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Physiological processes within the human body are regulated in approximately 24-h cycles known as circadian rhythms, serving to adapt to environmental changes. Bone rhythms play pivotal roles in bone development, metabolism, mineralization, and remodeling processes. Bone rhythms exhibit cell specificity, and different cells in bone display various expressions of clock genes. Multiple environmental factors, including light, feeding, exercise, and temperature, affect bone diurnal rhythms through the sympathetic nervous system and various hormones. Disruptions in bone diurnal rhythms contribute to the onset of skeletal disorders such as osteoporosis, osteoarthritis and skeletal hypoplasia. Conversely, these bone diseases can be effectively treated when aimed at the circadian clock in bone cells, including the rhythmic expressions of clock genes and drug targets. In this review, we describe the unique circadian rhythms in physiological activities of various bone cells. Then we summarize the factors synchronizing the diurnal rhythms of bone with the underlying mechanisms. Based on the review, we aim to build an overall understanding of the diurnal rhythms in bone and summarize the new preventive and therapeutic strategies for bone disorders., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Pseudoephedrine (PSE) is an agent that is contained in common cold medications. The agent, which is used to treat cold and cough, is the fourth most prescribed drug group in some countries. During pregnancy, expectant mothers use PSE for colds and other reasons. One out of every four expectant mothers use PSE alone or in combination with other medicines for various reasons. This study was aimed to investigate effects of PSE on long bones development in rat during fetal growth. Pregnant rats were divided into five groups: control and four experimental groups (25 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg PSE). Between 1 and 20 days of pregnancy, PSE was given to them by gavage. Weights and heights of fetuses isolated by cesarean on the 21st day were measured. Ossification of femur and humerus was examined by three different methods mentioned earlier. Depending on the dose increase, all morphometric data, ossification rate and bone length of the fetuses were decreased. Besides, it was determined that the amount of Calcium in the bone tissue decreased in the analyzes made with SEM-EDX Analysis. The data obtained from this study reveal that the use of PSE during pregnancy disrupts the existing balance in the bone and negatively affects ossification due to the dose increase. In conclusion, we present descriptive and novel data on the effects of PSE use during pregnancy on the bone development of rat fetal long bones., (© 2023. The Author(s), under exclusive licence to Japanese Association of Anatomists.)

Skeletal stem/progenitor cells (SSPCs) are tissue-specific stem/progenitor cells localized within skeletons and contribute to bone development, homeostasis, and regeneration. However, the heterogeneity of SSPC populations in mouse long bones and their respective regenerative capacity remain to be further clarified. In this study, we perform integrated analysis using single-cell RNA sequencing (scRNA-seq) datasets of mouse hindlimb buds, postnatal long bones, and fractured long bones. Our analyses reveal the heterogeneity of osteochondrogenic lineage cells and recapitulate the developmental trajectories during mouse long bone growth. In addition, we identify a novel Cd168 + SSPC population with highly replicating capacity and osteochondrogenic potential in embryonic and postnatal long bones. Moreover, the Cd168 + SSPCs can contribute to newly formed skeletal tissues during fracture healing. Furthermore, the results of multicolor immunofluorescence show that Cd168 + SSPCs reside in the superficial zone of articular cartilage as well as in growth plates of postnatal mouse long bones. In summary, we identify a novel Cd168 + SSPC population with regenerative potential in mouse long bones, which adds to the knowledge of the tissue-specific stem cells in skeletons., Competing Interests: Conflict of interest The authors declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1 + early mesenchymal cells surrounding the SOX9 + condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1 + cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1 + peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development., Competing Interests: Conflict of 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 © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To describe bone mineral density (BMD), bone structure, and fracture prevalence in adolescents with type 1 diabetes (T1D) and explore their associations with glycemic control and microvascular complications., Research Design and Methods: Cross sectional study of 64 adolescents (38 males) with T1D duration >10 years who underwent dual-energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), fracture survey, plantar fascia thickness, and microvascular complications assessment., Results: Mean age was 16.6 ± 2.1 years, diabetes duration 12.8 ± 2.2 years and HbA1c 8.9 ± 1.7% (74 mmol/mol). Fracture prevalence was 50%. DXA areal BMD (Z-score) was reduced for femoral neck (-0.5 ± 1.3, p = 0.008) and arm (-0.4 ± 1.0, p < 0.001), while total areal BMD and lumbar spine BMD were normal. In pQCT (Z-score), trabecular volumetric BMD (vBMD) was reduced for tibia (-0.4 ± 0.8, p < 0.001) and radius (-0.8 ± 1.4, p < 0.001) whereas cortical vBMD was increased at both sites (tibia: 0.5 ± 0.6, p < 0.001, radius: 0.7 ± 1.5, p < 0.001). Muscle cross-sectional area (CSA) was reduced for upper (-0.6 ± 1.2, p < 0.001) and lower (-0.4 ± 0.7, p < 0.001) limbs. DXA total areal BMD was positively correlated with BMI (p < 0.01) and age at T1D diagnosis (p = 0.04). Lower radial bone CSA, total and lumbar spine BMD were associated with autonomic nerve dysfunction. HbA1c, diabetes duration, fracture history and other microvascular complications were not significantly associated with bone parameters., Conclusions: Adolescents with childhood-onset T1D have site-specific bone deficits in upper and lower limbs but normal total and lumbar spine BMD. T1D appears to have differential effects on trabecular and cortical bone compartments. Future longitudinal analysis is warranted to examine whether these changes translate in to increased fracture risk., (© 2022 The Authors. Pediatric Diabetes published by John Wiley & Sons Ltd.)

Identification of skeletal maturity is of interest as a measure of species longevity and for identifying its maximal achievable size/mass. Measurement of age on the basis of growth arrest/accentuation lines and external fundamental system evidences cessation or at least extreme slowing of circumferential bone growth. Such intramembranous (periosteal)-derived growth is distinct from the endochondral ossification responsible for longitudinal growth and therefore achievable organismal size/mass. As subchondral transcortical channels are required for nourishment, their loss should identify cessation of longitudinal growth. Predicated on phylogenetic bracketing/relationship and shared anatomical structures with and without growth plates, birds represent an appropriate model for the study of dinosaur ontogeny. Persistence of transcortical subchondral channels in the long bones of birds are examined at ×100-200 magnification and correlated with bone length. Transcortical channels are present in subchondral articular surfaces, but disappear when terminal longitudinal growth is achieved. Articular vascular channels perforating articular surfaces from within the bone are detected. Loss of penetrating channels is interpreted as evidence of skeletal growth cessation, identifying the longitudinal bone length at which skeletal growth cessation has been achieved. The current study provides evidence that maximal bone length does correlate with endochondral cessation growth. Failure of circumferential growth reduction/cessation to correlate with bone length may be related to lack of synchronicity of periosteal-based circumferential growth with the endochondral process responsible for bone lengthening. Loss/closure of articular vascular channels may be the most reliable measure of a bird's achievement of maximal growth (indicating cessation of appendicular element lengthening)., (© 2022 American Association for Anatomy.)

