Operative treatment is an option for fractures when the fracture is unstable or the patient wishes to return early to daily life or social activities. Metal plates such as titanium and stainless steel are often used in fracture surgery, but the metal plate lacks bone-healing activity and is not bioabsorbable, requiring a second surgery to remove it after bone union. Here we show that a magnesium (Mg) plate made from an alloy of yttrium, zinc, and aluminum with magnesium as the main component in a long-period stacking ordered structure promotes bone formation in a rabbit tibia fracture model and is also bioabsorbable. We show that the Mg plate significantly promoted bone and callus formation compared to a titanium plate in the rabbit tibia fracture model. Moreover, the Mg plate was mostly bioabsorbed once bone union was achieved, but rabbits showed no evidence of biotoxic effects, such as weight loss or increased blood magnesium levels. We also demonstrate that treatment with exogenous magnesium significantly enhanced calcium deposition in an in vitro osteoblast culture system. Magnesium is an essential element, and its radiolucency facilitates observation of the fracture site during Mg plate fixation, while its lack of magnetic properties allows its use in patients who require MRI scans. Accordingly, we propose that a use of a Mg plate could be beneficial in treating bone fracture.

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel highly expressed in chondrocytes that supports cartilage development and homeostasis. Mutations in the channel can cause skeletal dysplasias, including the gain-of-function mutations V620I and T89I, which lead to brachyolmia and metatropic dysplasia, respectively. These mutations suppress hypertrophic differentiation, but the mechanisms by which they alter chondrocyte response to mechanical load remain to be elucidated. To determine the effect of these mutations on chondrocyte mechanotransduction, tissue-engineered cartilage was derived from differentiated clustered regularly interspaced short palindromic repeats (CRISPR)-edited human-induced pluripotent stem cells (hiPSCs) harboring the moderate V620I or severe T89I TRPV4 mutations. Wild-type and mutant tissue-engineered hiPSC-derived cartilage contructs were subjected to compressive mechanical loading at physiological levels, and transcriptomic signatures were assessed by RNA-sequencing. Our results demonstrate that the V620I and T89I mutations diminish the mechanoresponsiveness of chondrocytes, as evidenced by reduced gene expression downstream of TRPV4 activation, including those involved in endochondral ossification. Changes in the expression of genes involved in extracellular matrix production and organization were found to contribute toward the phenotype in V620I mutant chondrocytes, whereas dysregulated retinoic acid signaling was linked to T89I, and disrupted proliferation was common to both. Our findings suggest that dysfunctional mechanotransduction due to V620I and T89I mutations in TRPV4 contribute to the developmental phenotypes, supporting TRPV4 modulation as a potential pharmacologic target.NEW & NOTEWORTHY Gain-of-function mutations in TRPV4, a mechano- and osmosensitive ion channel, are linked to skeletal dysplasias, but their effects on chondrocyte mechanotransduction remain unknown. Using human iPSCs harboring skeletal dysplasia-causing mutations, we developed and mechanically loaded tissue-engineered cartilage. Our findings show that V620I and T89I mutations reduce chondrocyte mechanoresponsiveness, evidenced by decreased gene expression downstream of TRPV4 activation, providing insight into TRPV4-related skeletal disorders and potential pharmacological targets.

The MYST family histone acetyltransferase gene, KAT6B (MYST4, MORF, QKF) is mutated in two distinct human congenital disorders characterised by intellectual disability, facial dysmorphogenesis and skeletal abnormalities; the Say-Barber-Biesecker-Young-Simpson variant of Ohdo syndrome and Genitopatellar syndrome. Despite its requirement in normal skeletal development, the cellular and transcriptional effects of KAT6B in skeletogenesis have not been thoroughly studied. Here, we show that germline deletion of the Kat6b gene in mice causes premature ossification in vivo, resulting in shortened craniofacial elements and increased bone density, as well as shortened tibias with an expanded pre-hypertrophic layer, as compared to wild type controls. Mechanistically, we show that the loss of KAT6B in mesenchymal progenitor cells promotes transition towards an osteoblast-progenitor state with upregulation of gene targets of RUNX2, a master regulator of osteoblast development and concomitant downregulation of SOX9, a critical gene in chondrocyte development. Moreover, we find that compound heterozygosity at Kat6b and Runx2 loci partially rescues the reduction in ossification of Runx2 heterozygous, but not homozygous mice, suggesting that KAT6B may limit the action of RUNX2, possibly through a role in maintaining progenitors in an undifferentiated state. Moreover, our results show that KAT6B has essential roles in regulating the expression of a large number of genes involved in skeletogenesis and bone development.

