The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

Dietary Clostridium butyricum and 25-Hydroxyvitamin D3 modulate bone metabolism of broilers through the gut-brain axis.

Leg disorders have become increasingly common in broilers, leading to lower meat quality and major economic losses. This study evaluated the effects of dietary supplementation with Clostridium butyricum (C. butyricum) and 25-hydroxyvitamin D3 (25-OH-D3) on bone development by comparing growth performance, tibial parameters, Ca and P contents of tibial ash, bone development-related indicators' level, and cecal short-chain fatty acids in Cobb broilers. All birds were divided into four treatment groups, which birds fed either a basal diet (Con), basal diet + 75 mg chlortetracycline/kg (Anti), basal diet + C. butyricum at 109 CFU/kg (Cb), basal diet + C. butyricum at 109 CFU/kg and 25-OH-D3 at 25 µg/kg (CbD), or basal diet + 25-OH-D3 at 25 µg/kg (CD). Our results suggest that the dietary supplementation in Cb, CbD, and CD significantly increased the body weight (BW) and average daily gain (ADG), and reduced the feed-to-weight ratio (F/G) at different stages of growth (P < 0.05). Dietary supplementation in Cb, CbD, and CD prolonged (P < 0.05) the behavioral responses latency-to-lie (LTL) time, reduced (P < 0.05) the levels of osteocalcin (BGP) and peptide tyrosine (PYY), and increased (P < 0.05) serotonin (5-HT) and dopamine (DA). Treatment with Cb increased (P < 0.05) the levels of acetic acid, isobutyric acid, butyric acid, and isovaleric acid compared with those in Con group. The cecal metagenome showed that Alistipes spp. were significantly more abundant in Cb, CbD, and CD groups (P < 0.05). A total of 12 metabolic pathways were significantly affected by supplementation, including the signaling pathways of glucagon, insulin, and PI3K-AKT; primary and secondary bile acid biosynthesis; and P-type Ca 2+ transporters (P < 0.05). Hence, the CbD supplementation modulates bone metabolism by regulating the mediators of gut-brain axis, which may inform strategies to prevent leg diseases and improve meat quality in broilers.

The IFITM5 mutation in osteogenesis imperfecta type V is associated with an ERK/SOX9-dependent osteoprogenitor differentiation defect.

Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes in addition to bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in IFITM5. Here, we generated a conditional Rosa26 knock-in mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in OI type V patients. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with increase in the skeletal progenitor population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 showed decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupts early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V.

The specificities, influencing factors, and medical implications of bone circadian rhythms.

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.

Loss of ninein interferes with osteoclast formation and causes premature ossification.

Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human NINEIN gene have been linked to Seckel syndrome and to a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice, and that its absence reduces the fusion of precursor cells into syncytial osteoclasts, whereas the number of osteoblasts remains unaffected. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

Quantitative study of the ossification centers of the body of sphenoid bone in the human fetus.

The aim of the present study was to examine the growth dynamics of the two ossification centers of the body of sphenoid bone in the human fetus, based on their linear, planar and volumetric parameters. The examinations were carried out on 37 human fetuses of both sexes aged 18-30 weeks of gestation, which had been preserved in 10% neutral formalin solution. Using CT, digital image analysis software, 3D reconstruction and statistical methods, we evaluated the size of the presphenoid and postsphenoid ossification centers. The presphenoid ossification center grew proportionately in sagittal diameter, projection surface area and volume, and logarithmically in transverse diameter. The postsphenoid ossification center increased logarithmically in sagittal diameter, transverse diameter and projection surface area, while its volumetric growth followed proportionately. The numerical findings of the presphenoid and postsphenoid ossification centers may be considered age-specific reference values of potential relevance in monitoring the normal fetal growth and screening for congenital disorders in the fetus. The obtained results may contribute to a better understanding of the growing fetal skeleton, bringing new numerical information regarding its diagnosis and development.

Increased endothelial sclerostin caused by elevated DSCAM mediates multiple trisomy 21 phenotypes.

