRACT FROM AUTHOR]

Bone is susceptible to fluctuations in iron homeostasis, as both iron deficiency and overload are linked to poor bone strength in humans. In mice, however, inconsistent results have been reported, likely due to different diet setups or genetic backgrounds. Here, we assessed the effect of different high and low iron diets on bone in six inbred mouse strains (C57BL/6J, A/J, BALB/cJ, AKR/J, C3H/HeJ, and DBA/2J). Mice received a high (20,000 ppm) or low-iron diet (∼10 ppm) after weaning for 6–8 weeks. For C57BL/6J males, we used two dietary setups with similar amounts of iron, yet different nutritional compositions that were either richer ("TUD study") or poorer ("UCLA study") in minerals and vitamins. After sacrifice, liver, blood and bone parameters as well as bone turnover markers in the serum were analyzed. Almost all mice on the UCLA study high iron diet had a significant decrease of cortical and trabecular bone mass accompanied by high bone resorption. Iron deficiency did not change bone microarchitecture or turnover in C57BL/6J, A/J, and DBA/2J mice, but increased trabecular bone mass in BALB/cJ, C3H/HeJ and AKR/J mice. In contrast to the UCLA study, male C57BL/6J mice in the TUD study did not display any changes in trabecular bone mass or turnover on high or low iron diet. However, cortical bone parameters were also decreased in TUD mice on the high iron diet. Thus, these data show that cortical bone is more susceptible to iron overload than trabecular bone and highlight the importance of a nutrient-rich diet to potentially mitigate the negative effects of iron overload on bone. • Effects of high iron diet on bone are strain-depenent. • Cortical bone is more susceptible to dietary iron overload-induced bone loss than trabecular bone. • A nutrient-rich diet can potentially mitigate the deleterious effects of iron overload on bone. [ABSTRACT FROM AUTHOR]

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis. [Display omitted] • Bone-specific genes are silenced by closed chromatin in non-osteogenic cells. • Cell signaling and transcription regulate enzymes that modify DNA and histones. • Enzymes mediating acetylation and methylation of histones regulate osteogenesis. • Modifications of H3 lysines 4, 9 and 27 represent principal epigenetic marks. • The complexities of epigenetic control during osteogenesis have been insufficiently resolved. [ABSTRACT FROM AUTHOR]

This study investigated the efficacy of the two FDA-approved bone anabolic ligands of the parathyroid hormone receptor 1 (PTH1R), teriparatide or human parathyroid hormone 1–34 (PTH) and abaloparatide (ABL), to restoring skeletal health using a preclinical murine model of streptozotocin-induced T1-DM. Intermittent daily subcutaneous injections of equal molar doses (12 pmoles/g/day) of PTH (50 ng/g/day), ABL (47.5 ng/g/day), or vehicle, were administered for 28 days to 5-month-old C57Bl/6 J male mice with established T1-DM or control (C) mice. ABL was superior to PTH in increasing or restoring bone mass in control or T1-MD mice, respectively, which was associated with superior stimulation of trabecular and periosteal bone formation, upregulation of osteoclastic/osteoblastic gene expression, and increased circulating bone remodeling markers. Only ABL corrected the reduction in ultimate load, which is a measure of bone strength, induced by T1-DM, and it also increased energy to ultimate load. In addition, bones from T1-DM mice treated with PTH or ABL exhibited increased ultimate stress, a material index, compared to T1-DM mice administered with vehicle. And both PTH and ABL prevented the increased expression of the Wnt antagonist Sost/sclerostin displayed by T1-DM mice. Further, PTH and ABL increased to a similar extent the circulating bone resorption marker CTX and the bone formation marker P1NP in T1-DM after 2 weeks of treatment; however, only ABL sustained these increases after 4 weeks of treatment. We conclude that at equal molar doses, ABL is more effective than PTH in increasing bone mass and restoring the cortical and trabecular bone lost with T1-DM, due to higher and longer-lasting increases in bone remodeling. • Bone fragility associated with diabetes causes substantial morbidity and mortality. • Data regarding the efficacy of anabolic therapies in T1-DM patients are scarce. • T1-DM bone disease is characterized by a marked decrease in bone formation. • Standard of care with antiresorptives stops bone loss but does not increase bone formation. • PTH1R activation rebuilds bone, improves structure, and restores strength in T1-DM. [ABSTRACT FROM AUTHOR]

Chronic heavy alcohol consumption may influence the skeleton by suppressing intracortical bone remodeling which may impact the quality of bone and its mechanical properties. However, this aspect has not been thoroughly assessed in either humans or animal models whose cortical bone microstructure resembles the microstructure of human cortical bone. The current study is the first to investigate the effects of chronic heavy alcohol consumption on various mechanical properties of bone in a non-human primate model with intracortical remodeling. Male rhesus macaques (5.3 years old at the initiation of treatment) were induced to drink alcohol and then gi v en the choice to voluntarily self-administer water or ethanol (4 % w / v) for approximately 14 months, followed by three abstinence phases (lasting 34, 41, and 39–46 days) with approximately 3 months of ethanol access in between. During the initial 14 months of open-access, monkeys in the alcohol group consumed an average of 2.9 ± 0.8 g/kg/d ethanol (mean ± SD) resulting in a blood ethanol concentration of 89 ± 47 mg/dl in longitudinal samples taken at 7 h after the daily sessions began. To understand the impact of alcohol consumption on material properties, various mechanical tests were conducted on the distal tibia diaphysis of 2–5 monkeys per test group, including dynamic mechanical analysis (DMA) testing, nano-indentation, microhardness testing, compression testing, and fracture resistance curve (R -curve) testing. Additionally, compositional analyses were performed using Fourier-transform infrared (FTIR) spectroscopy. Significant differences in microhardness, compressive stress-strain response, and composition were not observed with alcohol consumption, and only minor differences were detected in hardness and elastic modulus of the matrix and osteons from nanoindentation. Furthermore, the R -curves of both groups overlapped, with similar crack initiation toughness, despite a significant decrease in crack growth toughness (p = 0.032) with alcohol consumption. However, storage modulus (p = 0.029) and loss factor (p = 0.015) from DMA testing were significantly increased in the alcohol group compared to the control group, while loss modulus remained unchanged. These results indicate that heavy alcohol consumption may have only a minor influence on the material properties and the composition of cortical bone in young adult male rhesus macaques. • First alcohol study investigating bone mechanical properties using rhesus macaques • DMA testing revealed increase in storage modulus and decrease in loss factor • Hardness, compressive stress-strain response, and bone composition were not affected • Crack initiation toughness was unchanged but growth toughness decreased [ABSTRACT FROM AUTHOR]