1. Valgus-varus deformity (VVD) is a disease that severely affects leg function in broilers and for which there is no effective control method current available. Although DNA methylation has an important impact on most physiological and pathological processes, its involvement in skeletal muscle growth and development in VVD broilers is unknown. In this study, genome-wide DNA methylation was analysed in VVD-affected and normal broilers using whole genome resulphite sequencing.2. The results showed that in the cytosine-phosphoric acid-guanine (CG) sequence environment there was a methylation rate of about 55% and 4,265 differentially methylated regions (DMRs) were found in the CG. Of these, 550 were located in the promoter, 547 in the exon region, and 1,718 in the intron region.3. All differentially methylated genes (DMGs) were analysed for enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The GO was enriched in pathways related to protein degradation such as proteasome complex, endopeptidase complex and extracellular region. The KEGG pathways were enriched in signalling pathways related to protein degradation and catabolism such as proteasome, nitrogen metabolism, adherens junction and alanine.4. Protein interactions analysis revealed that FOS, MYL9, and FRAS1 had a high degree of interactions, in which the DNA methylation level of the MYL9 promoter region was negatively correlated with mRNA expression level. Further studies showed that 5-azacytidine (5-AzaC) inhibited DNMT1 and DNMT3A gene expression and promoted MYL9 expression.5. This study systematically investigated overall DNA methylation patterns in the leg muscle of VVD and normal broilers. It screened common differential genes in conjunction with transcriptomic data to further identify genes associated with muscle growth and development. This study provides new insights to better understand the pathogenesis of VVD from an epigenetic perspective.

MicroRNAs (miRNAs) are critical regulators of the skeleton. In the growth plate, these small non-coding RNAs modulate gene networks that drive key stages of chondrogenesis, including proliferation, differentiation, extracellular matrix synthesis and hypertrophy. These processes are orchestrated through the interaction of pivotal pathways including parathyroid hormone-related protein (PTHrP), Indian hedgehog (IHH), and bone morphogenetic protein (BMP) signaling. This review highlights the miRNA-mRNA target networks essential for chondrocyte differentiation. Many miRNAs are differentially expressed in resting, proliferating and hypertrophic cartilage zones. Moreover, differential enrichment of specific miRNAs in matrix vesicles is also observed, providing means for chondrocytes to influence the function and differentiation of their neighbors by via matrix vesicle protein and RNA cargo. Notably, miR-1 and miR-140 emerge as critical modulators of chondrocyte proliferation and hypertrophy by regulating multiple signaling pathways, many of them downstream from their mutual target Hdac4. Demonstration that a human gain-of-function mutation in miR-140 causes skeletal dysplasia underscores the clinical relevance of understanding miRNA-mediated regulation. Further, miRNAs such as miR-26b have emerged as markers for skeletal disorders such as idiopathic short stature, showcasing the translational relevance of miRNAs in skeletal health. This review also highlights some miRNA-based therapeutic strategies, including innovative delivery systems that could target chondrocytes via cartilage affinity peptides, and potential applications related to treatment of physeal bony bridge formation in growing children. By synthesizing current research, this review offers a nuanced understanding of miRNA functions in growth plate biology and their broader implications for skeletal health. It underscores the translational potential of miRNA-based therapies in addressing skeletal disorders and aims to inspire further investigations in this rapidly evolving field.

Craniofacial abnormalities are among the most prevalent congenital defects, significantly affecting appearance, function, and quality of life. While the role of genetic mutations in craniofacial malformations is recognized, the underlying molecular mechanisms remain poorly understood. In this study, we investigate the role of p75 neurotrophin receptor (p75NTR) in craniofacial development by comparing wild-type (p75NTR+/+) mice against p75NTR-deficient (p75NTR-/-) knockout mice. We employed histology, micro-CT surface distance, volumetric analysis, and geometric morphometric analysis to assess craniofacial development and growth. On postnatal day 7 (P7), p75NTR-/- mice exhibited reduced skull length compared to wild-type controls. By P28, micro-CT analysis revealed significant reductions in calvarial bone volume and trabecular bone thickness in p75NTR-/- mice. Geometric morphometric analysis identified significant shape alterations in the nasal, parietal, and occipital regions, with p75NTR-/- mice showing a shortened cranium and tapered nasal bone morphology. These findings highlight the critical role of p75NTR in regulating postnatal craniofacial development. Disruption of p75NTR signaling impairs both the growth and morphological integrity of craniofacial structures, which may contribute to the pathogenesis of congenital craniofacial abnormalities. In the future, a better understanding of the molecular mechanisms through which p75NTR mediates craniofacial development may offer valuable insights for future targeted therapeutic strategies for craniofacial defects.

