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Kronenberg, H.M.: Developmental regulation of the growth plate. Nature 423, 332-336
Abstract
Vertebrates do not look like jellyfish because the bones of their skeletons are levers that allow movement and protect vital organs. Bones come in an enormous variety of shapes and sizes to accomplish these goals, but, with few exceptions, use one process--endochondral bone formation--to generate the skeleton. The past few years have seen an enormous increase in understanding of the signalling pathways and the transcription factors that control endochondral bone development.
... The resting zone contains slowly cycling cells, which, upon recruitment into the underlying proliferative zone, begin proliferating rapidly and arrange themselves into longitudinal columns of flat chondrocytes. Thereafter, these flat chondrocytes undergo further differentiation along with hypertrophy, forming the hypertrophic zone (6). Thereafter, the hypertrophic chondrocytes die or transdifferentiate (7), leaving a cartilaginous template on which spongy bone is built. ...
... JCI Insight 2024;9(6):e165226 https://doi.org/10.1172/jci.insight.165226 that controls the rate of chondrocyte differentiation (6). More specifically, Ihh produced by prehypertrophic and hypertrophic chondrocytes diffuses to the resting zone, where it stimulates expression of PTHrP, which in turn inhibits the hypertrophic differentiation of chondrocytes (6). ...
... that controls the rate of chondrocyte differentiation (6). More specifically, Ihh produced by prehypertrophic and hypertrophic chondrocytes diffuses to the resting zone, where it stimulates expression of PTHrP, which in turn inhibits the hypertrophic differentiation of chondrocytes (6). ...
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.
... The long bone originates from mesoderm-derived mesenchymal tissues, transiting a series of primary stages of chondrogenesis, chondrocyte hypertrophy, and osteogenesis, ultimately developing into a delicate and complex assembly of highly mineralized and vascularized solid connective tissues that construct the axial skeleton of the human body. This developmental process is also known as endochondral ossification [4,5]. In the middle of this process, a subpopulation of chondrocytes stops proliferation and undergoes hypertrophy; the hypertrophic chondrocytes (HCCs) play a central role in signaling for initiation of osteogenesis and induction of vascularization in the bulk of the preestablished cartilage template. ...
... The induction of vascularization by HCCs is executed via the expression of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), that mediate vascular intrusion through interactions with specific HCC receptors, such as neuropilins, and endothelial cells (EC) receptors, such as FLk-1 [17]. Following the invasion of ECs, infiltration of capillaries extends into the perichondrium and hypertrophic regions, from which abundant minerals and osteoclasts (OCs) are introduced [4]. Metallomatrix proteinases-13 (MMP13) are also expressed by HCCs to degrade the residues of the transient cartilage template, further facilitating osteogenic remodeling and vascularization [4,18]. ...
... Following the invasion of ECs, infiltration of capillaries extends into the perichondrium and hypertrophic regions, from which abundant minerals and osteoclasts (OCs) are introduced [4]. Metallomatrix proteinases-13 (MMP13) are also expressed by HCCs to degrade the residues of the transient cartilage template, further facilitating osteogenic remodeling and vascularization [4,18]. During this osteogenic process, the general ECM composition is dramatically altered, which is marked by upregulation of type X collagen and downregulation of the cartilage-specific proteoglycans and collagen types, namely type II and type IX collagen. ...
Endochondral ossification (ECO) is a form of bone formation whereby the newly deposited bone replaces the cartilage template. A decellularized artificial cartilage graft (dLhCG), which is composed of hyaline cartilage matrixes, has been developed in our previous study. Herein, the osteogenesis of bone marrow-derived MSCs in the dLhCG through chondrogenic differentiation, chondrocyte hypertrophy, and subsequent transdifferentiation induction has been investigated by simulating the physiological processes of ECO for repairing critical-sized bone defects. The MSCs were recellularized into dLhCGs and subsequently allowed to undergo a 14-day proliferation period (mrLhCG). Following this, the mrLhCG constructs were subjected to two distinct differentiation induction protocols to achieve osteogenic differentiation: chondrogenic medium followed by chondrocytes culture medium with a high concentration of fetal bovine serum (CGCC group) and canonical osteogenesis inducing medium (OI group). The formation of a newly developed artificial bone graft, ossified dLhCG (OsLhCG), as well as its capability of aiding bone defect reconstruction were characterized by in vitro and in vivo trials, such as mRNA sequencing, quantitative real-time PCR (qPCR), immunohistochemistry, the greater omentum implantation in nude mice, and repair for the critical-sized femoral defects in rats. The results reveal that the differentiation induction of MSCs in the CGCC group can realize in vitro ECO through chondrogenic differentiation, hypertrophy, and transdifferentiation, while the MSCs in the OI group, as expected, realize ossification through direct osteogenic differentiation. The angiogenesis and osteogenesis of OsLhCG were proved by being implanted into the greater omentum of nude mice. Besides, the OsLhCG exhibits the capability to achieve the repair of critical-size femoral defects.
... The peripheral mesenchymal cells of the condensations form the perichondrium, a precursor to the periosteum. Cells from different tissues, including cartilage, the perichondrium, and the vascular endothelium, participate in this spatiotemporal event through a complicated regulation [11]. Notably, lineage analyses have shown that the perichondrium is the source of all osteoblasts, including not only cortical osteoblasts but also trabecular osteoblasts, and regulates the initiation of vascular invasion [9]. ...
... PTHrP resides in the fibrous layer of periosteum and its receptor (PTHR1) is in the subjacent cambial layer [74]. Together, Ihh and PTHrP control the rate at which chondrocytes proliferate and differentiate, thereby driving linear bone growth through a feedback loop [11]. Ptpn11 deletion in Ctsk+ PDPCs upregulated Ihh/PTHrP signaling, leading to excessive proliferation and chondrocyte differentiation, finally causing chondroid neoplasm metachondromatosis [22]. ...