Conventional bone imaging methods primarily use X-ray techniques to assess bone mineral density (BMD), focusing exclusively on the mineral phase. This approach lacks information about the organic phase and bone water content, resulting in an incomplete evaluation of bone health. Recent research highlights the potential of ultrashort echo time magnetic resonance imaging (UTE MRI) to measure cortical porosity and estimate BMD based on signal intensity. UTE MRI also provides insights into bone water distribution and matrix organization, enabling a comprehensive bone assessment with a single imaging technique. Our study aimed to establish quantifiable UTE MRI-based biomarkers at clinical field strength to estimate BMD and microarchitecture while quantifying bound water content and matrix organization. Femoral bones from 11 cadaveric specimens (n = 4 males 67–92 yrs of age, n = 7 females 70–95 yrs of age) underwent dual-echo UTE MRI (3.0 T, 0.45 mm resolution) with different echo times and high resolution peripheral quantitative computed tomography (HR-pQCT) imaging (60.7 μm voxel size). Following registration, a 4.5 mm HR-pQCT region of interest was divided into four quadrants and used across the multi-modal images. Statistical analysis involved Pearson correlation between UTE MRI porosity index and a signal-intensity technique used to estimate BMD with corresponding HR-pQCT measures. UTE MRI was used to calculate T 1 relaxation time and a novel bound water index (BWI), compared across subregions using repeated measures ANOVA. The UTE MRI-derived porosity index and signal-intensity-based estimated BMD correlated with the HR-pQCT variables (porosity: r = 0.73, p = 0.006; BMD: r = 0.79, p = 0.002). However, these correlations varied in strength when we examined each of the four quadrants (subregions, r = 0.11–0.71). T 1 relaxometry and the BWI exhibited variations across the four subregions, though these differences were not statistically significant. Notably, we observed a strong negative correlation between T 1 relaxation time and the BWI (r = −0.87, p = 0.0006). UTE MRI shows promise for being an innocuous method for estimating cortical porosity and BMD parameters while also giving insight into bone hydration and matrix organization. This method offers the potential to equip clinicians with a more comprehensive array of imaging biomarkers to assess bone health without the need for invasive or ionizing procedures. • UTE MRI porosity index correlated with porosity measured by HRpQCT. • Estimated BMD based on UTE MRI signal intensity correlated with HRpQCT BMD. • UTE MRI could additionally quantify bound water. • T 1 relaxation time by UTE MRI varied across cortical bone specimens. • UTE MRI could provide a comprehensive imaging evaluation of bone health. [ABSTRACT FROM AUTHOR]

Proximal femoral (hip) strength computed by subject-specific CT scan-based finite element (FE) models has been explored as an improved measure for identifying subjects at risk of hip fracture. However, to our knowledge, no published study has reported the effect of loading condition on the association between incident hip fracture and hip strength. In the present study, we performed a nested age- and sex-matched case-control study in the Age Gene/Environment Susceptibility (AGES) Reykjavik cohort. Baseline (pre-fracture) quantitative CT (QCT) scans of 5500 older male and female subjects were obtained. During 4-7years follow-up, 51 men and 77 women sustained hip fractures. Ninety-seven men and 152 women were randomly selected as controls from a pool of age- and sex-matched subjects. From the QCT data, FE models employing nonlinear material properties computed FE-strength of the left hip of each subject in loading from a fall onto the posterolateral (FPL), posterior (FP) and lateral (FL) aspects of the greater trochanter (patent pending). For comparison, FE strength in stance loading (FStance) and total femur areal bone mineral density (aBMD) were also computed. For all loading conditions, the reductions in strength associated with fracture in men were more than twice those in women (p≤0.01). For fall loading specifically, posterolateral loading in men and posterior loading in women were most strongly associated with incident hip fracture. After adjusting for aBMD, the association between FP and fracture in women fell short of statistical significance (p=0.08), indicating that FE strength provides little advantage over aBMD for identifying female hip fracture subjects. However, in men, after controlling for aBMD, FPL was 424N (11%) less in subjects with fractures than in controls (p=0.003). Thus, in men, FE models of posterolateral loading include information about incident hip fracture beyond that in aBMD.

PurposeTo determine if important geographic differences exist in treatment rates for osteoporosis and whether this variation can be explained by regional variation in risk factors.MethodsThe Global Longitudinal Study of Osteoporosis in Women is an observational study of women ≥55 years sampled from primary care practices in 10 countries. Self-administered questionnaires were used to collect data on patient characteristics, risk factors for fracture, previous fractures, anti-osteoporosis medication, and health status.ResultsAmong 58,009 women, current anti-osteoporosis medication use was lowest in Northern Europe (16%) and highest in U.S.A. and Australia (32%). Between 48% (U.S.A., Southern Europe) and 68% (Northern Europe) of women aged ≥65 years with a history of spine or hip fracture since age 45 were untreated. Among women with osteoporosis, the percentage of treated cases was lowest in Europe (45-52% versus 62-65% elsewhere). Women with osteopenia and no other risk factors were treated with anti-osteoporosis medication most frequently in U.S.A. (31%) and Canada (31%), and least frequently in Southern Europe (12%), Northern Europe (13%), and Australia (16%). After adjusting for risk factors, U.S. women were threefold as likely to be treated with anti-osteoporosis medication as Northern European women (odds ratio 2.8; 95% confidence interval 2.5-3.1) and 1.5 times as likely to be treated as Southern European women (1.5, 1.4-1.6). Up to half of women reporting previous hip or spine fracture did not receive treatment.ConclusionsThe likelihood of being treated for osteoporosis differed between regions, and cannot be explained by variation in risk factors. Many women at risk of fracture do not receive prophylaxis.

Bone fracture repair represents an important clinical challenge with nearly 1 million non-union fractures occurring annually in the U.S. Gene expression differs between non-union and healthy repair, suggesting there is a pattern of gene expression that is indicative of optimal repair. Despite this, the gene expression profile of fracture repair remains incompletely understood. In this work, we used RNA-seq of two well-established murine fracture models to describe gene expression of intramembranous and endochondral bone formation. We used top differentially expressed genes, enriched gene ontology terms and pathways, callus cellular phenotyping, and histology to describe and contrast these bone formation processes across time. Intramembranous repair, as modeled by ulnar stress fracture, and endochondral repair, as modeled by femur full fracture, exhibited vastly different transcriptional profiles throughout repair. Stress fracture healing had enriched differentially expressed genes associated with bone repair and osteoblasts, highlighting the strong osteogenic repair process of this model. Interestingly, the PI3K-Akt signaling pathway was one of only a few pathways uniquely enriched in stress fracture repair. Full fracture repair involved a higher level of inflammatory and immune cell related genes than did stress fracture repair. Full fracture repair also differed from stress fracture in a robust downregulation of ion channel genes following injury, the role of which in fracture repair is unclear. This study offers a broad description of gene expression in intramembranous and endochondral ossification across several time points throughout repair and suggests several potentially intriguing genes, pathways, and cells whose role in fracture repair requires further study. • Inflammation is much higher in full fracture repair than stress fracture repair. • Stress fracture repair is more enriched for osteoblast related genes and GO terms. • PI3K-Akt signaling is enriched in stress fracture repair. • Voltage gated ion channels are downregulated in full fracture repair. [ABSTRACT FROM AUTHOR]