To explore the therapeutic efficacy of L-glutamine (L-Gln) on pathological progression and clinical symptoms of osteoarthritis (OA), and compare with glucosamine sulfate (GS), and celecoxib (CXB). Rats were administered sodium chloride, L-Gln, GS, or CXB via gavage for eight weeks starting from the fifth week after sham operation or Anterior Cruciate Ligament Transection (ACLT) + Medial Meniscectomy (MMx). Then the severity of knee OA in rats was evaluated by serological analysis, histological examination and imaging examination. In addition, patients with mild primary OA were administered L-Gln, GS, or CXB orally for 12 weeks in accordance with the randomization principle. The efficacy end points were the change from baseline to week 24 in the pain and physical function subscale scores of the Western Ontario and McMaster Universities OA Index (WOMAC), and Lequesne score. Treatment with L-Gln alleviated the increased concentration of serum cartilage degradation markers caused by OA in rats. Histological tests showed improvement in knee joint cartilage destruction after treatment. Three-dimensional CT scans and reconstructions revealed a reduction in osteophyte formation and subchondral bone loss. L-glutamine performed as well as or better than glucosamine sulfate and celecoxib in all comparative measures among the three treatment groups. In clinical trials, the WOMAC pain and physical function subscale scores, as well as the Lequesne score, decreased from baseline in all three patient groups during follow-up, with no significant differences observed between the groups. Our research indicates that L-Gln is comparable to GS and CXB in improving the pathological progression and clinical efficacy of OA, which makes it a promising drug for the treatment of osteoarthritis.

Longitudinal skeletal growth takes place in the cartilaginous growth plates. While growth plates are found at either end of conventional long bones, they occur at a variety of locations in the mammalian skeleton. For example, the metacarpals and metatarsals (MT) in the hands and feet form only a single growth plate at one end, and the pisiform in the wrist is the only carpal bone to contain a growth plate. We take advantage of this natural anatomical variation to test which components of the PTHrP/Ihh feedback loop, a fundamental regulator of chondrocyte differentiation, are specific to growth plate function.

Parathyroid hormone-like hormone (Pthlh), the gene that transcribes parathyroid hormone-related peptide (PTHrP), is expressed in the reserve zone of the growth plate-forming end of the MT. At the opposite end, the absence of a PTHrP+ reserve zone results in premature chondrocyte differentiation and Indian hedgehog (Ihh) expression. Pthlh is expressed in the reserve zone of the developing pisiform, confirming the existence of a true growth plate.

A pool of PTHrP+ reserve zone chondrocytes is a defining characteristic of growth plates, and its patterning may be key to evolved differences in growth plate location in the mammalian skeleton.

Marine-derived biomaterials are emerging as promising candidates for tissue regeneration due to their sustainability, biocompatibility, bioactivity, and unique chemical structure. This review provides an overview of different marine-derived inorganic and organic materials, such as calcium carbonate, magnesium salts, silica, polysaccharides, bioactive peptides, and lipid-based compounds, and their effects in promoting osteogenesis. Specifically, the osteoinductive, osteoconductive, and osteointegrative activities of traditional and innovative materials that influence key molecular pathways such as BMP/Smad and Wnt/β-catenin signaling underlying bone formation will be evaluated. This review also prospects innovative approaches, i.e., phage display technology, to optimize marine-derived peptides for targeted bone regeneration. In the context of innovative and sustainable materials, this review suggests some interesting applications of unusual materials able to overcome the limitations of conventional ones and stimulate cellular regeneration of bone tissue by activating specific molecular pathways.

Several cartilage and bone organoids have been developed in vitro and in vivo using adult mesenchymal stromal/stem cells (MSCs) or pluripotent stem cells (PSCs) to mimic different phases of endochondral ossification (ECO), as one of the main processes driving skeletal development and growth. While cellular and molecular features of growth plate-like structures have been observed through the generation and in vivo implantation of hypertrophic cartilage tissues, no functional analogue or model of the growth plate has yet been engineered. Herein, after a brief introduction about the growth plate architecture and function, we summarize the recent progress in dissecting the biology of the growth plate and indicate the knowledge gaps to better understand the mechanisms of its development and maintenance. We then discuss how this knowledge could be integrated with state-of-art bioengineering approaches to generate a functional in vitro growth plate model.

The resting zone (RZ) in mammalian growth plates is critical for maintaining and regulating chondrocyte turnover during longitudinal bone growth as a control tower and stem cell reservoir. Although recent lineage tracing studies have identified several markers for stem cells in the RZ, these markers only partially label chondrocytes in the RZ, suggesting that the resting chondrocytes (RCs) are a heterogeneous population with different types of stem cells. Since a comprehensive marker for RCs is still lacking, the RZ is generally determined based on ambiguous histological criteria, such as small and round chondrocytes without columnar formation, which may lead to inconsistencies among researchers. Therefore, in this study, we used single-cell RNA sequencing (scRNAseq) of growth plate chondrocytes followed by validation by fluorescence in situ hybridization (FISH) to precisely annotate cell clusters in scRNAseq and search for a marker of RCs. The scRNAseq analysis revealed that apolipoprotein E (Apoe) was the top-hit gene, which was ubiquitously expressed in the RC cluster. FISH confirmed that Apoe was exclusively localized to the histologically defined RZ. In newly generated ApoemCherry knock-in mice, we further confirmed that mCherry expression mirrored the distribution of Apoe-expressing chondrocytes in the RZ particularly after the formation of the secondary ossification center. These mCherry+ RCs were slow cycling in vivo and exhibited stem cell properties in vitro. Moreover, APOE was detected in human growth plate RCs. These findings suggest that apolipoprotein E is a novel pan-RC marker in both mouse and human growth plates.