Background: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging., Results: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation., Conclusions: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Bone mass in adulthood depends on growth and mineralization acquired during childhood and adolescence. It is well known that these stages of life are crucial for bone development, where genetic, nutritional, hormonal, and lifestyle factors play a significant role. Bone loss is normally a natural and slow process that begins years later after the peak bone mass is achieved and continues throughout the lifespan. Lifestyle choices in childhood and adolescence such as minimal physical activity, excessive caffeine or carbonated beverages intake, malnutrition, cigarette use, or high alcohol consumption and other factors like environmental pollutants can also negatively affect bone health and accelerate the bone loss process. The aim of this work is an overview of risk factors associated with inadequate bone health in early life., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bone biology has long been driven by the question as to what molecules affect cell differentiation or the functions of bone. Exploring this issue has been an extraordinarily powerful way to improve our knowledge of bone development and physiology. More recently, a second question has emerged: does bone have other functions besides making bone? Addressing this conundrum revealed that the bone-derived hormone osteocalcin affects a surprisingly large number of organs and physiological processes, including acute stress response. This review will focus on this emerging aspect of bone biology taking osteocalcin as a case study and will show how classical and endocrine functions of bone help to define a new functional identity for this tissue., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The severe impairment of bone development and quality was recently described as a new target for unbalanced ultra-processed food (UPF). Here, we describe nutritional approaches to repair this skeletal impairment in rats: supplementation with micro-nutrients and a rescue approach and switching the UPF to balanced nutrition during the growth period. The positive effect of supplementation with multi-vitamins and minerals on bone growth and quality was followed by the formation of mineral deposits on the rats' kidneys and modifications in the expression of genes involved in inflammation and vitamin-D metabolism, demonstrating the cost of supplementation. Short and prolonged rescue improved trabecular parameters but incompletely improved the cortical parameters and the mechanical performance of the femur. Cortical porosity and cartilaginous lesions in the growth-plate were still detected one week after rescue and were reduced to normal levels 3 weeks after rescue. These findings highlight bone as a target for the effect of UPF and emphasize the importance of a balanced diet, especially during growth.

A better understanding of the mechanical behaviour of child bone is essential to improve the diagnosis of pediatric bone disorders that may influence bone development. Even though the process of bone growth is well described, there are still lacks of knowledge on intrinsic material properties of child bone and particularly on child bone considered as "non-pathological". Geometry, material properties, microstructure and biochemical components are associated with child bone fragility and remain difficult to assess for two main reasons: the scarcity of the bone samples and their small dimensions. In this context, ultrasonic methods offer interesting possibilities by exploiting in particular their non-destructive character. In this chapter, the elasticity properties of Non Pathological Child Cortical Bone (NPCCB) obtained by ultrasonic methods are presented. The objective was to contribute to the construction of a reference database on NPCCB that would serve as a point of comparison for analyzing the effect of a pathology or treatment. After the presentation of the hypotheses on the elasticity and anisotropy of NPCCB, ultrasonic transmission-mode and resonance spectroscopy methods are described. Results are presented and discussed with respect to microstructural and biochemical properties., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The progression of chronic kidney disease (CKD) in children is associated with deregulated parathyroid hormone (PTH), growth retardation, and low bone accrual. PTH can cause both catabolic and anabolic impact on bone, and the activating transcription factor 4 (ATF4), a downstream target gene of PTH, is related to its anabolic effect. Osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) are PTH-dependent cytokines, which may play an important role in the regulation of bone remodeling. This study aimed to evaluate the impact of endogenous PTH and the bone RANKL/OPG system on bone growth, cross-sectional geometry and strength utilizing young, nephrectomized rats. The parameters of cross-sectional geometry were significantly elevated in rats with CKD during the three-month experimental period compared with the controls, and they were strongly associated with serum PTH levels and the expression of parathyroid hormone 1 receptor (PTH1R)/ATF4 genes in bone. Low bone soluble RANKL (sRANKL) levels and sRANKL/OPG ratios were also positively correlated with cross-sectional bone geometry and femoral length. Moreover, the analyzed geometric parameters were strongly related to the biomechanical properties of femoral diaphysis. In summary, the mild increase in endogenous PTH, its anabolic PTH1R/ATF4 axis and PTH-dependent alterations in the bone RANKL/OPG system may be one of the possible mechanisms responsible for the favorable impact on bone growth, cross-sectional geometry and strength in young rats with experimental CKD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Today's eating patterns are characterized by the consumption of unbalanced diets (UBDs) resulting in a variety of health consequences on the one hand, and the consumption of dietary supplements in order to achieve overall health and wellness on the other. Balanced nutrition is especially crucial during childhood and adolescence as these time periods are characterized by rapid growth and development of the skeleton. We show the harmful effect of UBD on longitudinal bone growth, trabecular and cortical bone micro-architecture and bone mineral density; which were analyzed by micro-CT scanning. Three point bending tests demonstrate the negative effect of the diet on the mechanical properties of the bone material as well. Addition of Spirulina algae or Pleurotus eryngii or Agaricus bisporus mushrooms, to the UBD, was able to improve growth and impaired properties of the bone. 16SrRNA Sequencing identified dysbiosis in the UBD rats' microbiota, with high levels of pro-inflammatory associated bacteria and low levels of bacteria associated with fermentation processes and bone related mechanisms. These results provide insight into the connection between diet, the skeletal system and the gut microbiota, and reveal the positive impact of three chosen dietary supplements on bone development and quality presumably through the microbiome composition.

Collagen-rich tissues have poor reparative capacity that predisposes to common age-related disorders such as osteoporosis and osteoarthritis. We used in vivo pulsed SILAC labelling to quantify new protein incorporation into cartilage, bone, and skin of mice across the healthy life course. We report dynamic turnover of the matrisome, the proteins of the extracellular matrix, in bone and cartilage during skeletal maturation, which was markedly reduced after skeletal maturity. Comparing young adult with older adult mice, new protein incorporation was reduced in all tissues. STRING clustering revealed changes in epigenetic modulators across all tissues, a decline in chondroprotective growth factors such as FGF2 and TGFβ in cartilage, and clusters indicating mitochondrial dysregulation and reduced collagen synthesis in bone. Several pathways were implicated in age-related disease. Fewer changes were observed for skin. This methodology provides dynamic protein data at a tissue level, uncovering age-related molecular changes that may predispose to disease., Competing Interests: YA, AS, RF, SD, RT, AW, TV none, PC None, (© 2021, Ariosa-Morejon et al.)