The periosteum is known as the thin connective tissue covering most bone surfaces. Its extrusive bone regeneration capacity was confirmed from the very first century-old studies. Recently, pluripotent stem cells in the periosteum with unique physiological properties were unveiled. Existing in dynamic contexts and regulated by complex molecular networks, periosteal stem cells emerge as having strong capabilities of proliferation and multipotential differentiation. Through continuous exploration of studies, we are now starting to acquire more insight into the great potential of the periosteum in bone formation and repair in situ or ectopically. It is undeniable that the periosteum is developing further into a more promising strategy to be harnessed in bone tissue regeneration. Here, we summarized the development and structure of the periosteum, cell markers, and the biological features of periosteal stem cells. Then, we reviewed their pivotal role in bone repair and the underlying molecular regulation. The understanding of periosteum-related cellular and molecular content will help enhance future research efforts and application transformation of the periosteum.
... hBM-MSC-seeded scaffolds cultured in BM under normoxia for 21 days showed that the SOX9 gene expression returned to the baseline, with low expression of COL2A1, and high expression of ACAN. These results may suggest that chondrocytes are turning hypertrophic as it is known that the SOX9 gene is turned off in hypertrophic chondrocytes, while low levels of Col2A1 are still expressed, and the ACAN expression, which is switched on during chondrogenic differentiation, continues during hypertrophy [72]. However, we show that RUNX2 [27,28], COL10A1 [73] and MMP13 [74,75], which are typical markers of matrix-mineralizing terminal hypertrophic chondrocytes [72], were expressed at similar levels as in undifferentiated/naïve control, which implies cells are not hypertrophic. ...
... These results may suggest that chondrocytes are turning hypertrophic as it is known that the SOX9 gene is turned off in hypertrophic chondrocytes, while low levels of Col2A1 are still expressed, and the ACAN expression, which is switched on during chondrogenic differentiation, continues during hypertrophy [72]. However, we show that RUNX2 [27,28], COL10A1 [73] and MMP13 [74,75], which are typical markers of matrix-mineralizing terminal hypertrophic chondrocytes [72], were expressed at similar levels as in undifferentiated/naïve control, which implies cells are not hypertrophic. ...
Biomaterials that can improve the healing of articular cartilage lesions are needed. To address this unmet need, we developed novel 3D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) hybrid scaffolds. Our aim was to carry out essential studies to advance this medical device towards functional validation in pre-clinical trials. First, we show that the chemical composition, microarchitecture and mechanical properties of these scaffolds were not affected by sterilisation with gamma irradiation. To evaluate the systemic and local immunogenic reactivity of the sterilised 3D printed hybrid scaffolds, they were implanted subcutaneously into Balb/c mice. The scaffolds did not trigger a systemic inflammatory response over one week of implantation. The interaction between the host immune system and the implanted scaffold elicited a local physiological reaction with infiltration of mononuclear cells without any signs of a chronic inflammatory response.
Then, we investigated how these 3D printed hybrid scaffolds direct chondrogenesis in vitro. Human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs) seeded within the 3D printed hybrid scaffolds were cultured under normoxic or hypoxic conditions, with or without chondrogenic supplements. Chondrogenic differentiation assessed by both gene expression and protein production analyses showed that 3D printed hybrid scaffolds support hBM-MSC chondrogenesis. Articular cartilage-specific extracellular matrix deposition within these scaffolds was enhanced under hypoxic conditions (1.7 or 3.7 fold increase in the median of aggrecan production in basal or chondrogenic differentiation media).
Our findings show that 3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffolds have the potential to support the regeneration of cartilage tissue.
... Ihh released by prehypertrophic cells stimulates nearby chondrocytes in the proliferating layer to undergo mitosis through PTHrP-Ihh negative feedback regulation, controlling the rate of hypertrophy and, so, the growth rate [133,134]. PTHrP, released by perichondrial cells and resting chondrocytes, binds to receptors expressed by proliferating chondrocytes and inhibits the hypertrophic terminal differentiation, sustaining the proliferative pool of chondrocytes [135]. As the source of PTHrP becomes distant, chondrocytes produce Ihh (prehypertrophic zone), stimulating its proliferation rate and hypertrophy and the differentiation of perichondrial cells into osteoblasts [135]. ...
... PTHrP, released by perichondrial cells and resting chondrocytes, binds to receptors expressed by proliferating chondrocytes and inhibits the hypertrophic terminal differentiation, sustaining the proliferative pool of chondrocytes [135]. As the source of PTHrP becomes distant, chondrocytes produce Ihh (prehypertrophic zone), stimulating its proliferation rate and hypertrophy and the differentiation of perichondrial cells into osteoblasts [135]. So, the crosstalk between PTHrP and Ihh paracrine signaling maintains long-term growth by balancing chondrocyte production and endochondral ossification [136]. ...
... Cartilage is crucial for bone and marrow development. Long bones and the vertebrate skeleton are formed by a multistep process called endochondral ossification (4). During endochondral ossification, MSCs condense to form a dense mass and undergo chondrogenesis, generating a primitive avascular cartilaginous anlage. ...
... In the process of ossification center formation, cartilage-specific ECMs are progressively degraded, generating a vascularized marrow space (3). In the long bone, there are different temporal-spatial mechanisms of endochondral ossification: an embryonal primary ossification center (POC) in the diaphysis and a postnatal secondary ossification center (SOC) in the epiphysis (4,(6)(7)(8). ...
Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9 , COL2A1 , and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes ( IHH , PTH1R, and COL10A1 ) and ossification ( SP7 , ALPL , and MMP13 ). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.
Highlights
Basement membrane extract (BME) enlarges MSC-based Cart-Org.
BME activates the SMAD pathway and inhibits the NK-kB pathway of the Cart-Org.
BME promotes cartilaginous maturation and further ossification of Cart-Org.
... The proliferation rate of cartilaginous cells in the growth plate is directly correlated with the longitudinal growth rate of the bone. Endochondral ossification begins with the condensation of loosely congregated, fibroblast-like mesenchymal stem cells (MSCs) [35,36]. Before developing into chondrocytes, this condensation process involves cell-to-cell contact, resulting in areas of high cell density. ...
... These cartilage templates are transformed into bone by endochondral ossification. As endochondral ossification progresses, chondrocytes gradually grow larger [35,[37][38][39], characterized by the synthesis of type X collagen and volumetric swelling. These hypertrophic chondrocytes stimulate the bone collar's development by enhancing the mineralization of the surrounding ECM, releasing vascular endothelial growth factor to direct blood vessels into adjacent ECM, and inducing mineralization of the surrounding ECM. ...