Ciliopathies encompass a broad spectrum of diseases stemming from dysfunction of the primary (non-motile) cilia, present on almost all cells in the human body. These disorders include autosomal dominant and recessive polycystic kidney diseases, nephronophthisis, and multisystem ciliopathies such as Joubert, Meckel, Bardet-Biedl, Alström, oral-facial-digital syndromes, and skeletal ciliopathies. The majority of these ciliopathies are associated with fibrocystic kidney disease resulting in progressive kidney dysfunction. In addition, many ciliopathies are associated with extra-renal manifestations including congenital hepatic fibrosis, retinal dystrophy, obesity, and brain and skeletal anomalies. The diagnoses may be challenging due to their overlapping clinical features and molecular heterogeneity. To date, over 190 genes encoding proteins that localize to the primary cilia have been identified as disease-causing. This review will discuss the clinical features of the most frequently encountered disorders of primary cilia.

Skeletal stem cells (SSCs) maintain the skeletal system via pluripotency and differentiation capacity. However, it remains largely unknown how these cells precisely regulate their function to maintain skeletal organization. Here, we delineate the RNA m6A modification landscape across skeletal cell populations in the mouse epiphysis. Our findings show that m6A modifications are prevalent in skeletal stem cell and progenitor populations and play critical roles in cell fate determination. Genetic deletion of Mettl3, the core catalytic subunit of the m6A-methyltransferase complex, in murine skeletal stem and progenitors impaired bone development, leading to shortened limbs, disrupted growth plate zonation, and decreased bone mass. Moreover, Mettl3 deficiency induced quiescence exit in SSCs, together with compromised self-renewal capacity and differentiation potential. Mechanistically, Mettl3-mediated m6A modification reduced mRNA stability of the Cul2-RING E3 ligase complex subunit Fem1b, which subsequently stabilizes Gli1 protein, a key transcription factor of Hedgehog pathway for maintaining SSC identity and function. Thus, we present a comprehensive RNA m6A modification landscape of skeletal cell hierarchy and uncover the essential function of epitranscriptomically-regulated proteostasis in maintaining SSCs quiescence and potency.

The temporomandibular joint (TMJ) is unique in both developmental origin and functional maintenance. The role of bone morphogenic protein (BMP) signaling in endochondral ossification has been widely investigated but not in the context of the TMJ. We employed a histomorphometric analysis approach to understand how augmented BMP signaling in the cranial neural crest affects the postnatal development of the TMJ. Our analysis showed that cartilage length in the mandibular condyle was reduced in Wnt1 Cre;caBmpr1a mice before the weaning stage (P17). However, following weaning, the mandibular condylar cartilage showed recovered length (P28 and P42). Furthermore, the changes in cartilage length coincide with alterations in cell death in the superficial region of the mandibular condyle. These results suggest that BMP signaling influences chondrocyte cell death and TMJ development in a timepoint-specific manner.

This meta-analysis aims to investigate the impact of abnormalities in mineral metabolic markers, including serum phosphate and calcium, intact parathyroid hormone (iPTH), and fibroblast growth factor 23 (FGF23) on the risk of fractures in patients with chronic kidney disease (CKD).

A systematic search was conducted across MEDLINE, Web of Science, EMBASE, ClinicalTrials.gov, and the Cochrane Central Register for Controlled Trials. The outcomes were association of mineral metabolic markers with the risk of fractures in patients with chronic kidney disease. Pooled risk estimates and 95% confidence intervals (CIs) were calculated using fixed-effects or random-effects models.

Thirty-two studies were included in the meta-analysis. High and low levels of serum phosphate in hemodialysis (HD) patients were both associated with an increased risk of fractures (RR = 1.08, 95% CI 1.02-1.15, P = 0.013; RR = 1.13, 95% CI 1.02-1.25, P = 0.022, respectively). Similarly, abnormal levels of iPTH in CKD patients, both high and low, were associated with increased fracture risk (RR = 1.25, 95% CI 1.20-1.31, P < 0.001; RR = 1.41, 95% CI 1.10-1.82, P = 0.007, respectively). Elevated FGF23 levels were also linked to an increased risk of fractures (RR = 1.32, 95% CI 1.06-1.66, P = 0.015). While a higher level of calcium exhibited a trend towards reduced fracture incidence without statistical significance (RR = 0.90, 95% CI 0.77-1.05, P = 0.181), lower calcium levels tended to increase fracture risk without statistical significance (RR = 1.11, 95% CI 0.99-1.24, P = 0.087). Notably, subjects treated with calcium and phosphorus modulating drugs demonstrated a statistically significant reduction in fractures among CKD patients undergoing dialysis (phosphate binders, RR = 0.79, 95% CI 0.70-0.89; cinacalcet, RR = 0.74, 95% CI 0.59-0.93; vitamin D analogues, RR = 0.82, 95% CI 0.74-0.92, respectively).