Histology-based skeletochronology is a widely used approach to determine the age of an individual, and is based on the assumption that temporal cessations or decelerations of bone growth lead to incremental growth marks (GM), reflecting annual cycles. We studied the reliability of histology-based skeletochronology in a variety of extant tetrapods by comparing two different approaches: petrographic ground sections versus stained microtomized sections. Each bone was cut into two corresponding halves at its growth centre in order to apply both approaches to one and the same sample. None of the samples unequivocally revealed the actual age of the specimens, but truly concerning is the fact that the majority of samples even led to conflicting age estimates between the two approaches. Although the microtomized sections tended to yield more GM and thus indicated an older age than the ground sections, the contrary also occurred. Such a pronounced ambiguity in skeletochronological data strongly challenges the value of the respective age determinations for both extant and extinct animals. We conclude that much more research on the fundamental methodological side of skeletochronology-especially regarding the general nature and microscopic recognition of GM-is required.

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research., Competing Interests: TA, RS, HS, MH, MM, LK, YW, WL, MM, NN, IW, ML, CC No competing interests declared, (© 2021, Ambrosi et al.)

Bone stroma contributes to the regulation of osteogenesis and hematopoiesis but also to fracture healing and disease processes. Mesenchymal stromal cells from bone (BMSCs) represent a heterogenous mixture of different subpopulations with distinct molecular and functional properties. The lineage relationship between BMSC subsets and their regulation by intrinsic and extrinsic factors are not well understood. Here, we show with mouse genetics, ex vivo cell differentiation assays, and transcriptional profiling that BMSCs from metaphysis (mpMSCs) and diaphysis (dpMSCs) are fundamentally distinct. Fate-tracking experiments and single-cell RNA sequencing indicate that bone-forming osteoblast lineage cells and dpMSCs, including leptin receptor-positive (LepR + ) reticular cells in bone marrow, emerge from mpMSCs in the postnatal metaphysis. Finally, we show that BMSC fate is controlled by platelet-derived growth factor receptor β (PDGFRβ) signaling and the transcription factor Jun-B. The sum of our findings improves our understanding of BMSC development, lineage relationships, and differentiation., Competing Interests: Declaration of interests R.H.A. is investigator on patent EP 2 860 243 A1 (Reprogramming bone endothelial cells for bone angiogenesis and osteogenesis)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Growth rates importantly determine developmental time and are, therefore, a key variable of a species' life history. A widely used method to reconstruct growth rates and to estimate age at death in extant and particularly in fossil vertebrates is the analysis of bone tissue apposition rates. Lines of arrested growth (LAGs) are of special interest here, as they indicate a halt in bone growth. However, although of great importance, the time intervals between, and particularly the reason of growth arrests remains unknown. Therefore, experiments are increasingly called for to calibrate growth rates with tissue types and life history events, and to provide reliable measurements of the time involved in the formation of LAGs. Based on in vivo bone labelling, we calibrated periods of bone tissue apposition, growth arrest, drift and resorption over the period from birth to post-weaning in a large mammal, the red deer. We found that bone growth rates tightly matched the daily weight gain curve, i.e. decreased with age, with two discrete periods of growth rate disruption that coincided with the life history events birth and weaning, that were visually recognisable in bone tissue as either partial LAGs or annuli. Our study identified for the first time in a large mammal a general pattern for juvenile bone growth rates, including periods of growth arrest. The tight correlation between daily weight gain and bone tissue apposition suggests that the red deer bone growth model is valid for ruminants in general where the daily weight gain curve is comparable.

Background: Radiographic markers of skeletal maturity are vital to the prediction and interpretation of skeletal growth patterns. Accurate predictions of skeletal maturity factor into the management of common musculoskeletal disorders. Bone age is conventionally measured using hand and wrist radiographs. The primary study objective was to optimize skeletal maturity estimates based on the morphology of markers at the hip, knee, and foot rather than conventional upper extremity radiographs., Methods: This was a retrospective analysis of children from the Bolton-Brush collection with anteroposterior radiographs of the hip and anteroposterior and lateral radiographs of the knee and foot, and heights recorded at the time of each radiograph. The percent growth achieved (%GA) was calculated as a function of final patient height. Poor quality radiographs were excluded, leaving 50 patients-32 females and 18 males-and 1068 radiographs for analysis. Skeletal maturity was evaluated using the Oxford bone, O'Connor knee, and calcaneal apophyseal scores. Interrater and intrarater reliability analyses were performed for hip and knee scores. Multiple linear regressions were conducted on these scores and chronologic age as predictors of %GA. Mean differences were calculated between actual and estimated %GAs. All analyses were performed in Prism 8.0., Results: Each lower extremity skeletal maturity score served as statistically significant, independent predictors of %GA, the accuracy and strength of which increased with the addition of chronologic age. The integration of all 3 systems and chronologic age yielded the most predictive, accurate model predictive of %GA, which can be used to determine percent growth remaining. However, this fully integrated system was not statistically superior to the combination of knee and foot scores and knee score and chronologic age, which yield similarly accurate %GA predictions. The hip and knee systems demonstrated good to excellent interrater and intrarater reliability., Conclusions: Integration of bone age scores across different regions of the lower extremity has the potential to facilitate orthopaedic decision-making using radiographs already obtained in the treatment of pediatric musculoskeletal conditions., Level of Evidence: Level IV., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Previous studies have suggested that bisphenol A (BPA) has a toxic effect on bone development; however, its pathological mechanism has not been fully elucidated. In the present study, pregnant Wistar rats were intragastrically administered BPA (10 μg/kg per day) during gestational days 14-21. Then, bone tissues were obtained from neonatal rats on postnatal day 1 for histological analysis, and the bone mass of adult rat offspring was analyzed by micro-CT at postnatal week 10. Furthermore, osteoprogenitors from neonatal rats were obtained and treated with various concentrations of BPA in vitro to clarify the associated mechanism. In vivo, we found that prenatal BPA exposure reduced body weight and body length in female neonatal rats but not in male neonatal rats. Meanwhile, BPA exposure during pregnancy delayed bone development and reduced bone mass only in female rat offspring. Moreover, BPA exposure during pregnancy inhibited osteogenic function and downregulated the transforming growth factor β (TGF β) signaling pathway in the bone tissue of female neonatal rats. Our in vitro findings further indicated that various concentrations of BPA suppressed the osteogenic function of osteoprogenitors by downregulating the TGFβ signaling pathway. Meanwhile, BPA downregulated H3K9ac and expression levels of TGFβ via the ERβ/HDAC5 signaling pathway. Collectively, this research revealed that prenatal BPA exposure impairs bone development and bone mass accumulation in female rat offspring, which was attributed to inhibitory osteogenic function via the ERβ/HDAC5/TGFβ signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Job syndrome is a rare genetic disorder caused by STAT3 mutations and primarily characterized by immune dysfunction along with comorbid skeleton developmental abnormalities including osteopenia, recurrent fracture of long bones, and scoliosis. So far, there is no definitive cure for the skeletal defects in Job syndrome, and treatments are limited to management of clinical symptoms only. Here, we have investigated the molecular mechanism whereby Stat3 regulates skeletal development and osteoblast differentiation. We showed that removing Stat3 function in the developing limb mesenchyme or osteoprogenitor cells in mice resulted in shortened and bow limbs with multiple fractures in long bones that resembled the skeleton symptoms in the Job Syndrome. However, Stat3 loss did not alter chondrocyte differentiation and hypertrophy in embryonic development, while osteoblast differentiation was severely reduced. Genome-wide transcriptome analyses as well as biochemical and histological studies showed that Stat3 loss resulted in down-regulation of Wnt/β-catenin signaling. Restoration of Wnt/β-catenin signaling by injecting BIO, a small molecule inhibitor of GSK3, or crossing with a Lrp5 gain of function (GOF) allele, rescued the bone reduction phenotypes due to Stat3 loss to a great extent. These studies uncover the essential functions of Stat3 in maintaining Wnt/β-catenin signaling in early mesenchymal or osteoprogenitor cells and provide evidence that bone defects in the Job Syndrome are likely caused by Wnt/β-catenin signaling reduction due to reduced STAT3 activities in bone development. Enhancing Wnt/β-catenin signaling could be a therapeutic approach to reduce bone symptoms of Job syndrome patients., Competing Interests: The authors declare no competing interest.