Trisomy 21 (T21), a recurrent aneuploidy occurring in 1:800 births, predisposes to congenital heart disease (CHD) and multiple extracardiac phenotypes. Despite a definitive genetic etiology, the mechanisms by which T21 perturbs development and homeostasis remain poorly understood. We compared the transcriptome of CHD tissues from 49 patients with T21 and 226 with euploid CHD (eCHD). We resolved cell lineages that misexpressed T21 transcripts by cardiac single-nucleus RNA sequencing and RNA in situ hybridization. Compared with eCHD samples, T21 samples had increased chr21 gene expression; 11-fold-greater levels (P = 1.2 × 10-8) of SOST (chr17), encoding the Wnt inhibitor sclerostin; and 1.4-fold-higher levels (P = 8.7 × 10-8) of the SOST transcriptional activator ZNF467 (chr7). Euploid and T21 cardiac endothelial cells coexpressed SOST and ZNF467; however, T21 endothelial cells expressed 6.9-fold more SOST than euploid endothelial cells (P = 2.7 × 10-27). Wnt pathway genes were downregulated in T21 endothelial cells. Expression of DSCAM, residing within the chr21 CHD critical region, correlated with SOST (P = 1.9 × 10-5) and ZNF467 (P = 2.9 × 10-4). Deletion of DSCAM from T21 endothelial cells derived from human induced pluripotent stem cells diminished sclerostin secretion. As Wnt signaling is critical for atrioventricular canal formation, bone health, and pulmonary vascular homeostasis, we concluded that T21-mediated increased sclerostin levels would inappropriately inhibit Wnt activities and promote Down syndrome phenotypes. These findings imply therapeutic potential for anti-sclerostin antibodies in T21.

Bone age and dental late effects in childhood cancer survivors: Radiographic findings in a Brazilian sample.

BACKGROUND: Changes in bone age and tooth development are late side effects of cancer therapy and can be identified by imaging examination. AIM: To evaluate the late effects of antineoplastic treatment on bone age and dental development in childhood cancer survivors. DESIGN: This is a retrospective case-control study on paediatric cancer survivors of both sexes who underwent antineoplastic treatment with 5-15 years of survival. Carpal radiographs were assessed for bone age and growth curve, and panoramic radiographs were used to evaluate dental development and alterations. Carpal radiographs were analyzed using the Greulich and Pyle inspection method, and the Martins and Sakima method was used to analyze the growth curve. All tests were applied with a confidence level of 95%. RESULTS: The study and control groups comprised 28 and 56 patients, respectively. There was no significant difference in bone age and growth curve between the study and control groups. Nonetheless, when sex was compared to chronological and bone ages, there was a significant difference in bone age (p = 0.019) and an underestimation in both groups and sexes in the Greulich and Pyle method. As to late dental effects, dental agenesia, microdontia, gyroversion, and unerupted teeth were found. Dental shape alterations mainly involve the root region. CONCLUSION: Close multidisciplinary collaboration is necessary during the follow-up period of young patients who have survived cancer.

RanGAP1 maintains chromosome stability in limb bud mesenchymal cells during bone development.

BACKGROUND: Bone development involves the rapid proliferation and differentiation of osteogenic lineage cells, which makes accurate chromosomal segregation crucial for ensuring cell proliferation and maintaining chromosomal stability. However, the mechanism underlying the maintenance of chromosome stability during the rapid proliferation and differentiation of Prx1-expressing limb bud mesenchymal cells into osteoblastic precursor cells remains unexplored. METHODS: A transgenic mouse model of RanGAP1 knockout of limb and head mesenchymal progenitor cells was constructed to explore the impact of RanGAP1 deletion on bone development by histomorphology and immunostaining. Subsequently, G-banding karyotyping analysis and immunofluorescence staining were used to examine the effects of RanGAP1 deficiency on chromosome instability. Finally, the effects of RanGAP1 deficiency on chromothripsis and bone development signaling pathways were elucidated by whole-genome sequencing, RNA-sequencing, and qPCR. RESULTS: The ablation of RanGAP1 in limb and head mesenchymal progenitor cells expressing Prx1 in mice resulted in embryonic lethality, severe cartilage and bone dysplasia, and complete loss of cranial vault formation. Moreover, RanGAP1 loss inhibited chondrogenic or osteogenic differentiation of mesenchymal stem cells (MSCs). Most importantly, we found that RanGAP1 loss in limb bud mesenchymal cells triggered missegregation of chromosomes, resulting in chromothripsis of chromosomes 1q and 14q, further inhibiting the expression of key genes involved in multiple bone development signaling pathways such as WNT, Hedgehog, TGF-ß/BMP, and PI3K/AKT in the chromothripsis regions, ultimately disrupting skeletal development. CONCLUSIONS: Our results establish RanGAP1 as a critical regulator of bone development, as it supports this process by preserving chromosome stability in Prx1-expressing limb bud mesenchymal cells.