Simple Summary
Tibial dyschondroplasia (TD) is an avian metabolic disease characterized by rapid apoptosis and reduced angiogenesis with compromised chondrocyte activity at the growth plate and, ultimately, poor osteogenesis. Thiram, a widely recognized fungicide used to preserve fruits and grains, is considered among one of the potential predisposing factors of TD through sonic hedgehog, notch-gridlock, and ephrin-B2/EphB4 pathways of angiogenesis. Current literature provides a comprehensive overview regarding the involvement of pro and anti-angiogenic proteins in the mechanism of TD development.
Abstract
Precise coupling of two fundamental mechanisms, chondrogenesis and osteogenesis via angiogenesis, plays a crucial role during rapid proliferation of growth plates, and alteration in their balance might lead to pathogenic conditions. Tibial dyschondroplasia (TD) is characterized by an avascular, non-mineralized, jade-white “cartilaginous wedge” with impaired endochondral ossification and chondrocyte proliferation at the proximal end of a tibial bone in rapidly growing poultry birds. Developing vascular structures are dynamic with cartilage growth and are regulated through homeostatic balance among pro and anti-angiogenic proteins and cytokines. Pro-angiogenic factors involves a wide spectrum of multifactorial mitogens, such as vascular endothelial growth factors (VEGF), platelet-derived growth factors (PDGF), basic fibroblast growth factor (bFGF), placental growth factors, transforming growth factor-β (TGF-β), and TNF-α. Considering their regulatory role via the sonic hedgehog, notch-gridlock, and ephrin-B2/EphB4 pathways and inhibition through anti-angiogenic proteins like angiostatin, endostatin, decoy receptors, vasoinhibin, thrombospondin, PEX, and troponin, their possible role in persisting inflammatory conditions like TD was studied in the current literature review. Balanced apoptosis and angiogenesis are vital for physiological bone growth. Any homeostatic imbalance among apoptotic, angiogenetic, pro-angiogenic, or anti-angiogenic proteins ultimately leads to pathological bone conditions like TD and osteoarthritis. The current review might substantiate solid grounds for developing innovative therapeutics for diseases governed by the disproportion of angiogenesis and anti-angiogenesis proteins.
... To test the role of cilia regulated HH-pathway repression in morphogenesis, we also utilized skeletal development in fore limb long bones and calvarium as paradigms for studying derepression phenotypes. Skeletal morphogenesis occurs by endochondral or intramembranous ossification, based on dependance or lack of dependance on an intermediate cartilaginous template, respectively [33,34]. During endochondral ossification, periarticular chondrocytes differentiate into columnar, prehypertrophic and hypertrophic chondrocytes. ...
... During endochondral ossification, periarticular chondrocytes differentiate into columnar, prehypertrophic and hypertrophic chondrocytes. IHH is secreted from prehypertrophic chondrocytes, and activates chondrocyte proliferation, differentiation of proliferating to hypertrophic chondrocytes and osteoblast differentiation in the perichondrium [33]. The chondrocytes are ciliated at all these stages [19,35]. ...
A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization—especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes—is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened , the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.
... Long bone growth occurs at specific locations known as growth plates, where chondrocytes undergo a well-regulated process of proliferation and differentiation to form cartilage, which is then replaced by bone via apoptosis of terminally differentiated hypertrophic chondrocytes, and invasion of blood vessels to provide bone cells for bone formation [1,2]. This endochondral bone growth process also occurs in the skull bones of the cranial base, upon which the brain sits. ...
... Hypophosphatasia is a rare inherited disorder caused by inactivating mutations in the gene (Alpl) that encodes the enzyme tissue non-specific alkaline phosphatase (TNAP). One primary and 2 well-established function of TNAP is to hydrolyze inorganic pyrophosphate (PPi) into inorganic phosphate (Pi) [5]. Because PPi inhibits matrix mineralization while Pi serves as a substrate for hydroxyapatite formation, TNAP activity promotes bone mineralization [6]. ...
Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to deficiency of Tissue Nonspecific Alkaline Phosphatase (TNAP, Alpl) enzyme activity. Abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global TNAP-/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout TNAP only in cranial neural crest derived tissues using TNAP flox mice. Here we show that TNAP deficiency in cranial neural crest leads to leads to skull shape defects and deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression is increased, indicative of premature differentiation in the absence of TNAP. Sox9 expression is increased in both resting and prehypertrophic zones of mutant mice. Expression of PTHrP and IHH were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that TNAP expression in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.
... The growth plate, a cartilage structure located between the metaphysis and epiphysis of long bones, is one of the most important factors influencing height linear growth in children and adolescents [10]. However, as the most widely used method to evaluate bone maturity in clinical practice, X-raybased assessment of BA cannot directly show the cartilage condition [11,12]. ...
... The association was assessed using multiple linear regressions. Values represented as mean (95% Confidence interval) GV 1-3 mom growth velocity in the first-to-third months after rhGH therapy, GV 4-6 mom growth velocity in the fourth-to-sixth months after rhGH therapy, UCGP unclosed growth plate, MCGP marginally closed growth plate, NCGP nearly closed growth plate, MRI magnetic resonance imaging, SDS standard deviation score, rhGH recombinant human growth hormone, AI aromatase inhibitor, factors affecting linear growth in children and adolescents [10]. Growth hormone can increase chondrogenesis in the growth plate by increasing the local production of IGF-1 to promote linear growth [1,39]. ...
There is no appropriate tool to predict recombinant human growth hormone (rhGH) response before therapy initiation in short-stature children in late puberty. The current study aimed to explore the associations between magnetic resonance imaging (MRI) stages of the knee growth plates and rhGH response in short-stature children in late puberty.
In this prospective cohort study, short-stature children in late puberty were treated with rhGH and followed up for 6 months. We proposed a novel knee MRI staging system according to the growth plate states of distal femurs or proximal tibias and divided the participants into three groups: unclosed growth plate group, marginally closed growth plate group, and nearly closed growth plate group. The primary outcomes were height gain and growth velocity (GV), which were assessed three months later.