Introduction: The determination of skeletal maturity stages is very important in orthodontic treatment planning, especially skeletal discrepancies in growing individuals. A hand-wrist radiograph is considered the most accurate approach for skeletal maturity detection. Dental calcification stages have been suggested as an alternative diagnostic method to decrease radiation exposure. The recent study is aimed at detecting the efficacy of dental calcification stages in assessing skeletal maturity during the prepubertal and pubertal growth periods., Methods: Patients' records were collected from the Aleppo Orthodontic Center. Dental maturity stages were assessed from a panoramic radiograph using the Demirjian method, while skeletal maturity stages were determined using the Björk method. Four permanent left mandibular teeth were included (canine, 1st premolar, 2nd premolar, and 2nd molar) for the study., Results: From 517 records, 295 records (145 males and 150 females) were included. The Spearman rank-order correlation coefficients between skeletal maturation and dental maturation were strong and statistically significant (ranging from 0.789 to 0.835). The highest correlation was between skeletal stages and the second molar ( r = 0.829 and 0.88 in males and females, respectively). Receiver operating characteristic (ROC ) curve suggested a high validity of the sum of dental stages for the four teeth in identifying MP3= stage (sensitivity was 70%, specificity was 92.77%, and ROC area was 0.81) but not for MP3cap (sensitivity was 50.85%, specificity was 81.36%, and ROC area was 0.66)., Conclusions: The correlation between the skeletal maturity stages and the dental calcification stages was high. The orthodontist can use the dental stages as a definite diagnostic tool for prepubertal growth period., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Ayah Jourieh et al.)

Skeletogenesis is a complex process that requires a rigorous control at multiple levels during osteogenesis, such as signaling pathways and transcription factors. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. In present study, we constructed a skeletogenesis database containing 4101, 3715, 2996, 3300, 3719 and 3737 genes in Danio rerio, Oryzias latipes, Gallus gallus, Xenopus tropicalis, Mus musculus and Homo sapiens genome, respectively. Then, we found over 55% of the genes are conserved in the six species. Notably, there are 181 specific-genes in the human genome without orthologues in the other five genomes, such as the ZNF family (ZNF100, ZNF101, ZNF14, CALML6, CCL4L2, ZIM2, HSPA6, etc); and 31 genes are identified explicitly in fish species, which are mainly involved in TGF-beta, Wnt, MAPK, Calcium signaling pathways, such as bmp16, bmpr2a, eif4e1c, wnt2ba, etc. Particularly, there are 20 zebrafish-specific genes (calm3a, si:dkey-25li10, drd1a, drd7, etc) and one medaka-specific gene (c-myc17) that may alter skeletogenesis formation in the corresponding species. The database provides the new systematic genomic insights into skeletal development from teleosts to mammals, which may help to explain some of the complexities of skeletal phenotypes among different vertebrates and provide a reference for the treatment of skeletal diseases as well as for applications in the aquaculture industry., (Copyright © 2021. Published by Elsevier Inc.)

The IL-23 receptor (IL-23R) signaling pathway has pleiotropic effects on the differentiation of osteoclasts and osteoblasts, since it can inhibit or stimulate these processes via different pathways. However, the potential role of this pathway in the regulation of bone homeostasis remains elusive. Therefore, we studied the role of IL-23R signaling in physiological bone remodeling using IL-23R deficient mice. Using µCT, we demonstrate that 7-week-old IL-23R -/- mice have similar bone mass as age matched littermate control mice. In contrast, 12-week-old IL-23R -/- mice have significantly lower trabecular and cortical bone mass, shorter femurs and more fragile bones. At the age of 26 weeks, there were no differences in trabecular bone mass and femur length, but most of cortical bone mass parameters remain significantly lower in IL-23R -/- mice. In vitro osteoclast differentiation and resorption capacity of 7- and 12-week-old IL-23R -/- mice are similar to WT. However, serum levels of the bone formation marker, PINP, are significantly lower in 12-week-old IL-23R -/- mice, but similar to WT at 7 and 26 weeks. Interestingly, Il23r gene expression was not detected in in vitro cultured osteoblasts, suggesting an indirect effect of IL-23R. In conclusion, IL-23R deficiency results in temporal and long-term changes in bone growth via regulation of bone formation.

Despite a well-established role of anterior cruciate ligament (ACL) anatomy on its biomechanics, little is known on how ACL anatomy develops and changes during skeletal growth. We hypothesized that ACL size and orientation will change by age during skeletal growth and maturation with different trends in males vs females. Magnetic resonance images of 269 unique knees (3-18 years old; 51% female) were used to measure ACL length, cross-sectional area, length-to-cross-sectional area ratio, and elevation angles. In both males and females, ACLs became longer, thicker, and more vertical in sagittal and coronal planes by increasing age (R 2  > 0.2; P < .001 for all associations). ACL cross-sectional area-to-length ratio increased by age only in males (R 2  = 0.06; P = .003). Despite similar ACL sizes between males and females at early age, adolescent males had significantly longer and thicker ACLs compared to the age-matched females (P < .05). There were no sex differences in ACL elevation angles (P > .2) except for larger coronal elevation in 7 to 10 years old females compared to age-matched males (P = .012). Observed changes in ACL cross-sectional area-to-length ratio indicate that age- and sex-dependent changes in ACL size are not homogenous. The trends seen in normalized ACL size measurements suggest that unlike ACL cross-sectional area, ACL length is primarily controlled by body size. Smaller ACLs and lower cross-sectional growth rates observed in females may be contributing factors to the higher risk of ACL injuries in females. Further investigations are required to identify the intrinsic and extrinsic factors responsible for these discrepancies., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Bone mineral density (BMD) is a highly heritable predictor of osteoporotic fracture. GWAS have identified hundreds of loci influencing BMD, but few have been functionally analyzed. In this study, we show that SNPs within a BMD locus on chromosome 14q32.32 alter splicing and expression of PAR-1a/microtubule affinity regulating kinase 3 (MARK3), a conserved serine/threonine kinase known to regulate bioenergetics, cell division, and polarity. Mice lacking Mark3 either globally or selectively in osteoblasts have increased bone mass at maturity. RNA profiling from Mark3-deficient osteoblasts suggested changes in the expression of components of the Notch signaling pathway. Mark3-deficient osteoblasts exhibited greater matrix mineralization compared with controls that was accompanied by reduced Jag1/Hes1 expression and diminished downstream JNK signaling. Overexpression of Jag1 in Mark3-deficient osteoblasts both in vitro and in vivo normalized mineralization capacity and bone mass, respectively. Together, these findings reveal a mechanism whereby genetically regulated alterations in Mark3 expression perturb cell signaling in osteoblasts to influence bone mass.