This meta-analysis underscores the association between abnormal mineral metabolic markers, including high serum phosphate, iPTH, and FGF23, and an increased risk of fractures in CKD patients. Notably, both elevated and decreased levels of phosphate and iPTH contribute to fracture risk. The efficacy of active vitamin D, phosphorus binders, and cinacalcet in preventing fractures was observed in HD patients but not in the non-dialysis CKD population.

PROSPERO CRD42023493951.

Endothelial cells coat blood vessels and release molecular signals to affect the fate of other cells. Endothelial cells can adjust their behavior in response to the changing microenvironmental conditions. During bone regeneration, bone tissue cells release factors that promote blood vessel growth. Notch is a key signaling that regulates cell fate decisions in many tissues and plays an important role in bone tissue development and homeostasis. Understanding the interplay between angiogenesis and osteogenesis is currently a focus of research efforts in order to facilitate and improve osteogenesis when needed. Our review explores the cellular and molecular mechanisms including Notch-dependent endothelial-MSC communication that drive osteogenesis-angiogenesis processes and their effects on bone remodeling and repair.

The skeleton is highly innervated by numerous nerve fibers. These nerve fibers, in addition to transmitting information within the bone and mediating bone sensations, play a crucial role in regulating bone tissue formation and regeneration. Traditional bone tissue engineering (BTE) often fails to achieve satisfactory outcomes when dealing with large-scale bone defects, which is frequently related to the lack of effective reconstruction of the neurovascular network. In recent years, increasing research has revealed the critical role of nerves in bone metabolism. Nerve fibers regulate bone cells through neurotransmitters, neuropeptides, and peripheral glial cells. Furthermore, nerves also coordinate with the vascular and immune systems to jointly construct a microenvironment favorable for bone regeneration. As a signaling driver of bone formation, neuroregulation spans the entire process of bone physiological activities from the embryonic formation to postmaturity remodeling and repair. However, there is currently a lack of comprehensive summaries of these regulatory mechanisms. Therefore, this review sketches out the function of nerves during bone formation and regeneration. Then, we elaborate on the mechanisms of neurovascular coupling and neuromodulation of bone immunity. Finally, we discuss several novel strategies for neuro-bone tissue engineering (NBTE) based on neuroregulation of bone, focusing on the coordinated regeneration of nerve and bone tissue.

Secondary ossification and maintenance of the growth plate are crucial aspects of long bone formation. Parathyroid hormone-related protein (PTHrP) has been implicated as a key factor in maintaining the growth plate, and studies suggest that PTHrP expression in the resting zone is closely related with formation of the secondary ossification center (SOC). However, details of the relationship between resting zone PTHrP expression and preservation of the growth plate remain unclear. In this study, we aim to investigate the role of resting zone PTHrP expression on maintenance of the growth plate using a computational method. We extend an existing continuum-based particle model of tissue morphogenesis to include PTHrP and Indian hedgehog (Ihh) signaling, allowing the model to capture biochemical and mechanical regulation of individual cell activities. Our model indicates that the timing of resting zone PTHrP expression-specifically the rate of increase in production at the onset of SOC formation-is potentially a crucial mechanism for maintenance of the growth plate.

Echolocation represents one of the most rapid adaptive sensorimotor modulation behaviors observed in mammals, establishing bats as one of the most evolutionarily successful mammals. Bats rely on high-frequency hearing for survival, but our understanding of its cellular molecular basis is scattered and segmented. Herein, we constructed the first single-cell transcriptomic landscape of the cochlea in Hipposideros armiger, a CF-FM bat, using a PacBio-optimized genome and compared it with the results obtained from unoptimized original genomes. Sixteen distinct cell types were distributed across five spatial regions of the cochlea. Notably, through hematoxylin and eosin staining and fluorescence in situ hybridization, we identified new types of spiral ganglion neuron (SGN) cells in the cochlea of H. armiger. These SGN cells are likely critical for auditory perception and may have driven the adaptive evolution of high-frequency hearing in this species. Furthermore, we uncovered the differentiation relationships of among specific cell types, such as the transition from supporting cells to hair cells. Using the cochlear cell atlas as a reference, cell types susceptible to deafness-associated genes (in the human) were also identified. In summary, this study provides novel insights into the cellular and molecular mechanisms underlying the adaptive high-frequency hearing in bats and highlights potential candidate cell types and genes for therapeutic interventions in hearing loss.