The microarchitecture of bone tissue presents many features that could act as stress concentrators for the initiation of bone microdamage. This was first identified by John Currey in a seminal paper in 1962 in which he presented the mechanical and biological evidence for stress concentrations at the bone surface, within the bone through the action of stiffness differentials between architectural features including between lamellae, and at the level of the lacunar and canalicular walls. Those early observations set the stage to consider how microscopic damage to bone tissue might affect the properties of bone at a time when most in the scientific community dismissed microcracks in bone as artifact. Evidence collected in the nearly 60 years since those important initial observations suggest that some of these architectural features in bone tissue are more effective as crack arrestors than as crack initiators. Sites of higher mineralization in the bone matrix, particularly interstitial sites in both cortical and trabecular bone, may serve preferentially as locations for crack initiation, whereas those boundaries identified by Currey as both stress concentrators and stress arrestors are more effective at stopping cracks than at initiating them. • John Currey identified stress concentrators and stress arrestors in bone nearly 60 years ago. • Evidence collected since indicate some of these architectural features are more effective crack arrestors than initiators. • Highly mineralized bone matrix, rather than stress concentration per se, may be effective stress concentrators. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) results in a dramatic increase in skeletal fracture risk. Bisphosphates (BP) are an effective treatment for reducing fracture risk but they are not recommended in advanced CKD. We have recently shown higher acute skeletal accumulation of fluorescently-tagged zoledronate (ZOL) in the setting of CKD but how this accumulation is retained/lost over time is unclear. Furthermore, it is unknown if alternative dosing approaches can modulate accumulation in the setting of CKD. To address these two questions normal (NL) and Cy/+ (CKD) rats were divided into control groups (no dosing), a single dose of a fluorescent-tagged ZOL (FAM-ZOL), a single dose of non-labelled zoledronate (ZOL) or ten weekly doses of FAM-ZOL each at 1/10th the dose of the single dose group. Half of the CKD animals in each group were provided water with 3% calcium in drinking water (CKD + Ca) to suppress PTH and remodeling. At 30 or 35 weeks of age, serum, tibia, ulna, radius, vertebra, femora, and mandible were collected and subjected to assessment methods including biochemistry, dynamic histomorphometry and multi-spectral fluorescence levels (using IVIS SpectrumCT). FAM-ZOL did not significantly reduce bone remodeling in either NL or CKD animals while Ca supplementation in CKD produced remodeling levels comparable to NL. At five- and ten-weeks post-dosing, both CKD and CKD + Ca groups had higher levels of FAM-ZOL in most, but not all, skeletal sites compared to NL with no difference between the two CKD groups suggesting that the rate of remodeling did not affect skeletal retention of FAM-ZOL. Fractionating the FAM-ZOL into ten weekly doses led to 20–32% less (p < 0.05) accumulation/retention of compound in the vertebra, radius, and ulna compared to administration as a single dose. The rate of bone turnover does not have significant effects on levels of FAM-ZOL accumulation/retention in animals with CKD. A lower dose/more frequent administration paradigm results in lower levels of accumulation/retention over time. These data provide information that could better inform the use of bisphosphonates in the setting of CKD in order to combat the dramatic increase in fracture risk. • Bisphosphonates effectively preventing bone loss in many conditions but are not used in CKD. • Levels of bisphosphonate accumulation and retention are studied in an animal model of CKD. • More frequent, lower doses of bisphosphonate result in lower overall accumulation/retention compared to single doses. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba -deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba −/− mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba -deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba −/− fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation. • GPR137b is a seven-pass transmembrane receptor expressed by osteoclasts • Decreasing Gpr137b in osteoclast precursors increased osteoclast differentiation and bone resorption activity in vitro • Loss-of-function gpr137ba zebrafish mutants demonstrated increased bone resorption • We propose that GPR137b is a conserved negative regulator of osteoclastogenesis in vertebrates [ABSTRACT FROM AUTHOR]

Within the dental alveolar socket, the sequence of events following tooth extraction involves deposition of a provisional connective tissue matrix that is later replaced by woven bone and eventually by lamellar bone. Bone regeneration within the dental alveolar socket is unique since the space occupied by the root(s) of a tooth does not originally contain any bone. However, extracellular matrix composition of the healing alveolar socket has not previously been investigated. Here, alveolar bone biopsies representing early (7–46 months, < 4y) and late (48–60 months; 4–5y) healing periods were investigated using Raman spectroscopy, X-ray micro-computed tomography and backscattered electron scanning electron microscopy. Partially or completely edentulous individuals and those with a smoking habit were not excluded. Between < 4y and 4–5y , mineral crystallinity and bone mineral density increase, phenylalanine, proline/hydroxyproline, and bone surface-to-volume ratio decrease, while the carbonate-to-phosphate ratio, the mineral-to-matrix ratio, and the collagen crosslink ratio remain relatively unchanged. Observed exclusively at 4–5y , hypermineralised osteocyte lacunae contain spherical and rhomboidal mineral nodules. Spearman correlation analysis reveals several significant, high (ρ = 0.7–0.9; p ≤ 0.01) and moderate (ρ = 0.5–0.7; p ≤ 0.01) correlations. Mineral crystallinity and proline/hydroxyproline, the carbonate-to-phosphate ratio and phenylalanine, mineral crystallinity and bone surface-to-volume ratio, the carbonate-to-phosphate ratio and bone surface-to-volume ratio, proline/hydroxyproline and bone mineral density, and bone mineral density and bone surface-to-volume ratio are negatively correlated. Mineral crystallinity and bone mineral density, and proline/hydroxyproline and bone surface-to-volume ratio are positively correlated. Although bone regeneration in the dental alveolar socket follows typical bone healing patterns, the compositional and microstructural patterns reveal mature bone at < 4y with indications of better mechanical competence at 4 – 5y. Unlabelled Image • Human alveolar bone biopsies representing early (< 4y; ~ 18 months) and late (4–5y; ~ 51 months) healing periods were studied • Between < 4y and 4–5y , mineral crystallinity increases, while phenylalanine and proline/hydroxyproline content decrease • The carbonate-to-phosphate ratio, mineral-to-matrix ratio, and collagen crosslink ratio remain constant between < 4y and 4–5y • Bone mineral density increases while bone surface-to-volume ratio decreases between < 4y and 4–5y • Observed exclusively at 4–5y, hypermineralised osteocyte lacunae contain spherical and rhomboidal mineral nodules [ABSTRACT FROM AUTHOR]

Proteins are an essential part of essentially all biological processes, and there is enormous variation in protein forms and concentrations that is not reflected in DNA or RNA. Recently there have been rapid advances in the ability to measure protein sequence, modification and concentration, particularly with methods based in mass spectrometry. Global measures of proteins in tissues or in the circulation provide a broad assessment of the proteome that can be extremely useful for discovery, and targeted proteomic measures can yield specific and sensitive assessments of specific peptides and proteins. While most proteomic measures are directed at the detection of consensus peptide sequences, mass spectrometry based proteomic methods also allow a detailed examination of the peptide sequence differences that result from genetic variants and that may have important effects on protein function. In evaluating proteomic data, a number of analytical considerations are important, including an understanding of missing data, the challenge of multiple testing and replication, and the use of rapidly evolving methods in systems biology. While proteomics has not yet had a major impact in skeletal research, interesting recent research has used these approaches in the study of bone cell biology and the discovery of biomarkers of skeletal disorders. Proteomics can be expected to have an increasing influence in the study of bone biology and pathophysiology. • Proteomics has not been extensively used in bone research, but these methods are becoming more commonly employed. • Major advances in proteomic measurement approaches have increased the ability to more comprehensively assess protein forms and concentrations. • Global proteomics approaches broadly survey the proteome in tissues or blood, while targeted proteomics assess specific proteins/peptides. • Mass spectrometry proteomics can determine both peptide sequence changes resulting from genetic variation and posttranslational modifications. • Key issues in assessing results include considerations of missing data, multiple testing, replication, and the use of systems biology. • Proteomics has not been extensively used in bone research, but proteomic approaches are becoming more commonly employed. [ABSTRACT FROM AUTHOR]