This study examines the functional model of bone development in peri-pubertal boys and girls. Specifically, we implemented a mixed-longitudinal design and hierarchical structural models to provide experimental evidence in support of the conceptual functional model of bone development, postulating that the primary mechanical stimulus of bone strength development is muscle force. To this end, we measured radial and tibial bone properties (speed of sound, SOS), isometric grip and knee extensors strength, bone resorption (urinary NTX concentration), body mass index (BMI), somatic maturity (years from peak height velocity) and skeletal maturity (bone age) in 180 children aged 8-16 years. Measurements were repeated 2-4 times over a period of 3 years. The multilevel structural equation modeling of 406 participant-session observations revealed similar results for radial and tibial SOS. Muscle strength (i.e., grip strength for the radial and knee extension for tibial model) and NTX have a significant direct effect on bone SOS (β = 0.29 and -0.18, respectively). Somatic maturity had a direct impact on muscle strength (β = 0.24) and both a direct and indirect effect on bone SOS (total effect, β = 0.30). Physical activity and BMI also had a significant direct impact on bone properties, (β = 0.06 and -0.18, respectively), and an additional significant indirect effect through muscle strength (β = 0.01 and 0.05, respectively) with small differences per bone site and sex. Muscle strength fully mediated the impact of bone age (β = 0.14) while there was no significant effect of energy intake on either muscle strength or bone SOS. In conclusion, our results support the functional model of bone development in that muscle strength and bone metabolism directly affect bone development while the contribution of maturity, physical activity, and other modulators such as BMI, on bone development is additionally modulated through their effect on muscle strength.

Osteoclasts are specialized cells of the hematopoietic lineage that are responsible for bone resorption and play a critical role in musculoskeletal disease. JAK2 is a key mediator of cytokine and growth factor signaling; however, its role in osteoclasts in vivo has yet to be investigated. To elucidate the role of JAK2 in osteoclasts, we generated an osteoclast-specific JAK2-KO (Oc-JAK2-KO) mouse using the Cre/Lox-P system. Oc-JAK2-KO mice demonstrated marked postnatal growth restriction; however, this was not associated with significant changes in bone density, microarchitecture, or strength, indicating that the observed phenotype was not due to alterations in canonical osteoclast function. Interestingly, Oc-JAK2-KO mice had reduced osteoclast-specific expression of IGF1, suggesting a role for osteoclast-derived IGF1 in determination of body size. To directly assess the role of osteoclast-derived IGF1, we generated an osteoclast-specific IGF1-KO mouse, which showed a similar growth-restricted phenotype. Lastly, overexpression of circulating IGF1 by human transgene rescued the growth defects in Oc-JAK2-KO mice, in keeping with a causal role of IGF1 in these models. Together, our data show a potentially novel role for Oc-JAK2 and IGF1 in the determination of body size, which is independent of osteoclast resorptive function.

The objective of this study was to investigate the effect of diet crude protein (CP) content and metabolisable energy (ME) intake on skeletal growth and associated parameters of growing steers prior to and during compensatory growth in weight and catch-up growth in skeletal elongation. The experiment was a factorial design with two cattle genotypes [Brahman crossbred (BX, 178 ± 6 kg) and Holstein-Friesian (HF, 230 ± 34 kg)] and three nutritional treatments; high CP content and high ME intake (HCP-HME), high CP content and low ME intake (HCP-LME) and low CP content and low ME intake (LCP-LME) with the ME intake of HCP-LME matched to that of LCP-LME. Nutritional treatments were imposed over a 103 d period (Phase 1), and after this, all steers were offered ad libitum access to the HCP-HME nutritional treatment for 100 d (Phase 2). Steers fed the high CP content treatment with a low ME intake, showed higher hip height gain (P = 0.04), larger terminal hypertrophic chondrocytes (P = 0.02) and a higher concentration of total triiodothyronine in plasma (P = 0.01) than steers with the same ME intake of the low CP content treatment. In addition, the low CP treatment resulted in significant decreases in bone volume (P = 0.03), bone surface area (P = 0.03) and the concentration of bone-specific alkaline phosphatase in plasma (P < 0.001) compared to steers fed the HCP-HME treatment. A significant interaction between genotype and nutritional treatment existed for the concentration of thyroxine (T4) in plasma where HF steers fed LCP-LME had a lower T4 concentration in plasma (P = 0.05) than BX steers. All steers with a restricted ME intake during Phase 1 demonstrated compensatory growth during Phase 2. However, HF steers fed the LCP treatment during Phase 1 showed a tendency (P = 0.07) for a greater LWG during Phase 2 without any increase in dry matter intake. Results observed at the growth plate and hip height growth suggest that catch-up growth in cattle may also be explained by the growth plate senescence hypothesis. Contrary to our initial hypothesis, the results demonstrate that greater CP intake during ME restriction does not increase compensatory gain in cattle during re-alimentation., Competing Interests: The authors have declared that no competing interests exist.

Background: Bone accrual impacts lifelong skeletal health, but genetic discovery has been primarily limited to cross-sectional study designs and hampered by uncertainty about target effector genes. Here, we capture this dynamic phenotype by modeling longitudinal bone accrual across 11,000 bone scans in a cohort of healthy children and adolescents, followed by genome-wide association studies (GWAS) and variant-to-gene mapping with functional follow-up., Results: We identify 40 loci, 35 not previously reported, with various degrees of supportive evidence, half residing in topological associated domains harboring known bone genes. Of several loci potentially associated with later-life fracture risk, a candidate SNP lookup provides the most compelling evidence for rs11195210 (SMC3). Variant-to-gene mapping combining ATAC-seq to assay open chromatin with high-resolution promoter-focused Capture C identifies contacts between GWAS loci and nearby gene promoters. siRNA knockdown of gene expression supports the putative effector gene at three specific loci in two osteoblast cell models. Finally, using CRISPR-Cas9 genome editing, we confirm that the immediate genomic region harboring the putative causal SNP influences PRPF38A expression, a location which is predicted to coincide with a set of binding sites for relevant transcription factors., Conclusions: Using a new longitudinal approach, we expand the number of genetic loci putatively associated with pediatric bone gain. Functional follow-up in appropriate cell models finds novel candidate genes impacting bone accrual. Our data also raise the possibility that the cell fate decision between osteogenic and adipogenic lineages is important in normal bone accrual.