Exosomes: A New Hope for Angiogenesis-Mediated Bone Regeneration.

Bone is a metabolically dynamic structure that is generally remodeled throughout the lifetime of an individual but often causes problems with increasing age. A key player for bone development and homeostasis, but also under pathological conditions, is the bone vasculature. This complex system of arteries, veins, and capillaries forms distinct structures where each subset of endothelial cells has important functions. Starting with the basic process of angiogenesis and bone-specific blood vessel formation, coupled with initial bone formation, the importance of different vascular structures is highlighted with respect to how these structures are maintained or changed during homeostasis, aging, and pathological conditions. After exemplifying the current knowledge on bone vasculature, this review will move on to exosomes, a novel hotspot of scientific research. Exosomes will be introduced starting from their discovery via current isolation procedures and state-of-the-art characterization to their role in bone vascular development, homeostasis, and bone regeneration and repair while summarizing the underlying signal transduction pathways. With respect to their role in these processes, especially mesenchymal stem cell-derived extracellular vesicles are of interest, which leads to a discussion on patented applications and an update on ongoing clinical trials. Taken together, this review provides an overview of bone vasculature and bone regeneration, with a major focus on how exosomes influence this intricate system, as they might be useful for therapeutic purposes in the near future.

The "toxic window" of amoxicillin exposure during pregnancy on long bone development in fetal mice.

AIMS: Amoxicillin is a broad-spectrum beta-lactam antibiotic used to treat infectious diseases in pregnant women. Studies have shown that prenatal amoxicillin exposure (PAmE) has developmental toxicity on fetal development. However, the effect of PAmE on long bone development has not been reported. This study aimed to investigate the "toxic window" of PAmE on long bone development and explore its possible mechanism in fetal mice. MATERIALS AND METHODS: Pregnant mice were administered amoxicillin by gavage at different stages (gestational day (GD)10-12 and GD16-18), different doses (150 and 300 mg/kg·d) and different courses (single and multiple courses). Fetal femurs were collected at GD18 and bone development related indicators were detected. KEY FINDINGS: The results showed that PAmE significantly reduced the length of the femur and primary ossification center of fetal mice, and inhibited the development of fetal growth plate. Meanwhile, PAmE inhibited the development of bone marrow mesenchymal stem cells, osteoclasts and endothelial cells in fetal long bone. Further, we found the fetal long bone developmental toxicity induced by PAmE was most significant at late-pregnancy (GD16-18), high dose (300 mg/kg·d) and multiple-course group. Besides, PAmE inhibited the expression of Wnt/ß-catenin signaling pathway in fetal long bone. The ß-catenin mRNA expression was significantly positively correlated with the development indexes of fetal long bone. SIGNIFICANCE: PAmE has toxic effects on long bone development, and there was an obvious "toxic window" of PAmE on the long bone development in fetal mice. The Wnt/ß-catenin signaling pathway may mediate PAmE-induced fetal long bone development inhibition.

Female rats consuming an iron and omega-3 fatty acid deficient diet preconception require combined iron and omega-3 fatty acid supplementation for the prevention of bone impairments in offspring.

We previously showed in rats that pre- and postnatal deficiencies in iron and omega-3 (n-3) fatty acids can impair bone development, with additive and potentially irreversible effects when combined. This study aimed to investigate, in female rats consuming a combined iron and n-3 fatty acid deficient (ID + n-3 FAD) diet preconception, whether supplementation with iron and docosahexaenoic/eicosapentaenoic acid (DHA/EPA), alone and in combination, can prevent bone impairments in offspring. Using a 2 × 2 factorial design, female Wistar rats consuming an ID + n-3 FAD diet preconception were randomised to receive an: 1) iron supplemented (Fe + n-3 FAD), 2) DHA/EPA supplemented (ID + DHA/EPA), 3) Fe + DHA/EPA, or 4) ID + n-3 FAD diet from gestational day 10 throughout pregnancy and lactation. Post-weaning, offspring (n = 24/group; male:female = 1:1) remained on the respective experimental diets for three weeks until postnatal day 42-45. Offspring born to female rats consuming a control diet preconception and an Fe+DHA/EPA diet throughout pregnancy and lactation served as non-deficient reference group (Control+Fe+DHA/EPA). Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry and bone strength using three-point bending tests. Only offspring in the Fe+DHA/EPA group had significantly higher spine and femur BMD, and higher femur stiffness than offspring in the ID + n-3 FAD group, and had similar spine BMD and femur stiffness as the Control + Fe + DHA/EPA group. Offspring in the Fe + DHA/EPA group further had significantly higher femur strength (ultimate load) than the other experimental groups, and a similar femur strength as the Control + Fe + DHA/EPA group. This study shows that only combined iron and DHA/EPA supplementation can prevent bone impairments in offspring of female rats consuming an iron and n-3 FA deficient diet preconception.