Osteoporosis and fracture risk are common complex diseases, caused by an interaction of numerous disease susceptibility genes and environmental factors. With the advances in genomic technologies, large-scale genome-wide association studies (GWAS) have been performed which have broadened our understanding of the genetic architecture and biological mechanisms of complex disease. Currently, more than ~90 loci have been found associated with DXA derived bone mineral density (BMD), over ~500 loci with heel estimated BMD and several others with other less widely available bone parameters such as bone geometry, shape, and microarchitecture. Notably, several of the pathways identified by the GWAS efforts correspond to pathways that are currently targeted for the treatment of osteoporosis. Overall, tremendous progress in the field of the genetics of osteoporosis has been achieved with the discovery of WNT16 , EN1 , DAAM2 , and GPC6 among others. Assessment of the function and biological mechanisms of the remaining genes may further untangle the complex genetic landscape of osteoporosis and fracture risk. With this review we aimed to provide a general overview of the existing GWAS studies on osteoporosis traits and fracture risk. • GWAS efforts have discovered >500 loci robustly associated with different bone parameters. • Pathways identified by the GWAS correspond to known-bone pathways currently targeted for the treatment of osteoporosis. • All fracture risk loci identified by GWAS are known BMD loci, underscoring the mediating role of BMD on fracture. • Heel BMD associated SNPs map near/to genes strongly enriched for positive control genes with pivotal role in bone biology. [ABSTRACT FROM AUTHOR]

Local muscle loss associated with open fractures remains an obstacle to functional recovery and bone healing. Muscle cells secrete bioactive myokines that elicit autocrine and paracrine effects and initiate signaling pathways for regenerating damaged muscle and bone. Mesenchymal stem/stromal cells (MSCs) are under investigation for the regeneration of both muscle and bone through their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit a more complex secretome with heightened therapeutic potential. We hypothesized that the osteogenic potential of myokines would be enhanced when myoblasts were exposed to the MSC spheroid secretome. Conditioned media from MSC spheroids increased osteogenic response of MC3T3 pre-osteoblasts compared to myokines from L6 myoblasts alone. This effect was synergistically enhanced when conditioned media of MSC spheroids was serially delivered to myoblasts and then osteoprogenitor cells in vitro. We then delivered myoblast-stimulated conditioned media in the presence or absence of syngeneic rat bone marrow stromal cells (rBMSCs) from alginate hydrogels to a rat critical-sized segmental defect. We observed increased bone formation in defects treated with conditioned media compared to rBMSCs alone, while bone formation was greatest in defects treated with both conditioned media and rBMSCs over 12 weeks. This foundational study demonstrates a novel approach for capitalizing on the paracrine signaling of muscle cells to promote bone repair and provides additional evidence of the synergistic interaction between muscle and bone. • Bone healing is impaired with concomitant muscle loss. • Muscle tissue secretes potent myokines that promote tissue repair. • Mesenchymal stem/stromal cell (MSC) spheroids induce changes in myoblast secretome. • Osteogenesis is enhanced when presenting myokines from MSC spheroid stimulation. • Bone union is attainable by presentation of myokines and osteoprogenitor cells. [ABSTRACT FROM AUTHOR]

The HOXA gene cluster is generally recognized as a pivotal mediator of positional identity in the skeletal system, expression of different orthologues conferring alternative locational phenotype of the vertebrate bone. Strikingly, however, the molecular mechanisms that regulate orthologue-specific expression of different HOXA cluster members in gestating osteoblasts remain largely obscure, but in analogy to the processes observed in acute lymphatic leukemia it is assumed that alternative methylation of HOXA promoter regions drives position specific expression patterns. In an effort to understand HOXA cluster gene expression in osteogenesis we characterize both expression and the epigenetic landscape of the HOXA gene cluster during in vitro osteoblast formation from mesenchymal precursors. We observe that osteoblast formation per se provokes strong upregulation of HOXA gene cluster expression, in particular of midcluster genes, and paradoxal downregulation of HOXA7 and HOXA10. These differences in expression appear related to promoter methylation. LnRNAs HOTAIR and HOTTIP, known to modulate HOXA expression, are also regulated by their promoter methylation processing, but do not correlate with HOXA cluster expression profile. We thus conclude that HOXA expression is profoundly regulated during osteoblast differentiation through canonical methylation-dependent mechanisms but not through the flanking lnRNAs. • Members of the HOXA gene cluster family are differentially expressed during osteoblast differentiation. • Methylation modifying enzymes are differentially expressed in osteoblasts. • Epigenetic machinery modulates HOXA genes during osteoblast differentiation. • DNMT drives HOXA gene activities during osteogenic phenotype acquisition. [ABSTRACT FROM AUTHOR]

Pigment epithelium-derived factor (PEDF) is known for its osteogenic properties, but its effects against primary and secondary bone tumors have not comprehensively been demonstrated. We show the ubiquitous expression of PEDF in murine embryonic tissue. Continuous administration of PEDF in pregnant mice for five days did not adversely affect foetal health, despite PEDF's known potent antiangiogenic properties. In the case of the devastating childhood bone cancer osteosarcoma, PEDF has direct anticancer activity per se , and protects against the toxicity of doxorubicin in the heart, small intestine and testes. PEDF demonstrated anti-proliferative and pro-apoptotic effects against human prostate and breast cancer cells, tumors which are known to metastasize to bone as the preferred secondary site. Caspase-2 was activated in both tumor cell types by PEDF. In models of prostate and breast cancer in bone, PEDF significantly reduced tumor volumes. When combined with zoledronic acid, continuously-administered PEDF significantly reduced breast tumor volume at the bone, and was able to preserve the quality of bone better than the combination therapy. These multiple positive findings make PEDF an ideal endogenous and safe biological for possible future clinical testing. In an animal model of the childhood bone cancer osteosarcoma, PEDF demonstrated direct anticancer activity per se , and protected against the toxicity of doxorubicin in the heart, small intestine and testes. PEDF demonstrated antiproliferative and pro-apoptotic effects against human prostate and breast cancer cells, tumors which are known to metastasize to bone as the preferred secondary site. When combined with zoledronic acid, continuously-administered PEDF significantly reduced breast tumor volume at the bone, and was able to preserve the quality of bone better than the combination therapy. Unlabelled Image • We show the ubiquitous expression of PEDF in murine embryonic tissue. • Continuous administration of PEDF in pregnant mice for five days did not adversely affect foetal health. • In the case of the devastating childhood bone cancer osteosarcoma, PEDF has direct anticancer activity per se. • In models of prostate and breast cancer in bone, PEDF significantly reduced tumor volumes. • When combined with zoledronic acid, PEDF significantly reduced breast tumor volume at the bone. [ABSTRACT FROM AUTHOR]

Previously we have demonstrated an association between maternal serum 25-hydroxyvitamin D (25(OH)D) during pregnancy and knee-heel length in offspring at birth. However, it is unknown whether maternal serum 25(OH)D is associated with bone measures in childhood. Thus, we aimed to examine associations between 25(OH)D at two stages of pregnancy and offspring bone measures at 11 years. Women were recruited from a single antenatal clinic in Victoria, Australia before 16 weeks gestation and provided two serum samples to determine 25(OH)D status at recruitment and 28–32 weeks gestation. Children and their mothers were followed up at 11 years of age. Children undertook dual energy X-ray absorptiometry scans at the lumbar spine and total body. Maternal 25(OH)D at recruitment (before 16 weeks gestation) was positively associated with the children's bone mineral content and density in boys, but not girls. In boys, a 10 nmol/L (4 ng/mL) increase in maternal 25(OH)D was associated with a median 0.5 g (95% CI 0.1,0.8) and 0.009 g/cm2 (95% CI 0.001,0.017) increase in bone mineral content and density at the spine, respectively, and a median 0.006 g/cm2 (95% CI 0.001,0.011) increase in at the total body. There was no sustained associations with 25(OH)D at the later timepoint (28–32 weeks) with any outcome. At age 11 years, maternal 25(OH)D levels during early pregnancy, but not late were positively associated with bone measures in boys, but not girls. • Maternal 25(OH)D in early pregnancy was associated with offspring bone measures at 11 years in a sexually dimorphic manner • Maternal 25(OH)D in early pregnancy was positively associated with bone density and content in boys, but not girls. • There were no clear maternal 25(OH)D cutpoints, though there appeared to be greater effect in those with 25(OH)D <28nmol/L [ABSTRACT FROM AUTHOR]