SOX9 is a crucial transcriptional regulator of cartilage development and homeostasis. Dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic dysplasia, acampomelic campomelic dysplasia, and scoliosis. Yet how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four pathogenic variants of SOX9 identified in a cohort of patients with congenital vertebral malformations. We report a pathogenic missense variant in the transactivation middle (TAM) domain of SOX9 associated with mild skeletal dysplasia and scoliosis. We isolated a Sox9 mutant mouse with an in-frame microdeletion in the TAM domain (Sox9Asp272del), which exhibits skeletal dysplasia including kinked tails, rib cage anomalies, and scoliosis in homozygous mutants. We find that both the human missense and the mouse microdeletion mutations resulted in reduced SOX9 protein stability in cell culture, while Sox9Asp272del mutant mice show decreased SOX9 expression in the growth plate and annulus fibrosus tissues of the spine. This reduction in SOX9 expression was correlated with the reduction of extracellular matrix components, such as tenascin-X and the Adhesion G-protein coupled receptor ADGRG6. In summary, our work identified and modeled a pathologic variant of SOX9 within the TAM domain and demonstrated its importance for SOX9 protein stability. Our work demonstrates that SOX9 stability is important for the regulation of ADGRG6 expression, which is a known regulator of postnatal spine homeostasis, underscoring the essential role of SOX9 dosage in a spectrum of axial skeleton dysplasia in humans.

A high prevalence of rotator cuff tears presents a major clinical challenge. A better understanding of the molecular mechanisms underlying enthesis development and healing is needed for developing treatments. We recently identified hedgehog (Hh)-lineage cells critical for enthesis development and repair. This study revealed cell-cell communication within the Hh-lineage cell population. To further characterize the role of Hh signaling, we used mouse models to activate and inactivate the Hh pathway in enthesis progenitors. Activation of Hh target genes during enthesis development increased its mineralization and mechanical properties. Activation of Hh signaling at the injured mature enthesis promoted fibrocartilage formation, enhanced mineralization, and increased expression of chondrogenic and osteogenic markers, which implies that Hh signaling drives cell differentiation to regenerate the damaged enthesis. Conversely, deletion of Hh target genes impaired enthesis healing. In summary, this study revealed a new strategy for enthesis repair via activation of Hh signaling in endogenous cells.

Osteoarthritis (OA) is a common joint disease characterized by pain and functional impairment, which severely impacts the quality of life of middle-aged and elderly individuals. During normal bone development, chondrocyte hypertrophy is a natural physiological process. However, in the progression of OA, chondrocyte hypertrophy becomes one of its key pathological features. Although there is no definitive evidence to date confirming that chondrocyte hypertrophy is the direct cause of OA, substantial experimental data indicate that it plays an important role in the disease's pathogenesis. In this review, we first explore the mechanisms underlying chondrocyte hypertrophy in OA and offer new insights. We then propose strategies for inhibiting chondrocyte hypertrophy from the perspectives of targeting signaling pathways and tissue engineering, ultimately envisioning the future prospects of OA treatment.

Recombinant human IGF-1 is used to treat severe primary IGF-1 deficiency, but this treatment requires twice-daily injection, often does not fully correct the growth deficit, and has important off-target effects. We therefore sought to target IGF-1 to growth plate cartilage by generating fusion proteins combining IGF-1 with single-chain human antibody fragments that target matrilin-3, a cartilage matrix protein. We previously showed that this cartilage-targeting IGF-1 fusion protein (CV1574-1) promoted growth plate function in a GH-deficient (lit) mouse model. Here, we studied CV1574-1 in a second mouse model, C57BL/6 wild-type mice treated with pegvisomant to induce GH resistance. In this model, once-daily injections of CV1574-1 for 5 days partially restored the pegvisomant-induced decrease in growth plate height without increasing kidney cell proliferation. Furthermore, we found that subcutaneous CV1574-1 showed significantly reduced hypoglycemic effect compared to injection of IGF-1 itself. Lastly, to gain mechanistic insights into the role of matrilin-3 targeting, we assessed the ability of CV1574-1 to activate AKT signaling in vitro and found that CV1574-1 caused a prolonged increase in AKT signaling compared to IGF-1 and that this effect was dependent on matrilin-3. Taken together, our findings provide further evidence that cartilage-targeted therapy could provide new pharmacological approaches for the treatment of childhood growth disorders, such as GH-insensitivity syndrome.

Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5 % uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500-1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100-200 nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined. STATEMENT OF SIGNIFICANCE: We do not fully understand the process by which epiphyseal cartilage mineralizes - a vital step in endochondral bone formation. Previous work has proposed that mitochondria and intracellular vesicles are storage sites for the delivery of mineral to collagen fibrils. However, these concepts are founded on results from in vitro models of mineralization; no prior work has observed mineral-containing intracellular vesicles or mitochondria in developing epiphyseal cartilage. Here we developed a new cryofixation preparation route for transmission electron microscopy (TEM) imaging that has disclosed a cell-regulated process of mineralization in epiphyseal cartilage. High resolution TEM images revealed an involvement of mitochondria and intracellular and extracellular vesicles in delivering transient mineral phases to the collagen fibrils to promote cartilage mineralization.