Growth hormone (GH) and its mediator, the insulin-like growth factor-1 (IGF-1) regulate somatic growth, metabolism and many aspects of aging. As such, actions of GH/IGF have been studied in many tissues and organs over decades. GH and IGF-1 are part of the hypothalamic/pituitary somatotrophic axis that consists of many other regulatory hormones, receptors, binding proteins, and proteases. In humans, GH/IGF actions peak during pubertal growth and regulate skeletal acquisition through stimulation of extracellular matrix production and increases in bone mineral density. During aging the activity of these hormones declines, a state called somatopaguss, which associates with deleterious effects on the musculoskeletal system. In this review, we will focus on GH/IGF-1 action in bone and cartilage. We will cover many studies that have utilized congenital ablation or overexpression of members of this axis, as well as cell-specific gene-targeting approaches used to unravel the nature of the GH/IGF-1 actions in the skeleton in vivo., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pathological new bone formation is a typical pathological feature in ankylosing spondylitis (AS), and the underlying molecular mechanism remains elusive. Previous studies have shown that the calcium-sensing receptor (CaSR) is critical for osteogenic differentiation while also being highly involved in many inflammatory diseases. However, whether it plays a role in pathological new bone formation of AS has not been reported. Here, we report the first piece of evidence that expression of CaSR is aberrantly upregulated in entheseal tissues collected from AS patients and animal models with different hypothetical types of pathogenesis. Systemic inhibition of CaSR reduced the incidence of pathological new bone formation and the severity of the ankylosing phenotype in animal models. Activation of PLCγ signalling by CaSR promoted bone formation both in vitro and in vivo. In addition, various inflammatory cytokines induced upregulation of CaSR through NF-κB/p65 and JAK/Stat3 pathways in osteoblasts. These novel findings suggest that inflammation-induced aberrant upregulation of CaSR and activation of CaSR-PLCγ signalling in osteoblasts act as mediators of inflammation, affecting pathological new bone formation in AS., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The heparan sulfate proteoglycan Syndecan-1, a mediator of signals between the extracellular matrix and cells involved is able to interact with OPG, one of the major regulators of osteoclastogenesis. The potential of osteoblasts to induce osteoclastogenesis is characterized by a switch of OPG (low osteoclastogenic potential) towards RANKL production (high osteoclastogenic potential). In the present study, we investigated the influence of endogenous Syndecan-1 on local bone-cell-communication via the RANKL/OPG-axis in murine osteoblasts and osteoclasts in wild type and Syndecan-1 lacking cells. Syndecan-1 expression and secretion was increased in osteoblasts with high osteoclastogenic potential. Syndecan-1 deficiency led to increased OPG release by osteoblasts that decreased the availability of RANKL. In co-cultures of Syndecan-1 deficient osteoblasts with osteoclast these increased OPG in supernatant caused decreased development of osteoclasts. Syndecan-1 and RANKL level were increased in serum of aged WT mice, whereas Syndecan-1 deficient mice showed high serum OPG concentration. However, bone structure of Syndecan-1 deficient mice was not different compared to wild type. In conclusion, Syndecan-1 could be regarded as a new modulator of bone-cell-communication via RANKL/OPG axis. This might be of high impact during bone regeneration or bone diseases like cancer where Syndecan-1 expression is known to be even more prevalent.

Vertebrates have evolved flexible and hard dermal armor, but there is often a tradeoff between mobility and protection. Skeletal modifications include fish scales, the shells of tortoises and turtles, and the cranial projections that are found in marine iguanas and lionfish. Sexual dimorphisms of cranial structures are prominent in fishes; we hypothesized that cranial projections of Pterois volitans will change over ontogeny and are sexually dimorphic. The goal of this study is to quantify differences in the cranial armor of P. volitans over a range of sizes (juveniles to adults) to understand allometric growth and possible sexual dimorphisms using specimens from invaded ranges. We found no difference in the total number of cranial spines between sexes, suggesting that the number of cranial spines is not a sexually dimorphic trait in P. volitans. We found positive allometry for total cranial spine number, and length and width of the three largest cranial spines, with respect to the fish total length. Here, we also document another skeletal modification in P. volitans, which is the addition of spines on the caudal fin. Overall, these data show that P. volitans' develop robust dermal modifications as they grow larger, and we suggest that phenotypic plasticity of this species should be explored in their native and invaded ranges., (© 2020 Wiley Periodicals LLC.)

The skeleton is well-innervated, but only recently have the functions of this complex network in bone started to become known. Although our knowledge of skeletal sensory and sympathetic innervation is incomplete, including the specific locations and subtypes of nerves in bone, we are now able to reconcile early studies utilizing denervation models with recent work dissecting the molecular signaling between bone and nerve. In total, sensory innervation functions in bone much as it does elsewhere in the body-to sense and respond to stimuli, including mechanical loading. Similarly, sympathetic nerves regulate autonomic functions related to bone, including homeostatic remodeling and vascular tone. However, more study is required to translate our current knowledge of bone-nerve crosstalk to novel therapeutic strategies that can be effectively utilized to combat skeletal diseases, disorders of low bone mass, and age-related decreases in bone quality., (Copyright © 2020 Tomlinson, Christiansen, Giannone and Genetos.)

Connecting the selective forces that drive the evolution of phenotypes to their underlying genotypes is key to understanding adaptation, but such connections are rarely tested experimentally. Threespine stickleback (Gasterosteus aculeatus) are a powerful model for such tests because genotypes that underlie putatively adaptive traits have been identified. For example, a regulatory mutation in the Ectodysplasin (Eda) gene causes a reduction in the number of bony armor plates, which occurs rapidly and repeatedly when marine sticklebacks invade freshwater. However, the source of selection on plate loss in freshwater is unknown. Here, we tested whether dietary reduction of phosphorus can account for selection on plate loss due to a growth advantage of low-plated fish in freshwater. We crossed marine fish heterozygous for the 16 kilobase freshwater Eda haplotype and compared the growth of offspring with different genotypes under contrasting levels of dietary phosphorus in both saltwater and freshwater. Eda genotype was not associated with growth differences in any treatment, or with mechanisms that could mitigate the impacts of phosphorus limitation, such as differential phosphorus deposition, phosphorus excretion, or intestine length. This study highlights the importance of experimentally testing the putative selective forces acting on phenotypes and their underlying genotypes in the wild., (© 2020 The Authors. Evolution © 2020 The Society for the Study of Evolution.)