Diabetes increases the risk of fracture, impairs fracture healing and causes rapid loss of the fracture callus cartilage, which was linked to increased FOXO1 expression in chondrocytes. We recently demonstrated that deletion of FOXO1 in chondrocytes blocked the premature removal of cartilage associated with endochondral bone formation during fracture healing. However, the ultimate impact of this deletion on mechanical strength was not investigated and remains unknown. Closed fractures were induced in Col2α1Cre+.FOXO1L/L mice with lineage specific deletion of FOXO1 in chondrocytes compared to littermate controls. Type 1 diabetes was induced by multiple low dose streptozotocin treatment. Thirty-five days after fracture micro CT analysis showed that diabetes significantly reduced callus volume and bone volume (P < 0.05), both which were reversed by FOXO1 deletion in chondrocytes. Diabetes significantly reduced mechanical strength measured by maximum torque, stiffness, modulus of rigidity and toughness and FOXO1 deletion in diabetic mice rescued each parameter (P < 0.05). Diabetes also reduced both bone volume and mechanical strength in non-fractured femurs. However, FOXO1 deletion did not affect bone volume or strength in non-fractured bone. These results point to the important effect that diabetes has on chondrocytes and show for the first time that the premature removal of cartilage induced by FOXO1 in chondrocytes has a significant impact on the mechanical strength of the healing bone. • Diabetes impairs fracture healing and reduces mechanical strength of the facture callus. • Deletion of FOXO1 in chondrocytes rescues the negative effect of the diabetes on reduced bone formation. • FOXO1 deletion in chondrocytes rescues the negative effect of diabetes on reduced mechanical strength of the hard callus. [ABSTRACT FROM AUTHOR]

Stress fractures are common overuse injuries caused by repetitive bone loading. These fractures are of particular concern for military recruits and athletes resulting in attrition in up to 60% of recruits that sustain a fracture. Army and Navy recruits supplemented with daily calcium and vitamin D (Ca + D) demonstrated improved bone strength and reduced stress fractures. The aim of the current study was to evaluate whether Ca + D supplementation improves measures of bone health in recruits undergoing United States Marine Corps initial military training (IMT), and whether the effect of supplementation on indices of bone health varied by season. One-hundred ninety-seven Marine recruits (n = 107 males, n = 90 females, mean age = 18.9 ± 1.6 y) were randomized to receive either Ca + D fortified snack bars (2000 mg Ca and 1000 IU vitamin D per day) or placebo divided into twice daily doses during 12 weeks of IMT. Anthropometrics, fasted blood samples, and peripheral quantitative computed tomography (pQCT) scans of the tibial metaphysis and diaphysis were collected upon entrance to- and post-training (12 weeks later). Half of the volunteers entered training in July and the other half started in February. Time-by-group interactions were observed for vitamin D status (25OHD) and the bone turnover markers, BAP, TRAP and OCN. 25OHD increased and BAP, TRAP and OCN all decreased in the Ca + D group (p <.05). Training increased distal tibia volumetric BMD (+1.9 ± 2.8%), BMC (+2.0 ± 3.1%), and bone strength index (BSI; +4.0 ± 4.0%) and diaphyseal BMC (+1.0 ± 2.2%) and polar stress strain index (SSIp; +0.7 ± 2.1%) independent of Ca + D supplementation (p <.05 for all). When analyzed by season, change in BSI was greater in the Ca + D group as compared to placebo in the summer iteration only (T*G; p <.05). No other effects of supplementation on bone tissue were observed. When categorized by tertile of percent change in BSI, recruits demonstrating the greatest changes in BSI and 25OHD entered training with the lowest levels of 25OHD (p <.05). Over all, these results suggest that Ca + D supplementation reduced some markers of bone formation and resorption and the decline in 25OHD over training in volunteers that started training in the summer was prevented by supplementation. Baseline 25OHD and trajectory may impact bone responses to IMT, but little effect of Ca + D supplementation was observed at the investigated doses. • Season may impact bone responses to calcium and vitamin D in Marine recruits. • Supplementation prevented 25OHD decline in those starting training in the summer. • Daily calcium and vitamin D did not improve most pQCT indices of the tibia. • Recruits with the greatest bone strength increases also exhibited 25OHD increases. [ABSTRACT FROM AUTHOR]

Chronic exposure to combined fluoride (F) and arsenic (As) continues to be a major public health problem worldwide, attracting the attention of an increasing number of researchers. While bone is the main target organ of syndrome of endemic arsenic poisoning and fluorosis (SEAF), the specific mechanism and targeted intervention remains uncertain. The first question in this study sought to determine the interaction of F and As on the Wnt signaling pathway and its role in osteogenic differentiation in the SEAF population. As can be seen from the data, with the increase in exposure to F, the content of Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1) gradually decreased, but the expression of glycogen synthase kinase-3β (GSK3β), β-catenin and the osteogenic differentiation indicators pro-collagen I alpha 1 (COL1A1) and bone alkaline phosphatase (BALP) were increased. Next, we grouped the SEAF population according to urinary As and found that As can upregulate the GSK3β, β-catenin level and the bone formation bio-marker BALP in serum. But the experiments did not detect any evidence that As can change the content of DKK1 in serum. To better understand the combined effects of F and As on the Wnt signaling pathway, we performed further interaction analysis. These results suggest that the interaction of F and As can inhibit the GSK3β, β-catenin, COL1A1 and BALP. And DKK1 is mainly manifested by the independent effect of F. To further study the role of DKK1 and GSK3β in fluoride-arsenic pollution combined with osteogenic differentiation, we attempted to silence the DKK1 and GSK3β gene in hFOB 1.19 cells. The results show that F, As alone and in combination exposure can up-regulate GSK catenin transcription and protein expression levels and down-regulate DKK1, and COL1A1 and ALP are significantly increased, after silenced the DKK1. The same results did not appear after silenced the GSK3β. F and As alone and in combination exposure did not reverse the inhibition of GSK3β and β-catenin by GSK3β silencing, and COL1A1 and ALP are significantly decreased. The results indicate that silencing GSK3β instead of DKK1 can inhibit osteogenic differentiation caused by co-exposure to fluoride and arsenic. This study can provide a scientific basis for further understanding the causes of bone formation caused by F and As and the improvement of targeted intervention strategies. Unlabelled Image • Wnt signaling is involved in the osteogenic differentiation caused by co-exposure to F and As. • Silencing GSK3β can inhibit osteogenic differentiation caused by co-exposure to F and As. • Silencing DKK1 cannot inhibit osteogenic differentiation caused by co-exposure to F and As. • The interaction between F and As of the Wnt signaling occurs mainly as antagonism. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a disease of the whole joint characterized by cartilage loss and subchondral bone remodeling. The role of microcracks in cartilage integrity and subchondral bone homeostasis is not fully understood. The main goal of this work was to evaluate microcrack density in both calcified cartilage and subchondral bone plate in relation to cartilage damage in humans and to better define the association of microcracks and osteocyte density in subchondral bone. We investigated 18 bone cores from cadaveric human knees that were stained with En-Bloc Basic Fuchsin. We quantified microcrack density, osteocyte density, cartilage surfaces and cartilage damage. The presence of microcracks was confirmed for each bone core by scanning electron microscopy. Finally, trabecular subchondral bone parameters were measured by micro-CT. Microcracks were detected in both calcified cartilage and subchondral bone plate. The density of microcracks in both calcified cartilage (CC) and subchondral bone plate (SBP) was negatively correlated with cartilage damage (r = −0.45, p < 0.05). The presence of microcracks in SBP was associated with a lower histological OA score. Osteocytes formed a dendrite network that abruptly stopped at the border of calcified cartilage. Osteocyte density in subchondral bone plate was increased in the presence of microcracks in calcified cartilage. Subchondral bone plate microcracks might be required for maintaining cartilage homeostasis. Microcracks in calcified cartilage may trigger osteocyte density in subchondral bone plate with subsequent regulation of subchondral bone remodeling to prevent cartilage damage. • Subchondral bone contains a high density of osteocytes, the dendrites of which are bordering the calcified cartilage • Microcracks are negatively correlated with cartilage damage, suggesting that they might be required for cartilage homeostasis • Microcracks in the calcified cartilage are associated with increased osteocyte density in the subchondral bone • Microcracks in the calcified cartilage might regulate subchondral bone remodeling [ABSTRACT FROM AUTHOR]