The growth plate is the primary site of longitudinal bone growth with chondrocytes playing a pivotal role in endochondral bone development. Chondrocytes undergo a series of differentiation steps, resulting in the formation of a unique hierarchical columnar structure comprising round, proliferating, pre-hypertrophic, and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic stages, are a critical cell population in the growth plate. However, the molecular basis of pre-hypertrophic chondrocytes remains largely undefined. Here, we employed scRNA-seq analysis on fluorescently labeled growth plate chondrocytes for their molecular characterization. Serine incorporator 5 (Serinc5) was identified as a marker gene for pre-hypertrophic chondrocytes. Histological analysis revealed that Serinc5 is specifically expressed in pre-hypertrophic chondrocytes, overlapping with Indian hedgehog (Ihh). Serinc5 represses cell proliferation and Col2a1 and Acan expression by inhibiting the transcriptional activity of Sox9 in primary chondrocytes. Chromatin profiling using ChIP-seq and ATAC-seq revealed an active enhancer of Serinc5 located in intron 1, with its chromatin status progressively activated during chondrocyte differentiation. Collectively, our findings suggest that Serinc5 regulates sequential chondrocyte differentiation from proliferation to hypertrophy by inhibiting Sox9 function in pre-hypertrophic chondrocytes, providing novel insights into the mechanisms underlying chondrocyte differentiation in growth plates.

In most cases of bone metabolic disorders, such as osteoporosis and osteomalacia, these conditions are often attributed to dysfunctional osteoclasts, leading to their common characterization as "destructors." In addition to the widely documented regulatory process where osteoblasts direct osteoclastic bone resorption, there is increasing evidence suggesting that osteoclasts also in turn influence osteoblastic bone formation through direct and indirect mechanisms. It is well-known that differentiation of osteoclasts involves several stages, each characterized by specific cellular features and functions. Stage-specific key molecules secreted during these stages play a critical role in mediating osteoclast-osteoblast communication. In this review, we described the different stages of osteoclast differentiation and reviewed stage-specific key molecules involved in osteoclasts-osteoblasts communication. We highlighted that a detailed understanding of these processes and molecular mechanism could facilitate the development of novel treatments for bone metabolic disorders.

Bone homeostasis relies on several contributing factors, encompassing growth factors and mechanical stimuli. While bone morphogenetic protein (BMP) signaling is acknowledged for its essential role in skeletal development, its specific impact on mandibular morphogenesis remains unexplored. Here, we investigated the involvement of BMP signaling and mechanical loading through mastication in postnatal mandibular morphogenesis.

We employed conditional deletion of Bmpr1a in osteoblasts and chondrocytes via Osterix-Cre. Cre activity was induced at birth for the 3-week group and at three weeks for the 9-week and 12-week groups, respectively. The conditional knockout (cKO) and control mice were given either a regular diet (hard diet, HD) or a powdered diet (soft diet, SD) from 3 weeks until sample collection, followed by micro-CT and histological analysis.

The cKO mice exhibited shorter anterior lengths and a posteriorly inclined ramus across all age groups compared to the control mice. The cKO mice displayed an enlarged hypertrophic cartilage area along with fewer osteoclast numbers in the subchondral bone of the condyle compared to the control group at three weeks, followed by a reduction in the cartilage area in the posterior region at twelve weeks. Superimposed imaging and histomorphometrical analysis of the condyle revealed that BMP signaling primarily affects the posterior part of the condyle, while mastication affects the anterior part.

Using 3D landmark-based geometric morphometrics and histological assessments of the mandible, we demonstrated that BMP signaling and mechanical loading reciprocally contribute to the morphological alterations of the mandible and condyle during postnatal development.

The central role of the endothelial microenvironment in orchestrating bone marrow (BM) homeostasis and hematopoietic support has been confirmed at various developmental stages and in adult life. The BM vasculature is crucial in mediating communication between BM parenchyma and circulating blood, displaying remarkable heterogeneity in structure and function. While vascular cell diversity in other tissues has long been recognized, the molecular basis of this phenomenon in BM is just now emerging. Over the past decade, single-cell approaches and microscopic observations have expanded our understanding of BM vasculature. While solely characterized for their paracrine properties in the past, recent advances have revolutionized our perception of endothelial function, revealing distinct anatomical locations associated with diverse endothelial cell states. The identification of phenotypic differences between normal and pathological conditions has therefore deepened our understanding of vascular dynamics and their impact on hematopoiesis in health and disease. In this review, we highlight key milestones and recent advances in understanding vascular heterogeneity within BM microenvironment during development, adulthood and aging. We also explore how leukemia affects this heterogeneity and how we can take this knowledge forward to improve clinical practices. By synthesizing existing literature, we aim to address unresolved questions and outline future research directions.