Background: Spontaneous catch-up (CU) growth occurs when a growth-restricting factor is resolved. However, its efficiency is sometimes inadequate and growth deficits remain permanent. The therapeutic toolbox for short stature is currently very limited, thus, finding new regulatory pathways is important for the development of novel means of treatment. Our previous studies using a nutrition-induced CU growth model showed that the level of sirtuin-1 (Sirt1) was significantly increased in food-restricted animals and decreased during CU growth., Aim: This study sought to investigate the role of Sirt1 in modulating the response of the epiphyseal growth plate (EGP) to nutritional manipulation., Method: Collagen type II-specific Sirt1 knockout (CKO) mice were tested for response to our CU growth model consisting of a period of food restriction followed by re-feeding., Results: The transgenic CKO mice weighed more than the control (CTL) mice, their EGP was higher and less organized, specifically at the resting and proliferative zones, leading to shorter bones. Ablation of Sirt1 in the chondrocytes was found to have a dramatic effect on bone mineralization on micro-CT analysis. The CKO mice were less responsive to the nutritional manipulation, and their CU growth was less efficient. They remained shorter than the CTL mice who corrected the food restriction-induced growth deficit during the re-feeding period., Conclusions: Sirt1 appears to be important for normal regulation of the EGP. In its absence, the EGP is less organized and CU growth is less efficient. These results suggest that SIRT1 may serve as a novel therapeutic target for short stature., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Primiparous ewes and multiparous ewes show physiological differences during pregnancy, which can have an impact on the development of their offspring. The objective of this study was to compare the changes in the metabolic profile and in the size of some foetal bones throughout gestation between primiparous and multiparous ewes. Twelve primiparous (PM) ewes and 14 multiparous (MT) ewes were used. According to the dates of lambing, two groups of ewes were formed: Group 1 (G1, n = 6 PM and n = 7 MT) and Group 2 (G2, n = 6 PM and n = 7 MT). The body weight, body condition score, metabolic and foetal morphometric parameters were determined from before conception until the end of gestation. After lambing, the body weight and survival rate during the first 72 hr of life of lambs, as well as the maternal behaviour score were recorded. The PM ewes were lighter (p < .01) and had a greater mobilization of body reserves during gestation, mainly evidenced by a greater serum concentration of NEFAs and lower serum concentration of total proteins (p < .05) compared with the MT ewes. The parity did not affect the foetal morphometric variables. The lambs of MT ewes were heavier at parturition (p = .002) and tended to have a greater survival rate than those lambs of PM ewes (p = .09). In conclusion, PM ewes and MT ewes differ in their metabolic profile throughout the gestation. However, in the present study, we did not find parity differences in the dimensions of foetal bones during growth in gestation., (© 2020 Blackwell Verlag GmbH.)

Pluripotent stem cells (PSCs) are attractive regenerative therapy tools for skeletal tissues. However, a deep understanding of skeletal development is required in order to model this development with PSCs, and for the application of PSCs in clinical settings. Skeletal tissues originate from three types of cell populations: the paraxial mesoderm, lateral plate mesoderm, and neural crest. The paraxial mesoderm gives rise to the sclerotome mainly through somitogenesis. In this process, key developmental processes, including initiation of the segmentation clock, formation of the determination front, and the mesenchymal-epithelial transition, are sequentially coordinated. The sclerotome further forms vertebral columns and contributes to various other tissues, such as tendons, vessels (including the dorsal aorta), and even meninges. To understand the molecular mechanisms underlying these developmental processes, extensive studies have been conducted. These studies have demonstrated that a gradient of activities involving multiple signaling pathways specify the embryonic axis and induce cell-type-specific master transcription factors in a spatiotemporal manner. Moreover, applying the knowledge of mesoderm development, researchers have attempted to recapitulate the in vivo development processes in in vitro settings, using mouse and human PSCs. In this review, we summarize the state-of-the-art understanding of mesoderm development and in vitro modeling of mesoderm development using PSCs. We also discuss future perspectives on the use of PSCs to generate skeletal tissues for basic research and clinical applications.

Bone growth and the maintenance of bone structure are controlled by multiple endocrine and paracrine factors, including cytokines expressed locally within the bone microenvironment and those that are elevated, both locally and systemically, under inflammatory conditions. This review focuses on those bone-active cytokines that initiate JAK-STAT signaling, and outlines the discoveries made from studying skeletal defects caused by induced or spontaneous modifications in this pathway. Specifically, this review describes defects in JAK1, STAT3, and SOCS3 signaling in mouse models and in humans, including mutations designed to modify these pathways downstream of the gp130 coreceptor. It is shown that osteoclast formation is generally stimulated indirectly by these pathways through JAK1 and STAT3 actions in inflammatory and other accessory cells, including osteoblasts. In addition, in bone remodeling, osteoblast differentiation is increased secondary to stimulated osteoclast formation through an IL-6-dependent pathway. In growth plate chondrocytes, STAT3 signaling promotes the normal differentiation process that leads to bone lengthening. Within the osteoblast lineage, STAT3 signaling promotes bone formation in normal physiology and in response to mechanical loading through direct signaling in osteocytes. This activity, particularly that of the IL-6/gp130 family of cytokines, must be suppressed by SOCS3 for the normal formation of cortical bone.

Purpose of Review: The main goal of this narrative review is to assess whether physical activity (PA) influences peak bone mass and fracture risk., Recent Findings: Several randomized controlled trials (RCT) show that short-term PA intervention programs in childhood improve the accrual of bone mineral. There are now also long-term controlled PA intervention studies demonstrating that both boys and girls with daily school PA through puberty gain higher bone mineral content (BMC) and bone mineral density (BMD) and greater bone size than boys and girls with school PA 1-2 times/week. These benefits seem to be followed by a gradual reduction in expected fracture rates, so that in children with daily school PA, the incidence rate ratio (IRR) after 8 years is less than half that expected by age. Daily school PA from before to after puberty is associated with beneficial gains in bone traits and gradually lower relative fracture risk.

There is ample evidence that cholinergic actions affect the health status of bones in vertebrates including man. Nicotine smoking, but also exposure to pesticides or medical drugs point to the significance of cholinergic effects on bone status, as reviewed here in Introduction. Then, we outline processes of endochondral ossification, and review respective cholinergic actions. In Results, we briefly summarize our in vivo and in vitro studies on bone development of chick and mouse [1,2], including (i) expressions of cholinergic components (AChE, BChE, ChAT) in chick embryo, (ii) characterisation of defects during skeletogenesis in prenatal ChE knockout mice, (iii) loss-of-function experiments with beads soaked in cholinergic components and implanted into chicken limb buds, and finally (iv) we use an in vitro mesenchymal 3D-micromass model that mimics cartilage and bone formation, which also had revealed complex crosstalks between cholinergic, radiation and inflammatory mechanisms [3]. In Discussion, we evaluate non-cholinergic actions of cholinesterases during bone formation by considering: (i) how cholinesterases could function in adhesive mechanisms; (ii) whether and how cholinesterases can form bone-regulatory complexes with alkaline phosphatase (ALP) and/or ECM components, which could regulate cell division, migration and adhesion. We conclude that cholinergic actions in bone development are driven mainly by classic cholinergic, but non-neural cycles (e.g., by acetylcholine); in addition, both cholinesterases can exert distinct ACh-independent roles. Considering their tremendous medical impact, these results bring forward novel research directions that deserve to be pursued., (Copyright © 2020 Elsevier B.V. All rights reserved.)