Abstract Obese children grow faster than their normal-weight peers. Insulin resistance and hyperinsulinemia have been associated with obesity-related growth acceleration. To determine whether obesity-associated hyperinsulinemia promotes bone growth by activating the insulin receptor in the growth plate, we generated TamCart IR flox / flox mice. The injection of 4 doses of tamoxifen in these mice (beginning at postnatal day 5th with 2 days interval between injections) resulted in the Insulin Receptor (IR) gene excision exclusively in the cartilage. TamCart IR flox / flox tamoxifen-treated mice (KO mice) and their IR flox / flox control littermates (C mice) at 3 weeks of age were exposed to a standard or hypercaloric (high-fat) diet for 4 weeks. At the end of study, C and KO mice fed with a high-fat diet exhibited greater weight gain than the respective strains fed with a standard diet. Body and tibial growth and growth plate height of C mice fed with high-fat diet were greater than those of standard-diet-fed C mice; however, no difference was observed between KO mice fed with standard or high-fat diet with respect to body and tibial growth and growth plate height. Circulating levels of insulin, IGF-1 and leptin were significantly higher in C and KO mice exposed to high-fat diet compared to those in the same strain exposed to standard diet. Increased phosphorylation of Akt (one of the intracellular mediators of insulin action in bone) in the growth plate of C mice on high-fat diet (compared to those on standard diet) suggests that high-fat-mediated increased circulating insulin levels may directly affect growth plate function and bone growth. High-fat diet was not associated with any change of Akt phosphorylation in KO mice. In addition, in vitro studies in cultured primary chondrocytes revealed that Akt mediates the stimulatory effects of insulin on chondrocyte proliferation and differentiation. In conclusion, the activation of the insulin receptor in the growth plate of mice fed with a hypercaloric diet stimulates skeletal growth and growth plate chondrogenesis. Highlights • Insulin resistance and hyperinsulinemia in humans and experimental animal models are associated with growth acceleration. • High-fat diet stimulated bone growth in WT mice, but it did not in KO mice lacking the insulin receptor in chondrocytes. • High-fat-fed WT mice exhibited increased growth plate cell proliferation and differentiation, unlike high-fat-fed KO mice. • In cultured chondrocytes, Akt mediates the stimulatory effects of insulin on chondrocyte proliferation and differentiation. • Insulin receptor activation in the growth plate of high-fat-fed mice accelerates bone growth and growth plate function. [ABSTRACT FROM AUTHOR]

Abstract Cellular senescence refers to a process induced by various types of stress that causes irreversible cell cycle arrest and distinct cellular alterations, including profound changes in gene expression, metabolism, and chromatin organization as well as activation/reinforcement of anti-apoptotic pathways and development of a pro-inflammatory secretome or senescence-associated secretory phenotype (SASP). However, because of challenges and technical limitations in identifying and characterizing senescent cells in living organisms, only recently have some of the diverse in vivo roles of these unique cells been discovered. New findings indicate that senescent cells and their SASP can have acute beneficial functions, such as in tissue regeneration and wound healing. However, in contrast, when senescent cells accumulate in excess chronically at sites of pathology or in old tissues they drive multiple age-associated chronic diseases. Senotherapeutics that selectively eliminate senescent cells ("senolytics") or inhibit their detrimental SASP ("senomorphics") have been developed and tested in aged preclinical models. These studies have established that targeting senescence is a powerful anti-aging strategy to improve "healthspan" – i.e. , the healthy period of life free of chronic disease. The roles of senescence in mediating age-related bone loss have been a recent focus of rigorous investigation. Studies in mice and humans demonstrate that with aging, at least a subset of most cell types in the bone microenvironment become senescent and develop a heterogeneous SASP. Furthermore, age-related bone loss can be alleviated in old mice, with apparent advantages over anti-resorptive therapy, by reducing the senescent cell burden genetically or pharmacologically with the first class of senolytics or a senomorphic. Collectively, these findings point to targeting senescence as a transformational strategy to extend healthspan, therefore providing strong rationale for identifying and optimizing senotherapeutics to alleviate multiple chronic diseases of aging, including osteoporosis, and set the stage for translating senotherapeutics to humans, with clinical trials currently ongoing. Highlights • Cellular senescence is process induced by various types of stress. • Senescent cells develop a unique senescence-associated secretory phenotype (SASP). • With aging, at least a subset of most cell types in bone becomes senescent. • Eliminating senescent cells or disrupting the SASP in old mice compress morbidity. • Targeting senescent cells in old mice prevent age-related bone loss and frailty. [ABSTRACT FROM AUTHOR]