The primary cilium is a cellular antenna to orchestrate cell growth and differentiation. Deficient or dysfunctional cilia are frequently linked to skeletal abnormalities. Previous research demonstrated that ciliary proteins regulating axoneme elongation are essential for skeletogenesis. However, the role of the ciliary proteins responsible for initiating cilium assembly in skeletal development remains unknown. Here, we investigate the function of centrosomal protein of 164 kDa (CEP164), a key ciliogenesis regulator that localizes at the distal appendages of the mother centriole, during skeletal development in mice. Interestingly, the mesodermal cell-specific Cep164 deletion resulted in severe bone defects and osteoblast-specific deletion of Cep164 affected bone development. In contrast, chondrocyte-specific Cep164 deletion did not cause overt skeletal abnormalities, indicating that CEP164 functions in a cell type-specific manner within skeletal tissues. Importantly, Cep164-mutant osteoblasts not only displayed a lack of cilia but also showed an increased number of γH2AX-positive cells, indicating the involvement of defective DNA damage response in the etiology of skeletal lesions of Cep164-mutant mice. These results demonstrate that CEP164 has both ciliary and non-ciliary functions to control osteoblast growth and survival. Our study therefore reveals a novel understanding of the pathogenesis of skeletal ciliopathies associated with CEP164 dysfunction.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in children's daily necessities and foods, and their health hazards to children have attracted particular attention. Childhood is a critical time for accelerated bone growth and development. Current studies revealed that TiO2 NPs exposure causes bone damage in juvenile rats; however, the underlying mechanism is unknown. Ghrelin is a polypeptide hormone that is considered to be a candidate factor for regulating bone growth and development. In this research, 3-week-old juvenile male rats were administered 0, 100 or 200 mg/kg TiO2 NPs and 50 μg/kg ghrelin for 4 weeks to explore the underlying mechanism of TiO2 NPs-induced bone damage, and the protective effect of ghrelin. Our results revealed that TiO2 NPs resulted in decreased synthesis of bone growth-related hormones, disturbed bone metabolism, and destruction of bone structure. Further mechanism studies showed that TiO2 NPs inhibited Wnt/β-catenin pathway, reduced collagen synthesis, inhibited chondrocyte proliferation and differentiation, promoted chondrocyte apoptosis, and inhibited endochondral osteogenesis, ultimately leading to long bone longitudinal growth retardation and osteoporosis. Ghrelin alleviated the negative effects of TiO2 NPs-induced bone growth in juvenile rats by upregulating the Wnt/β-catenin signaling pathway. This study provided a reference for the clinical treatment of growth retardation and idiopathic short stature in juvenile children caused by environmental pollutants.

Tibial dyschondroplasia (TD) is a kind of metabolic bone disease in fast-growing broilers, which seriously restricts the development of poultry industry. Our previous studies have revealed a significant upregulation of miR-205a in TD cartilage tissue, suggesting its potential role as a regulatory factor in the pathogenesis of TD. However, the precise function implications and underlying regulatory mechanism remain elusive. Therefore, this study aims to elucidate the biological functions and regulatory mechanisms of miR-205a in the progression of TD by employing mehtodologies such as qRT-PCR, CCK-8 assay, EdU assays, and flow cytometry. The findings demonstrated that the transfection of miR-205a overexpression plasmid reduced chondrocytes growth and development in TD while enhancing apoptosis; conversely, blocking miR-205a had opposite effects. RUNX2 was identified as a target gene of miR-205a through biosynthesis and dual luciferase assays, and its overexpression helps chondrocytes in TD grow and develop. However, when both miR-205a and RUNX2 were overexpressed, the regulatory effect of RUNX2 was significantly suppressed. In conclusion, miR-205a plays a role in slowing the growth and development of chondrocytes in TD by targeting and reducing RUNX2 expression, which helps to initiate and progress TD.

Interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signalling pathways play important roles in the complex process of bone formation and bone remodelling. However, whether IL-4/IL-4Rα participates in skeletogenesis during embryonic development is not completely understood. We used the anti-IL-4Rα monoclonal antibody (anti-IL-4Rα mAb) as a powerful investigational tool to evaluate the potential roles of IL-4/IL-4Rα in the chondrogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Simultaneously, we explored the effect of IL-4/IL-4Rα on bone ossification during rat embryo-fetal development. In this study, we found that, compared to the control group, IL-4 can significantly promote the chondrogenic differentiation of BMSCs. Furthermore, following exposure to anti-IL-4Rα mAb in pregnant rats, unexpected phenomena were observed in fetal bone development, including non-ossification of the fetal sternum, an incomplete ossification centre in long bones and a reduced number of ossification points in digit (toe) bones. To further investigate the underlying mechanism of the phenotype, we studied the rat sternum as the target organ, starting from different time points of sternum development in the embryonic stage. The results indicated that the retardation mainly occurred in the middle and late stages of embryonic development. This retardation was characterized by the inhibition of the differentiation process of mesenchymal stem cells into chondrocytes, resulting in reduced angiogenesis near the ossification centre, failure of osteoblasts to invade the centre of the cartilage body with the blood vessels and delayed formation of the primary ossification centre (POC). Overall, our study demonstrated the significant function of IL-4/IL-4Rα in chondrogenic differentiation of BMSCs and bone ossification during embryo-fetal development.

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2-/- mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2-/- (double knockout) mice resembled Phlpp1-/- mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1-/- and Phlpp1/2-/- chondrocytes had similarities and were significantly different from WT and Phlpp2-/- chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.