Fifty participants were enrolled, including 23 boys and 27 girls. GV and height gain after 6 months of rhGH therapy decreased successively in the three groups with an increased degree of growth plate fusion, especially when grouped by proximal tibias (GV1-3 mon from 9.38 to 6.08 to 4.56 cm/year, GV4-6 mon from 6.75 to 4.92 to 3.25 cm/year, and height gain from 4.03 to 2.75 to 1.95 cm, all P < 0.001). Moreover, the MRI stages of growth plates independently served as a significant variable for GV and height gain after therapy, especially when grouped by proximal tibias (all P < 0.01).
The MRI staging method is expected to be an effective tool for predicting rhGH response before therapy initiation in short-stature children in late puberty.
... Mammalian bone tissue is formed through two distinct processes: intramembranous and endochondral bone formation [1]. Endochondral bone formation is the main process which forms most mammalian bones, including the skull, longitudinal and appendage bones [1][2][3]. The differentiation and maturation of chondrocytes play a key role in the process of endochondral bone formation, in which chondro-cytes promote longitudinal growth through proliferation, extracellular matrix secretion, and hypertrophy [4]. ...
... Our transcriptomic analysis showed that Wnt/β-catenin, Hedgehog and Notch4 signaling pathways, which are known to play key roles in cartilage development and regulate the function of chondrocytes, 37,38 were upregulated in dexamethasone + LiCl group as compared to the dexamethasone alone group. We have previously shown that glucocorticoids trigger apoptosis in chondrocytes, 5 leading to severe growth retardation both in ex vivo and in vivo models. ...
Background
Glucocorticoids are commonly used in children with different chronic diseases. Growth failure represents a so far untreatable undesired side-effect. As lithium chloride (LiCl) is known to induce cell renewal in various tissues, we hypothesized that LiCl may prevent glucocorticoid-induced growth failure.
Methods
We monitored growth of fetal rat metatarsals cultured ex-vivo with dexamethasone and/or LiCl, while molecular mechanisms were explored through RNA sequencing by implementing the differential gene expression and gene set analysis. Quantification of β-catenin in human growth plate cartilage cultured with dexamethasone and/or LiCl was added for verification.
Results
After 14 days of culture, the length of dexamethasone-treated fetal rat metatarsals increased by 1.4 ± 0.2 mm compared to 2.4 ± 0.3 mm in control bones ( p < 0.001). The combination of LiCl and dexamethasone led to bone length increase of 1.9 ± 0.3 mm ( p < 0.001 vs. dexamethasone alone). By adding lithium, genes for cell cycle and Wnt/β-catenin, Hedgehog and Notch signaling, were upregulated compared to dexamethasone alone group.
Conclusions
LiCl has the potential to partially rescue from dexamethasone-induced bone growth impairment in an ex vivo model. Transcriptomics identified cell renewal and proliferation as candidates for the underlying mechanisms. Our observations may open up the development of a new treatment strategy for bone growth disorders.
Impact
LiCl is capable to prevent glucocorticoid-induced growth failure in rat metatarsals in vitro.
The accompanying drug-induced transcriptomic changes suggested cell renewal and proliferation as candidate underlying mechanisms.
Wnt/beta-catenin pathway could be one of those novel mechanisms.
... The epiphyseal plate is a characteristic feature of bones in children. As the epiphyseal plate is mainly composed of cartilage [37], it is easily damaged. Injury to the epiphyseal plate may result in growth arrest or deformity [38]. ...
Background
Fractures of hands and feet are common in children, but relevant epidemiological studies are currently lacking. We aim to study the epidemiological characteristics of hand and foot fractures and growth plate injuries in children and provide a theoretical basis for their prevention, diagnosis, and treatment.
Methods
We retrospectively analyzed the data of children with hand and foot fractures who were hospitalized at Shenzhen Children’s Hospital between July 2015 and December 2020. Data on demographic characteristics, fracture site, treatment method, etiology of injury, and accompanying injuries were collected. The children were divided into four age groups: infants, preschool children, school children, and adolescents. The fracture sites were classified as first-level (the first–fifth finger/toe, metacarpal, metatarsal, carpal, and tarsal) and second-level (the first–fifth: proximal phalanx, middle phalanx, distal phalanx, metacarpal, and metatarsal) sites. The changing trends in fracture locations and injury causes among children in each age group were analyzed.
Results
Overall, 1301 children (1561 fractures; 835 boys and 466 girls) were included. The largest number of fractures occurred in preschool children (n = 549, 42.20%), with the distal phalanx of the third finger being the most common site (n = 73, 15.57%). The number of fractures in adolescents was the lowest (n = 158, 12.14%), and the most common fracture site was the proximal phalanx of the fifth finger (n = 45, 29.61%). Of the 1561 fractures, 1143 occurred in the hands and 418 in the feet. The most and least common first-level fracture sites among hand fractures were the fifth (n = 300, 26.25%) and first (n = 138, 12.07%) fingers, respectively. The most and least common first-level foot fracture locations were the first (n = 83, 19.86%) and fourth (n = 26, 6.22%) toes, respectively. The most common first-level and second level etiologies were life related injuries (n = 1128, 86.70%) and clipping injuries (n = 428, 32.90%), respectively. The incidence of sports injuries gradually increased with age, accounting for the highest proportion in adolescents (26.58%). Hand and foot fractures had many accompanying injuries, with the top three being nail bed injuries (570 cases, 36.52%), growth plate injuries (296 cases, 18.96%), and distal severed fracture (167 cases, 10.70%). Among the 296 growth plate injuries, 246 occurred on the hands and 50 on the feet.
Conclusions
In contrast to previous epidemiological studies on pediatric hand and foot fractures, we mapped the locations of these fractures, including proximal, shaft, distal, and epiphyseal plate injuries. We analyzed the changing trends in fracture sites and injury etiologies with age. Hand and foot fractures have many accompanying injuries that require attention during diagnosis and treatment. Doctors should formulate accident protection measures for children of different ages, strengthen safety education, and reduce the occurrence of accidental injuries.