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Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

Skeletal stem cells in bone development, homeostasis and disease.

Tissue-resident stem cells are essential for development and repair, and in the skeleton this function is fulfilled by recently identified skeletal stem cells (SSCs). However, recent work has identified that SSCs are not monolithic, with long bones, craniofacial sites, and the spine being formed by distinct stem cells. Recent studies have utilized techniques such as fluorescence-activated cell sorting (FACS), lineage tracing and single-cell sequencing to investigate the involvement of SSCs in bone development, homeostasis and disease. These investigations have allowed researchers to map the lineage commitment trajectory of SSCs in different parts of the body and at different time points. Furthermore, recent studies have shed light on the characteristics of SSCs in both physiological and pathological conditions. This review focuses on discussing the spatiotemporal distribution of SSCs and enhancing our understanding of the diversity and plasticity of SSCs by summarizing recent discoveries.

Ptip safeguards the epigenetic control of skeletal stem cell quiescence and potency in skeletogenesis.

Stem cells remain in a quiescent state for long-term maintenance and preservation of potency; this process requires fine-tuning regulatory mechanisms. In this study, we identified the epigenetic landscape along the developmental trajectory of skeletal stem cells (SSCs) in skeletogenesis governed by a key regulator, Ptip (also known as Paxip1, Pax interaction with transcription-activation domain protein-1). Our results showed that Ptip is required for maintaining the quiescence and potency of SSCs, and loss of Ptip in type II collagen (Col2)+ progenitors causes abnormal activation and differentiation of SSCs, impaired growth plate morphogenesis, and long bone dysplasia. We also found that Ptip suppressed the glycolysis of SSCs through downregulation of phosphoglycerate kinase 1 (Pgk1) by repressing histone H3K27ac at the promoter region. Notably, inhibition of glycolysis improved the function of SSCs despite Ptip deficiency. To the best of our knowledge, this is the first study to establish an epigenetic framework based on Ptip, which safeguards skeletal stem cell quiescence and potency through metabolic control. This framework is expected to improve SSC-based treatments of bone developmental disorders.

Exploring epigenetic strategies for the treatment of osteoporosis.

The worldwide trend toward an aging population has resulted in a higher incidence of chronic conditions, such as osteoporosis. Osteoporosis, a prevalent skeletal disorder characterized by decreased bone mass and increased fracture risk, encompasses primary and secondary forms, each with distinct etiologies. Mechanistically, osteoporosis involves an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Current pharmacological interventions for osteoporosis, such as bisphosphonates, denosumab, and teriparatide, aim to modulate bone turnover and preserve bone density. Hormone replacement therapy and lifestyle modifications are also recommended to manage the condition. While current medications offer therapeutic options, they are not devoid of limitations. Recent studies have highlighted the importance of epigenetic mechanisms, including DNA methylation and histone modifications, in regulating gene expression during bone remodeling. The use of epigenetic drugs, or epidrugs, to target these mechanisms offers a promising avenue for therapeutic intervention in osteoporosis. In this review, we comprehensively examine the recent advancements in the application of epidrugs for treating osteoporosis.

Knocking out FAM20C in pre-osteoblasts leads to up-regulation of osteoclast differentiation to affect long bone development.