Abstract Serum bone turnover markers show diurnal variation in humans, suggesting that circadian rhythms contribute to normal bone physiology. This conclusion is corroborated by bone phenotypes in mice with genetic disruption of the circadian molecular clock mechanism, for instance via deletion of the transcription factor Brain and Muscle Arntl-like 1 (Bmal1). To dissect the contribution of circadian molecular clocks in individual bone cell types, we generated mice with conditional deletion of Bmal1 in osteoclasts (Ctsk-cre) and in mesenchymal cells of the limbs (Prx1-cre). We report that deletion of Bmal1 in osteoclasts had no effect on trabecular or cortical bone parameters in vivo or on osteoclast differentiation in vitro. In contrast, Bmal1 f/f .Prx1-cre mice had significantly less trabecular and cortical bone than Bmal1 f/f littermate controls, recapitulating the bone phenotype of Bmal1 germline deficient mice. The number of osteoblast precursors in the bone marrow of Bmal1 f/f .Prx1-cre mice was similar to wild-type controls, while the in vitro differentiation capacity of Bmal1 -deficient osteoblast precursors, measured as induction of alkaline phosphatase activity, was significantly lower. Despite this, serum procollagen type 1 N-terminal propeptide (P1NP), a measure of bone formation in vivo , was higher in Bmal1 f/f .Prx1-cre mice than in Bmal1 f/f mice. Consistent with a high bone turnover state in the mutant mice, the bone resorption marker serum C-terminal telopeptides of Type I collagen (CTX-I) was also elevated, and Bmal1 f/f .Prx1-cre mice had a higher number of tartrate resistant acid phosphatase (TRAP) positive osteoclasts than Bmal1 f/f controls. These results demonstrate that adult bone mass in mice is controlled by the intrinsic circadian molecular clock in mesenchymal cells but not osteoclasts. The effect of the mesenchymal cell clock on bone turnover appears to involve osteoblast-osteoclast cross-talk. Highlights • Bmal1 −/− mice with genetic disruption of the circadian molecular clock have a low bone mass phenotype. • The bone phenotype of Bmal1 −/− mice is recapitulated in mice with conditional Bmal1 deletion in mesenchymal cells (Bmal1 f/f .Prx1-cre). • Bmal1 f/f .Prx1-cre mice exhibit high bone turnover with enhanced bone resorption and bone formation. [ABSTRACT FROM AUTHOR]

Abstract Chronic PTH deficiency has a marked effect on the skeleton, leading to characteristic decreases in bone remodeling and increases in bone mass. An effect on fracture risk has not been demonstrated, although biochemical, imaging, and histomorphometric data indicate abnormalities in skeletal properties1,21,21,21,2. Replacement with PTH leads to a new skeletal state that is maintained with long-term treatment. Highlights • Low bone turnover and increased bone density are typically seen hypoparathyroidism. • Histomorphometric data show abnormalities in skeletal properties. • There is a lack of evidence on increased fracture risk. • Treatment with rhPTH (1–84) leads to skeletal changes that are maintained. [ABSTRACT FROM AUTHOR]

Abstract Until recently, it was assumed that the only interaction between muscle and bone is mechanical, that the muscle acts as a pulley and the bone as a lever to move the organism. A relatively new concept is that muscle, especially contracted muscle, acts as a secretory organ, regulating metabolism. An even newer concept is that bone, especially the osteocytes in bone, act as endocrine cells targeting other organs such as kidney and more recently, muscle. These two new concepts logically led to the third concept: that muscle and bone communicate via soluble factors. Crosstalk occurs through muscle factors such as myostatin, irisin, and a muscle metabolite, β-aminoisobutyric acid, BAIBA, and through bone factors such as osteocalcin, transforming growth factor beta, TGFβ, Prostaglandin E2, PGE 2 and Wnts. Some of these factors have positive and some negative effects on the opposing tissue. One feature both bone and muscle have in common is that their tissues are mechanically loaded and many of their secreted factors are regulated by load. This mechanical loading, also known as exercise, has beneficial effects on many systems leading to the hypothesis that muscle and bone factors can be responsible for the beneficial effects of exercise. Many of the characteristics of aging and diseases associated with aging such as sarcopenia and osteoporosis and neurological conditions such as Alzheimer's disease and dementia, are delayed by exercise. This beneficial effect has been ascribed to increased blood flow increasing oxygen and nutrients, but could also be due to the secretome of the musculoskeletal system as outlined in this review. Highlights • The aging of the musculoskeletal system is strongly associated with health or morbidity. • Muscle and bone not only interact mechanically but through secreted biochemical signaling molecules. • The musculoskeletal system secretome most likely has effects on metabolism and function of other organs. • The musculoskeletal system is the mediator of the positive effects of exercise. [ABSTRACT FROM AUTHOR]

Abstract Purpose Adequate dietary protein intake is important for the maintenance of bone health; however, data in this area is ambiguous with some suggestion that high protein intake can have deleterious effects on bone health. The aim of the current study was to explore the associations of protein intake with bone mineral density (BMD). Methods We used baseline data from the UK Biobank (participants aged 40–69 years) to examine the association of protein intake with BMD (measured by ultrasound). These associations were examined, in women (n = 39,066) and men (n = 31,149), after adjustment for socio-demographic and lifestyle confounders and co-morbidities. Results Protein intake was positively and linearly associated with BMD in women (β-coefficient 0.010 [95% CI 0.005; 0.015, p < 0.0001]) and men (β-coefficient 0.008 [95% CI 0.000; 0.015, p = 0.044]); per 1.0 g/kg/day increment in protein intake, independently of socio-demographics, dietary factors and physical activity. Conclusions The current data have demonstrated that higher protein intakes are positively associated with BMD in both men and women. This indicates that higher protein intakes may be beneficial for both men and women. Highlights • A higher dietary protein intake was associated with a higher bone mineral density. • These associations were adjustment for socio-demographic and lifestyle confounders and co-morbidities • A sex by age interaction was observed, but stratified analysis found little difference in associations between age groups. [ABSTRACT FROM AUTHOR]

Abstract Bone marrow contains numerous different cell types arising from hematopoietic stem cells (HSCs) and non-hematopoietic mesenchymal/skeletal stem cells, in addition to other cell types such as endothelial cells- these non-hematopoietic cells are commonly referred to as stromal cells or microenvironment cells. HSC function is intimately linked to complex signals integrated by their niches, formed by combinations of hematopoietic and stromal cells. Studies of hematopoietic cells have been significantly advanced by flow cytometry methods, enabling the quantitation of each cell type in normal and perturbed situations, in addition to the isolation of these cells for molecular and functional studies. Less is known, however, about the specific niches for distinct developing hematopoietic lineages, or the changes occurring in the niche size and function in these distinct anatomical sites in the bone marrow under stress situations and ageing. Significant advances in imaging technology during the last decade have permitted studies of HSC niches in mice. Additional imaging technologies are emerging that will facilitate the study of human HSC niches in trephine BM biopsies. Here we provide an overview of imaging technologies used to study HSC niches, in addition to highlighting emerging technology that will help us to more precisely identify and characterize HSC niches in normal and diseased states. [ABSTRACT FROM AUTHOR]

X-linked hypophosphatemia (XLH) is an inherited disorder caused by inactivating mutations in the PHEX gene leading to renal phosphate wasting, rickets and osteomalacia. XLH is also associated with dentoalveolar mineralization defects in tooth enamel, dentin and cementum, and in alveolar bone, which lead to an increased prevalence of dental abscesses, periodontal disease and tooth loss. Genetic mouse experiments, and deficiencies in XLH patient therapies where treatments do not fully ameliorate mineralization defects, suggest that other pathogenic mechanisms may exist in XLH. The mineralization-inhibiting, secreted extracellular matrix phosphoprotein osteopontin (OPN, gene Spp1) is a substrate for the PHEX enzyme whereby extensive and inactivating degradation of inhibitory OPN by PHEX facilitates mineralization. Conversely, excess OPN accumulation in skeletal and dental tissues – for example in XLH where inactivating mutations in the PHEX gene limit degradation of inhibitory OPN, or as occurs in Fgf23 -null mice – contributes to mineralization defects. We hypothesized that Spp1 /OPN ablation in Hyp mice (a mouse model for XLH) would reduce dentoalveolar mineralization defects. Immunostaining revealed increased OPN in Hyp vs. wild-type (WT) alveolar bone, particularly in osteocyte lacunocanalicular networks where Hyp mice have characteristic hypomineralized peri-osteocytic lesions (POLs). Micro-computed tomography and histology showed that ablation of Spp1 in Hyp mice (Hyp;Spp1 −/− ) on a normal diet did not ameliorate bulk defects in enamel, dentin, or alveolar bone. On a high-phosphate diet, both Hyp and Hyp;Spp1 −/− mice showed improved mineralization of enamel, dentin, and alveolar bone. Silver staining indicated Spp1 ablation did not improve alveolar or mandibular bone osteocyte POLs in Hyp mice; however, they were normalized by a high-phosphate diet in both Hyp and Hyp;Spp1 −/− mice, although inducing increased OPN. Collectively, these data indicate that despite changes in OPN content in the dentoalveolar mineralized tissues, there exist other compensatory mineralization mechanisms that arise from knockout of Spp1 /OPN in the Hyp background. • Levels of osteopontin (Spp1 /OPN), a mineralization-inhibiting non-collagenous matrix protein, are increased in dentoalveolar tissues of the Hyp mutant mouse model of X-linked hypophosphatemia (XLH) • Genetic ablation of Spp1 /OPN in Hyp mice did not reduce mineralization defects in most dentoalveolar tissues • High phosphate diet markedly improved many mineralization measurements in Hyp and Hyp ; Spp1 double deficient mouse dentoalveolar tissues, with some indication of interaction between genotype and diet [ABSTRACT FROM AUTHOR]