... The timely cartilage replacement with bone and BM at sites of endochondral ossification is required for normal skeletal development, growth, and shaping. Essential steps in the substitution process include apoptosis of hypertrophic chondrocytes, resorption of mineralized ECM, and homing of blood vesselassociated progenitor cells producing bone, marrow stroma, and marrow adipose tissue (16,21). Accordingly, available studies on the mechanisms underlying the abnormal retention of cartilage observed in MPS IH endochondrally formed bone (12)(13)(14)(22)(23)(24) are mainly focused on the effects of GAG accumulation on this sequence of events (10,14,25). ...
Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet-based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.
... The epiphyseal plate is a characteristic feature of bones in children. As the epiphyseal plate is mainly composed of cartilage [35], it is easily damaged. Injury to the epiphyseal plate may result in growth arrest or deformity [36]. ...
Background
Fractures of hands and feet are common in children, but relevant epidemiological studies are currently lacking. We aim to study the epidemiological characteristics of hand and foot fractures and growth plate injuries in children and provide a theoretical basis for their prevention, diagnosis, and treatment.
Methods
We retrospectively analyzed the data of children with hand and foot fractures who were hospitalized at Shenzhen Children's Hospital between July 2015 and December 2020. Data on demographic characteristics, fracture site, treatment method, etiology of injury, and accompanying injuries were collected. The children were divided into four age groups: infants, preschool children, school children, and adolescents. The fracture sites were classified as first-level (the first–fifth finger/toe, metacarpal, metatarsal, carpal, and tarsal) and second-level (the first–fifth: proximal phalanx, middle phalanx, distal phalanx, metacarpal, and metatarsal) sites. The changing trends in fracture locations and injury causes among children in each age group were analyzed.
Results
Overall, 1301 children (1561 fractures; 835 boys and 466 girls) were included. The largest number of fractures occurred in preschool children (n = 549, 42.20%), with the distal phalanx of the third finger being the most common site (n = 73, 15.57%). The number of fractures in adolescents was the lowest (n = 158, 12.14%), and the most common fracture site was the proximal phalanx of the fifth finger (n = 45, 29.61%). Of the 1561 fractures, 1143 occurred in the hands and 418 in the feet. The most and least common first-level fracture sites among hand fractures were the fifth (n = 300, 26.25%) and first (n = 138, 12.07%) fingers, respectively. The most and least common first-level foot fracture locations were the first (n = 83, 19.86%) and fourth (n = 26, 6.22%) toes, respectively. The most common first-level and second level etiologies were life related injuries (n = 1128, 86.70%) and clipping injuries (n = 428, 32.90%), respectively. The incidence of sports injuries gradually increased with age, accounting for the highest proportion in adolescents (26.58%). Hand and foot fractures had many accompanying injuries, with the top three being nail bed injuries (570 cases, 36.52%), growth plate injuries (296 cases, 18.96%), and distal severed fracture (167 cases, 10.70%). Among the 296 growth plate injuries, 246 occurred on the hands and 50 on the feet.
Conclusions
In contrast to previous epidemiological studies on pediatric hand and foot fractures, we mapped the locations of these fractures, including proximal, shaft, distal, and epiphyseal plate injuries. We analyzed the changing trends in fracture sites and injury etiologies with age. Hand and foot fractures have many accompanying injuries that require attention during diagnosis and treatment. Doctors should formulate accident protection measures for children of different ages, strengthen safety education, and reduce the occurrence of accidental injuries.
... The negative-feedback regulation of chondrocyte maturation also occurs naturally, often limited by the range of paracrine signaling (e.g., PTHrP/IHH feedback loop). [54][55][56] Free from the dependence on paracrine signaling, cisCXp-shRunx2 gene circuits allow chondrocytes to resist maturation based their internal tendency to undergo hypertrophy, measured by RUNX2 activity. As a gatekeeper, RUNX2 mediates the signaling of many molecular and biophysical cues in chondrocytes. ...
The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue‐engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp‐shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1‐like promoter (cisCXp). The cisCXp‐shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp‐shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp‐shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell‐derived (hMSC‐derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC‐derived chondrocytes in response to the microenvironment of the degenerative joint.
... The chondrocytes originate from the resting or germinal zone to proliferate where they start to be arranged in a columnar pattern (proliferate zone) until they increase in size (hypertrophic zone) and excrete extracellular matrix as part of osteogenesis. Both the chondrocytes in the proliferate and hypertrophic zones as well as the newly formed bone in the metaphysis have a symmetrical columnar pattern (2). The columnar pattern creates an anisotropic diffusion in the growth plate, especially in the zone of provisional calcification in the hypertrophic zone. ...
How tall will I be?” Every paediatrician has been asked this during their career.
The growth plate is the main site of longitudinal growth of the long bones. The chondrocytes in the growth plate have a columnar pattern detectable by diffusion tensor imaging (DTI). DTI shows the diffusion of water in a tissue and whether it is iso- or anisotropic. By detecting direction and magnitude of diffusion, DTI gives information about the microstructure of the tissue. DTI metrics include tract volume, length, and number, fractional anisotropy (FA)and mean diffusivity. DTI metrics, particularly tract volume, provide quantitative data regarding skeletal growth and, in conjunction with the fractional anisotropy, be used to determine whether a growth plate is normal. Tractography is a visual display of the diffusion, depicting its direction and amplitude. Tractography gives a more qualitative visualization of cellular orientation in a tissue and reflects the activity in the growth plate. These two components of DTI can be used to assess the growth plate without ionizing radiation or pain. Further refinements in DTI will improve prediction of post-imaging growth and growth plate closure, and assessment of the positive and negative effect of treatments like cis-retinoic acid and growth hormone administration.
... IACS administration markedly reduced HO formation without causing abnormalities in the heart, liver, kidney, and lung (Fig S8A and B). Physiological endochondral ossification, a crucial process for longitudinal growth, occurs in growth plates at the ends of the long tubular bones of growing limbs (Kronenberg, 2003). Disease onset in most FOP patients occurs when the growth plate is still open. ...