Family with sequence similarity 20 member C (FAM20C) is a Golgi casein kinase that phosphorylates extracellularly-secreted regulatory proteins involved in bone development and mineralization, but its specific role in bone development is still largely unknown. In this study, to examine the specific mechanisms that FAM20C influences bone development, we cross-bred Osx-Cre with FAM20Cflox/flox mice to establish a Osx-Cre; FAM20Cflox/flox knockout (oKO) mouse model; FAM20C was KO in pre-osteoblasts. oKO development was examined at 1-10 weeks, in which compared to control FAM20Cflox/flox, they had lower body weights and bone tissue mineralization. Furthermore, oKO had lower bone volume fractions, thickness, and trabecular numbers, along with higher degrees of trabecular separation. These mice also had decreased femoral metaphyseal cartilage proliferation layer, along with thickened hypertrophic layer and increased apoptotic cell counts. Transcriptomic analysis found that differentially-expressed genes in oKO were concentrated in the osteoclast differentiation pathway, in line with increased osteoclast presence. Additionally, up-regulation of osteoclast-related, and down-regulation of osteogenesis-related genes, were identified, in which the most up-regulated genes were signal regulatory protein ß-1 family (Sirpb1a-c) and mitogen-activated protein kinase 13. Overall, FAM20C KO in pre-osteoblasts leads to abnormal long bone development, likely due to subsequent up-regulation of osteoclast differentiation-associated genes.

Stimulation of skeletal stem cells in the growth plate promotes linear bone growth.

Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (epiphyseal skeletal stem cells, epSSCs), but their function in connection with linear bone growth remains unknown. Here, we explore the possibility that modulating the number of epSSCs can correct differences in leg length. First, we examined regulation of the number and activity of epSSCs by Hedgehog (Hh) signaling. Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched1 (Ptch1) ablation in Pthrp-creER Ptch1fl/fl tdTomato mice promoted proliferation of epSSCs and clonal enlargement. Transient intra-articular administration of SAG also elevated the number of epSSCs. When SAG-containing beads were implanted into the femoral secondary ossification center of 1 leg of rats, this leg was significantly longer 1 month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced 2 and 6 months after implantation. We conclude that Hh signaling activates growth plate epSSCs, which effectively leads to increased longitudinal growth of bones. This opens therapeutic possibilities for the treatment of differences in leg length.

Gender-affirming hormone therapy preserves skeletal maturation in young mice via the gut microbiome.

Gender-affirming hormone therapy (GAHT) is often prescribed to transgender (TG) adolescents to alleviate gender dysphoria, but the effect of GAHT on the growing skeleton is unclear. We found GAHT to improve trabecular bone structure via increased bone formation in young male mice and not to affect trabecular structure in female mice. GAHT modified gut microbiome composition in both male and female mice. However, fecal microbiota transfers (FMTs) revealed that GAHT-shaped gut microbiome was a communicable regulator of bone structure and turnover in male, but not in female mice. Mediation analysis identified 2 species of Bacteroides as significant contributors to the skeletal effects of GAHT in male mice, with Bacteroides supplementation phenocopying the effects of GAHT on bone. Bacteroides have the capacity to expand Treg populations in the gut. Accordingly, GAHT expanded intestinal Tregs and stimulated their migration to the bone marrow (BM) in male but not in female mice. Attesting to the functional relevance of Tregs, pharmacological blockade of Treg expansion prevented GAHT-induced bone anabolism. In summary, in male mice GAHT stimulated bone formation and improved trabecular structure by promoting Treg expansion via a microbiome-mediated effect, while in female mice, GAHT neither improved nor impaired trabecular structure.

Chondrocyte hypertrophy in the growth plate promotes stress anisotropy affecting long bone development through chondrocyte column formation.

The length of long bones is determined by column formation of proliferative chondrocytes and subsequent chondrocyte hypertrophy in the growth plate during bone development. Despite the importance of mechanical loading in long bone development, the mechanical conditions of the cells within the growth plate, such as the stress field, remain unclear owing to the difficulty in investigating spatiotemporal changes within dynamically growing tissues. In this study, the mechanisms of longitudinal bone growth were investigated from a mechanical perspective through column formation of proliferative chondrocytes within the growth plate before secondary ossification center formation using continuum-based particle models (CbPMs). A one-factor model, which simply describes essential aspects of a biological signaling cascade regulating cell activities within the growth plate, was developed and incorporated into CbPM. Subsequently, the developmental process and maintenance of the growth plate structure and resulting bone morphogenesis were simulated. Thus, stress anisotropy in the proliferative zone that affects bone elongation through chondrocyte column formation was identified and found to be promoted by chondrocyte hypertrophy. These results provide further insights into the mechanical regulation of multicellular dynamics during bone development.