AHNAK has been reported to be involved in actin cytoskeleton rearrangement of some cell types, calcium homeostasis, and activation of T cells. Although the functional role of AHNAK in muscle cells, epidermis, and brain has been determined, its association with apparent clinical impairment has not been found yet. During phenotypic analysis of AHNAK knock out (KO) mice for many years, we observed that AHNAK KO mice showed very slow growth. Snouts of these animals were very short, and their bones were easily broken compared to normal mice. It is known that AHNAK is closely related to calcium. However, intensive morphological studies on phenotypes of bone have yet been reported for AHNAK. Thus, the objective of the present study was to analyze the morphology of skull, mandibular, limbs, and caudal bones of AHNAK KO mice intensively using micro-CT with many factors for various ages of these mice (6 weeks, 18 weeks, and 40 weeks). As a result, it was found that the facial region of AHNAK KO mouse showed a large difference in mandible than skull. Their both femur and tibia were shortened, and bone strength was also significantly decreased compared to normal mice. Particularly, the tail bone of AHNAK KO mice exhibited morphological abnormality by age. Taken together, these results suggest that AHNAK plays an important role in bone shape, development, and metabolism. Although our results demonstrated that AHNAK has a distinct role in bone, further investigations are needed to determine various features of bone metabolism related to AHNAK in the future.

In recent years, growth hormone deficiency in children has been treated with hormone therapy despite the possible significant side effects. Therefore, it was deemed beneficial to develop functional foods or dietary supplements for safely improving children's growth. Spirulina platensis is known for its high antioxidant, anti-aging, anti-cancer, and immunity-enhancing properties, as well as its high digestibility and high protein content, but little has been reported about its influence on bone development in children with a normal supply of protein. In this study, we evaluated the effects of spirulina on the bone metabolism and antioxidant profiles of three-week-old growing male rats. The animals were divided into four groups ( n = 17 per group) and were fed AIN93G diets with 0% (control), 30% (SP30), 50% (SP50), and 70% (SP70) of casein protein replaced by spirulina, respectively, for seven weeks. We observed that spirulina enhanced bone growth and bone strength by stimulating parathyroid hormone and growth hormone activities, as well its increased antioxidant activity. These results indicate that spirulina provides a suitable dietary supplement and alternative protein source with antioxidant benefits for growth improvement in early developmental stages.

Hematopoietic stem cells (HSCs) and skeletal stem cells (SSCs) cohabit in the bone marrow. KITL (C-KIT ligand) from LEPR + adult bone marrow stromal cells is pivotal for HSC maintenance. In contrast, it remains unclear whether KITL/C-KIT signaling also regulates SSCs. Here, we lineage traced C-KIT + cells and found that C-KIT was expressed by fetal, but not postnatal skeletal progenitors. Fetal C-KIT + cells gave rise to 20% of LEPR + stromal cells in adult bone marrow, forming nearly half of all osteoblasts. Disruption of mTOR signaling in fetal C-KIT + cells impaired bone formation. Notably, conditional deletion of Kitl from PRX1 + fetal bone marrow stromal cells, but not LEPR + adult bone marrow stromal cells, significantly increased bone formation. Thus, our work identified C-KIT + skeletal progenitors as an important source of bones formed during development., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteoporosis and osteoporotic fractures lead to decreased life quality and high healthcare costs. Current treatments prevent losses in bone mass and fractures to some extent but have side effects. Therefore, better therapies are needed. This study investigated whether the transcription factor Jun has a specific pro-osteogenic potency and whether modulating Jun could serve as a novel treatment for osteoporosis-associated fractures. We demonstrate that ectopically transplanted whole bones and distinct osteoprogenitors increase bone formation. Perinatal Jun induction disturbs growth plate architecture, causing a striking phenotype with shortened and thickened bones. Molecularly, Jun induces hedgehog signaling in skeletal stem cells. Therapeutically, Jun accelerates bone growth and healing in a drilling-defect model. Altogether, these results demonstrate that Jun drives bone formation by expanding osteoprogenitor populations and forcing them into the bone fate, providing a rationale for future clinical applications., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Microtubule actin crosslinking factor 1 (MACF1) is a large crosslinker that contributes to cell integrity and cell differentiation. Recent studies show that MACF1 is involved in multiple cellular functions such as neuron development and epidermal migration, and is the molecular basis for many degenerative diseases. MACF1 is highly abundant in bones, especially in mesenchymal stem cells; however, its regulatory role is still less understood in bone formation and degenerative bone diseases. In this study, we found MACF1 expression in mesenchymal stem cells (MSCs) of osteoporotic bone specimens was significantly lower. By conditional gene targeting to delete the mesenchymal Macf1 gene in mice, we observed in MSCs decreased osteogenic differentiation capability. During early stage bone development, the MACF1 conditional knockout (cKO) mice exhibit significant ossification retardation in skull and hindlimb, and by adulthood, mesenchymal loss of MACF1 attenuated bone mass, bone microarchitecture, and bone formation capability significantly. Further, we showed that MACF1 interacts directly with SMAD family member 7 (SMAD7) and facilitates SMAD7 nuclear translocation to initiate downstream osteogenic pathways. Hopefully these findings will expand the biological scope of the MACF1 gene, and provide an experimental basis for targeting MACF1 in degenerative bone diseases such as osteoporosis., Competing Interests: The authors declare no conflict of interest.

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

This current study was conducted to investigate whether bone tissue impairment induced by early life exposure to cadmium (Cd) during postnatal development could result from disruption to zinc (Zn) metabolism. For this reason, the offspring from mothers receiving either tap water, Cd, Zn or Cd + Zn during gestation and lactation periods were euthanized at PND21 and PND70. At the end of the lactation period (PND21), our results showed that exposure to Cd increased Cd accumulation and Zn depletion in the femur. Furthermore, calcium (Ca) level was reduced. At the molecular level, Cd induced an increase of MT-1 expression and caused an upregulation of ZIP2 accompanied with a down-regulation of ZnT5. Runx2, ALP, colα-1 and Oc mRNA levels were also decreased. In plasma, IGF-1 and osteocalcin concentrations were decreased. Further, Cd altered femoral growth by generating changes in the growth plate. Consequently, the toxic effect of Cd persisted at adult age (PND70) by decreasing bone volume (%BV/TV), bone mineral density (BMD) and Ca content and by increasing trabecular separation (Tb.Sp) in the distal femur. Interestingly, Zn supply provided total or partial corrections of several toxic effects of Cd. These data suggest that the increases of Zn bioavailability as well as the reduction of Cd accumulation in the femur following the changes in ZIP2 and ZnT5 expression are part of the mechanism involved in Zn protection against Cd toxicity on bone tissue., Competing Interests: The authors declare no conflict of interest.