Heterotopic ossification (HO) is a non-physiological bone formation where soft tissue progenitor cells differentiate into chondrogenic cells. In fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by progressive and systemic HO, the Activin A/mutated ACVR1/mTORC1 cascade induces HO in progenitors in muscle tissues. The relevant biological processes aberrantly regulated by activated mTORC1 remain unclear, however. RNA-sequencing analyses revealed the enrichment of genes involved in oxidative phosphorylation (OXPHOS) during Activin A–induced chondrogenesis of mesenchymal stem cells derived from FOP patient–specific induced pluripotent stem cells. Functional analyses showed a metabolic transition from glycolysis to OXPHOS during chondrogenesis, along with increased mitochondrial biogenesis. mTORC1 inhibition by rapamycin suppressed OXPHOS, whereas OXPHOS inhibitor IACS-010759 inhibited cartilage matrix formation in vitro, indicating that OXPHOS is principally involved in mTORC1-induced chondrogenesis. Furthermore, IACS-010759 inhibited the muscle injury–induced enrichment of fibro/adipogenic progenitor genes and HO in transgenic mice carrying the mutated human ACVR1. These data indicated that OXPHOS is a critical downstream mediator of mTORC1 signaling in chondrogenesis and therefore is a potential FOP therapeutic target.
... In response to regional mechanical stresses and paracrine signaling cues, newly laid down bone is continuously remodeled by bone resorbing osteoclasts and bone-forming osteoblasts to shape the developing long bone. Later, linear growth takes place in the specialized epiphyseal growth plates located at the proximal and distal extremities of long bones using the same ordered mechanism (43)(44)(45). ...
... In vertebrate skeletal development, condensation of mesenchymal stem cells drives endochondral bone formation during limb bud growth. The cells of most condensations differentiated into chondrocytes, which sequentially undergo resting, proliferative, prehypotrophic, and hypertrophic stages before apoptosis (Chagin and Newton 2020;Kronenberg 2003;Olsen et al. 2000;Shimizu et al. 2007). Disarrangement in any stage would result in a failure of endochondral bone elongation causing skeletal dysplasia. ...
Ras homology enriched in the brain (Rheb) is well established as a critical regulator of cell proliferation and differentiation in response to growth factors and nutrients. However, the role of Rheb1 in limb development remains unknown. Here, we found that Rheb1 was dynamically expressed during the proliferation and differentiation of chondrocytes in the growth plate. Given that Prrx1⁺ limb-bud-like mesenchymal cells are the source of limb chondrocytes and are essential for endochondral ossification, we conditionally deleted Rheb1 using Prrx1-Cre and found a limb dwarfism in Prrx1-Cre; Rheb1fl/fl mice. Normalized to growth plate height, the conditional knockout (cKO) mice exhibited a significant decrease in column count of proliferative zones which was increased in hypertrophic zones resulting in decreased growth plate size, indicating abnormal endochondral ossification. Interestingly, although Rheb1 deletion profoundly inhibited the transcription factor Sox9 in limb cartilage; levels of runx2 and collagen type 2 were both increased. These novel findings highlight the essential role of Rheb1 in limb growth and indicate a complex regulation of Rheb1 in chondrocyte proliferation and differentiation.
... The extracellular matrix of chondrocytes serves as a scaffold for cellular structural organization, providing signals for proliferation and differentiation that are essential for normal endochondral ossification [30,47]. In this study, we identified significant alterations in amino acid and glucose metabolism, partly reflecting the responses of intracellular metabolites to porcine chondrogenic differentiation and maturation. ...
Porcine body length is closely related to meat production, growth, and reproductive performance, thus playing a key role in the profitability of the pork industry. Cartilage development is critical to longitudinal elongation of individual vertebrae. This study isolated primary porcine vertebral chondrocytes (PVCs) to clarify the complex mechanisms of elongation. We used transcriptome and target energy metabolome technologies to confirm crucial genes and metabolites in primary PVCs at different differentiation stages (0, 4, 8, and 12 days). Pairwise comparisons of the four stages identified 4566 differentially expressed genes (DEGs). Time-series gene cluster and functional analyses of these DEGs revealed four clusters related to metabolic processes, cartilage development, vascular development, and cell cycle regulation. We constructed a transcriptional regulatory network determining chondrocyte maturation. The network indicated that significantly enriched transcription factor (TF) families, including zf-C2H2, homeobox, TF_bZIP, and RHD, are important in cell cycle and differentiation processes. Further, dynamic network biomarker (DNB) analysis revealed that day 4 was the tipping point for chondrocyte development, consistent with morphological and metabolic changes. We found 24 DNB DEGs, including the TFs NFATC2 and SP7. Targeted energy metabolome analysis showed that most metabolites were elevated throughout chondrocyte development; notably, 16 differentially regulated metabolites (DRMs) were increased at three time points after cell differentiation. In conclusion, integrated metabolome and transcriptome analyses highlighted the importance of amino acid biosynthesis in chondrocyte development, with coordinated regulation of DEGs and DRMs promoting PVC differentiation via glucose oxidation. These findings reveal the regulatory mechanisms underlying PVC development and provide an important theoretical reference for improving pork production.
... 1,2 Osteogenesis, which is critical for the maintenance of a healthy skeletal system, is coupled to angiogenic growth of blood vessels in the developing skeletal system. 3 Bone is a highly vascularized tissue; the formation and maintenance of healthy bone is dependent on the bone vascular network throughout the lifespan. In contrast, declining bone blood flow to bone may be an important contributor to agerelated bone loss. ...
The magnitude of bone formation and remodeling is linked to both the magnitude of strain placed on the bone and the perfusion of bone. It was previously reported that an increase in bone perfusion and bone density occurs in the femur of old rats with moderate aerobic exercise training. This study determined the acute and chronic effects of static muscle stretching on bone blood flow and remodeling. Old male Fischer 344 rats were randomized to either a naïve or stretch-trained group. Static stretching of ankle flexor muscles was achieved by placement of a dorsiflexion splint on the left ankle for 30 min/day, 5d/wk for 4wks. The opposite hindlimb served as a contralateral control (nonstretched) limb. Bone blood flow was assessed during and after acute stretching in naïve rats, and at rest and during exercise in stretch-trained rats. Vascular reactivity of the nutrient artery of the proximal tibia was also assessed in stretch-trained rats. MicroCT analysis was used to assess bone volume and micro-architecture of the trabecular bone of both tibias near that growth plate. In naïve rats, static stretching increased blood flow to the proximal tibial metaphasis. Blood flow to the proximal tibial metaphysis during treadmill exercise was higher in the stretched limb after 4 weeks of daily stretching. Daily stretching also increased tibial bone weight and increased total volume in both the proximal and distal tibial metaphyses. In the trabecular bone immediately below the proximal tibial growth plate, total volume and bone volume increased, but bone volume/total volume was unchanged and trabecular connectivity decreased. In contrast, intravascular volume increased in this region of the bone. These data suggest that blood flow to the tibia increases during bouts of static stretching of the hindlimb muscles, and that 4 weeks of daily muscle stretching leads to bone remodeling and an increase in intravascular volume of the tibial bone.