Basic Multicellular Units (BMUs) conduct bone remodeling, a critical process of tissue turnover which, if imbalanced, can lead to disease, including osteoporosis. Parathyroid hormone (PTH 1–34; Teriparatide) is an osteoanabolic treatment for osteoporosis; however, it elevates the rate of intra-cortical remodeling (activation frequency) leading, at least transiently, to increased porosity. The purpose of this study was to test the hypothesis that PTH not only increases the rate at which cortical BMUs are initiated but also increases their progression (Longitudinal Erosion Rate; LER). Two groups (n = 7 each) of six-month old female New Zealand white rabbits were both administered 30 μg/kg of PTH once daily for a period of two weeks to induce remodeling. Their distal right tibiae were then imaged in vivo by in-line phase contrast micro-CT at the Canadian Light Source synchrotron. Over the following two weeks the first group (PTH) received continued daily PTH while the second withdrawal group (PTHW) was administrated 0.9 % saline. At four weeks all animals were euthanized, their distal tibiae were imaged by conventional micro-CT ex vivo and histomorphometry was performed. Matching micro-CT datasets (in vivo and ex vivo) were co-registered in 3D and LER was measured from 612 BMUs. Counter to our hypothesis, mean LER was lower (p < 0.001) in the PTH group (30.19 ± 3.01 μm/day) versus the PTHW group (37.20 ± 2.77 μm/day). Despite the difference in LER, osteonal mineral apposition rate (On.MAR) did not differ between groups indicating the anabolic effect of PTH was sustained after withdrawal. The slowing of BMU progression by PTH warrants further investigation; slowed resorption combined with elevated bone formation rate, may play an important role in how PTH enhances coupling between resorption and formation within the BMU. Finally, the prolonged anabolic response following withdrawal may have utility in terms of optimizing clinical dosing regimens. • In-line phase contrast Micro-CT tracks rabbit cortical bone remodeling events in 4D. • PTH 1–34 slows Basic Multicellular Units (BMU) advancement in rabbit cortical bone. • Osteonal mineral apposition remains elevated 2 weeks after PTH withdrawal in rabbits. [ABSTRACT FROM AUTHOR]

Physical exercise is a well-known modality for maintaining healthy locomotor mechanism. A detailed preclinical research on physical exercise effect on bone healing kinetics could help to improve the rehabilitation process after fracture treatment and bone remodeling. Our aim was to evaluate the effect of early post-operative exercise effect on bone microstructural changes in a rat model. Twenty Sprague Dawley male rats underwent bi-cortical 1.6 mm hole drilling in both femur diaphysis, after which (n = 10) underwent continuous treadmill training (TR) over two weeks, while the other group of rats (n = 10) was assigned to non-training (NT) control group. New bone formation labeling was performed by subcutaneous fluorochrome injections at day 5, 14 and 31. In vivo micro-computed tomography (μCT) scans were performed once a week during the 6-week post-operative period. Ten animals (five from each group) were euthanized at 3rd week while remaining animals were euthanized at 6th week. Femur samples were extracted and underwent ex vivo μCT and histological evaluation, while serum was used for evaluating alkaline phosphatase (ALP). μCT data demonstrated increased volume and surface of newly formed bone in defect area of TR group. Bone volume/Tissue volume (BV/TV) ratio and number of osteocytes showed an increase in TR group after 3-week period. Fluorochrome distances were increased between day 5 and 14 within the training group. Serum ALP level increased in both groups over 3- and 6-weeks. Post-operative exercise increases the bone healing kinetics and stimulates the new bone formation during and after the training protocol has ended. [Display omitted] • Early post-operative exercise increases kinetics of femoral bone defect healing in rats. • Volume of newly formed was higher in the training group after one week. • Training group exhibited higher BV/TV after three-week period. • Number of osteocytes was seen to be increased in training group after three-weeks. [ABSTRACT FROM AUTHOR]

Osteoblast differentiation is epigenetically suppressed by the H3K27 methyltransferase EZH2, and induced by the morphogen BMP2 and transcription factor RUNX2. These factors also regulate distinct G protein coupled receptors (GPRCs; e.g., PTH1R, GPR30/GPER1). Because GPRCs transduce many physiological stimuli, we examined whether BMP2 or EZH2 inhibition (i.e., GSK126) regulates other GPRC genes in osteoblasts. RNA-seq screening of >400 mouse GPRC-related genes showed that many GPRCs are downregulated during osteogenic differentiation. The orphan receptor GPRC5C, along with a small subset of other GPRCs, is induced by BMP2 or GSK126 during Vitamin C dependent osteoblast differentiation, but not by all-trans retinoic acid. ChIP-seq analysis revealed that GSK126 reduces H3K27me3 levels at the GPRC5C gene locus in differentiating MC3T3-E1 osteoblasts, consistent with enhanced GPRC5C mRNA expression. Loss of function analyses revealed that shRNA-mediated depletion of GPRC5C decreases expression of bone markers (e.g., BGLAP and IBSP) and mineral deposition in response to BMP2 or GSK126. GPRC5C mRNA was found to be reduced in the osteopenic bones of KLF10 null mice which have compromised BMP2 signaling. GPRC5C mRNA is induced by the bone-anabolic activity of 17β-estradiol in trabecular but not cortical bone following ovariectomy. Collectively, these findings suggest that GPRC5C protein is a key node in a pro-osteogenic axis that is normally suppressed by EZH2-mediated H3K27me3 marks and induced during osteoblast differentiation by GSK126, BMP2, and/or 17β-estradiol. Because GPRC5C protein is an understudied orphan receptor required for osteoblast differentiation, identification of ligands that induce GPRC5C signaling may support therapeutic strategies to mitigate bone-related disorders. [Display omitted] • The methyltransferase EZH2 epigenetically suppresses osteoblast differentiation. • Inhibition of EZH2 and activation of BMP2 co-stimulate osteogenesis. • This co-stimulation activates expression of the G-protein coupled receptor GPRC5C. • GPRC5C is a key orphan receptor required for osteoblastogenesis. • GPRC5C is induced by 17β-estradiol in trabecular bone of ovariectomized mice. [ABSTRACT FROM AUTHOR]