... Generally, bone regeneration modes present two distinct approaches: intramembranous ossification (IMO) and endochondral ossification (ECO). [22][23][24][25] For most craniofacial and long bones, previous researches have reported that the IMO mode often leads to avascular necrosis and degradation in central regions due to insufficient angiogenesis and poor nutrient perfusion, particularly in harsh microenvironment. [24,[26][27][28] In comparison, the ECO mode, a time-dependent process starting with an initially cartilaginous template followed by hypertrophy and mineralization, is extremely tolerant to avascular and hypoxic microenvironments due to the early-stage chondrogenic formation. ...
Tissue‐engineered bone has emerged as a promising alternative for bone defect repair due to the advantages of regenerative bone healing and physiological functional reconstruction. However, there is very limited breakthrough in achieving favorable bone regeneration due to the harsh osteogenic microenvironment after bone injury, especially the avascular and hypoxic conditions. Inspired by the bone developmental mode of endochondral ossification, a novel strategy is proposed for tolerant and rapid endochondral bone regeneration using framework‐enhanced 3D biomineralized matrix hydrogels. First, it is meticulously designed 3D biomimetic hydrogels with both hypoxic and osteoinductive microenvironment, and then integrated 3D‐printed polycaprolactone framework to improve their mechanical strength and structural fidelity. The inherent hypoxic 3D matrix microenvironment effectively activates bone marrow mesenchymal stem cells self‐regulation for early‐stage chondrogenesis via TGFβ/Smad signaling pathway due to the obstacle of aerobic respiration. Meanwhile, the strong biomineralized microenvironment, created by a hybrid formulation of native‐constitute osteogenic inorganic salts, can synergistically regulate both bone mineralization and osteoclastic differentiation, and thus accelerate the late‐stage bone maturation. Furthermore, both in vivo ectopic osteogenesis and in situ skull defect repair successfully verified the high efficiency and mechanical maintenance of endochondral bone regeneration mode, which offers a promising treatment for craniofacial bone defect repair.
... SOX9, a transcription factor, is widely recognized to possess a substantial influence on every stage of the chondrocyte lineage, encompassing the early condensation phase in addition to the process of chondrocyte proliferation, there is also the transformation of these cells into hypertrophic chondrocytes. 18 Col X is produced through hypertrophic chondrocyte activity. 15 Through the current investigation, T-2 toxin decreased Runx2, SOX9, and Col X expressions at the levels of mRNA and protein, while these changes can be inhibited by FGFR3 inhibitor (Figure 4). ...
This study evaluated the effect of fibroblast growth factor receptor 3 (FGFR3) on damaged hypertrophic chondrocytes of Kashin–Beck disease (KBD). Immunohistochemical staining was used to evaluate FGFR3 expression in growth plates from KBD rat models and engineered cartilage. In vitro study, hypertrophic chondrocytes were pretreated by FGFR3 binding inhibitor (BGJ398) for 24 h before incubation at different T-2 toxin concentrations. Differentiation -related genes (Runx2, Sox9, and Col Ⅹ) and ECM degradation -related genes (MMP-13, Col Ⅱ) in the hypertrophic chondrocytes were analyzed using RT-PCR, and the corresponding proteins were analyzed using western blotting. Hypertrophic chondrocytes death was detected by the Annexin V/PI double staining assay. The integrated optical density of FGFR3 staining was increased in knee cartilage of rats and engineered cartilage treated with T-2 toxin. Both protein and mRNA levels of Runx2, Sox9, Col Ⅱ, and Col Ⅹ were decreased in a dose-dependent manner when exposed to the T-2 toxin and significantly upregulated by 1 μM BGJ398. The expression of MMP-1, MMP-9, and MMP-13 increased in a dose-dependent manner when exposed to T-2 toxin and significantly reduced by 1 μM BGJ398. 1 μM BGJ398 could prevent early apoptosis and necrosis induced by the T-2 toxin. Inhibiting the FGFR3 signal could alleviate extracellular matrix degradation, abnormal chondrocytes differentiation, and excessive cell death in T-2 toxin-induced hypertrophic chondrocytes.
... Exogenous Sema3A also inhibited the intrachondrocyte concentration of PTH-R1, which functions as a PTHrP protein receptor and aids in the regulation of chondrocyte proliferation ( Figure 5). This inhibiting impact may hasten the endochondral ossification (Kronenberg, 2003;Kajii et al., 2018). Collectively, these observations suggest that Sema3A plays a pivotal role in mechanotransduction within cartilage, whether under physiological or pathological conditions. ...
Semaphorin 3A (Sema3A) is a neuroinformatic protein molecule with widespread expression across various tissues and organs. Recent investigations have unveiled its pivotal role in the skeletal system, primarily through its binding interactions with two co-receptors, neuropilin-1 (Nrp-1) and members of the plexin family. Prior research has confirmed the expression of Sema3A and its receptors in both osteocytes and chondrocytes. Beyond its expression patterns, Sema3A plays a multifaceted role in regulating bone and cartilage metabolism via employing diverse signaling pathways. Additionally, it engages in collaborative interactions with the immune and nervous systems, contributing to the pathophysiological processes underlying a spectrum of bone and joint diseases. In this paper, we undertake a comprehensive review of recent research developments in this field. Our objective is to deepen the understanding of Sema3A within the context of skeletal physiology and pathology. Furthermore, we aim to furnish a valuable reference for potential therapeutic interventions in the realm of bone and joint diseases.
... Besides cells, the ECM comprises a nanostructured organic phase rich in collagen (mainly type 1, mucopolysaccharides, and water, providing flexibility and tensile strength and an inorganic mineral phase presenting nanocrystalline hydroxyapatite (HA) (calcium phosphate), ensuring bone mechanical rigidity and compressive strength [18][19][20][21][22][23]. ...
Osteoporosis is characterized by low bone mass and structural deterioration of bone tissue with increased risk of fracture. The population that is most at risk for primary osteoporosis in-cludes post-menopausal women. Among different approaches, nutraceuticals are increasingly popular in developed countries since can contribute to bone health and can delay the onset of pathological bone loss. Our review covers three main aspects. Firstly, we discuss the current state of the two con-ditions, osteopenia, and osteoporosis. Secondly, we focus on the latest developments in natural Nutraceuticals and their effectiveness in reducing bone loss. Finally, we conduct a Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis to examine the pros and cons of Nutraceuticals assumption. Nutraceuticals hold great promise, but we must address challenges such as regulatory control and experimental evidence to fully realize their potential. Other challenges include identifying the source of raw materials, ensuring the purity of the compound, avoiding contamination or the presence of other active compounds, maintaining product quality, and preventing interactions with other elements or drugs. By addressing these challenges, the scientific community can ensure more reliable indica-tions for the use of Nutraceuticals and help people lead healthier lives.
... The mammalian growth plate is an excellent example for this concept, as its complex architecture is the engine driving longitudinal bone growth (7)(8)(9)(10)(11). The growth plate, which is located at both ends of developing long bones, drives bone elongation by a tightly regulated process of cell proliferation and differentiation, which involves increase in cell size and their organization along the proximal-distal (P-D) axis (12)(13)(14)(15). The growth plate comprises four zones. ...
Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. However, despite decades of research, column structure has thus far been studied only in two dimensions. To fill this knowledge gap, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of both ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Our finding that embryonic growth plates function without forming columns suggests that longitudinal bone growth is regulated by different cellular mechanisms during pre- and postnatal development. Moreover, the observed complex columnar and cluster arrangements may serve other, yet unknown morphogenetic functions. More broadly, our findings provide a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.
... Long bone growth occurs at specific locations known as growth plates, where chondrocytes undergo a well-regulated process of proliferation and differentiation to form cartilage, which is then replaced by bone via apoptosis of terminally differentiated hypertrophic chondrocytes, and invasion of blood vessels to provide bone cells for bone formation [1,2]. This endochondral bone growth process also occurs in the skull bones of the cranial base, upon which the brain sits. ...
Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl−/− mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.
Bones are vital for anchoring muscles, tendons, and ligaments, serving as a fundamental element of the human skeletal structure. However, our understanding of bone development mechanisms and the maintenance of bone homeostasis is still limited. Extracellular signal-related kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, plays a critical role in the pathogenesis and progression of various diseases, especially neoplasms. Recent studies have highlighted ERK5’s significant role in both bone development and bone-associated pathologies. This review offers a detailed examination of the latest research on ERK5 in different tissues and diseases, with a particular focus on its implications for bone health. It also examines therapeutic strategies and future research avenues targeting ERK5.
Background: In the domain of plastic surgery, nasal cartilage regeneration is of significant importance. The extracellular matrix (ECM) from porcine nasal septum cartilage has shown potential for promoting human cartilage...
BACKGROUND
Regular physical activity during childhood and adolescence is beneficial to bone development, as evidenced by the ability to increase bone density and peak bone mass by promoting bone formation.
AIM
To investigate the effects of exercise on bone formation in growing mice and to investigate the underlying mechanisms.
METHODS
20 growing mice were randomly divided into two groups: Con group (control group, n = 10) and Ex group (treadmill exercise group, n = 10). Hematoxylin-eosin staining, immunohistochemistry, and micro-CT scanning were used to assess the bone formation-related indexes of the mouse femur. Bioinformatics analysis was used to find potential miRNAs targets of long non-coding RNA H19 (lncRNA H19). RT-qPCR and Western Blot were used to confirm potential miRNA target genes of lncRNA H19 and the role of lncRNA H19 in promoting osteogenic differentiation.
RESULTS
Compared with the Con group, the expression of bone morphogenetic protein 2 was also significantly increased. The micro-CT results showed that 8 wk moderate-intensity treadmill exercise significantly increased bone mineral density, bone volume fraction, and the number of trabeculae, and decreased trabecular segregation in the femur of mice. Inhibition of lncRNA H19 significantly upregulated the expression of miR-149 and suppressed the expression of markers of osteogenic differentiation. In addition, knockdown of lncRNA H19 significantly downregulated the expression of autophagy markers, which is consistent with the results of autophagy-related protein changes detected in mouse femurs by immunofluorescence.
CONCLUSION
Appropriate treadmill exercise can effectively stimulate bone formation and promote the increase of bone density and bone volume in growing mice, thus enhancing the peak bone mass of mice. The lncRNA H19/miR-149 axis plays an important regulatory role in osteogenic differentiation.
The repair of critical-sized bone defect caused by trauma or disease poses a remarkable clinical challenge. Even though conventional methods such as autografts and allografts have been commonly used, they each have corresponding shortcomings limiting their clinical application. Generally, existing biomaterials such as metals and cements cannot meet the clinical needs. Therefore, there is a critical need for the development of biomimetic and bioactive three-dimensional (3D) scaffolds for bone regeneration. The use of hydrogels is considered a promising strategy in bone tissue engineering due to their highly aqueous 3D network structure and functional characteristics. This chapter focuses on use of natural and synthetic hydrogel scaffolds inspired by extracellular matrix (ECM) biology and structure for bone tissue engineering. Firstly, we discuss the requirements of hydrogel substitutes, the type of hydrogels used and their properties, as well as the combination of hydrogels with growth factors, cells, drugs, and inorganic components. Afterwards, current fabrication strategies are presented, with the emphasis on the implantable hydrogels and injectable hydrogels. Finally, the current challenges, considerations, and future directions are discussed in order to provide new directions for the design and clinical use of hydrogel scaffolds in bone regeneration. We believe that recent advances in the multifunctional bioactive hydrogels offer important information to researchers in the field and clinicians to generate new perspectives on bone repair.
Background and Purpose
MRI is an emerging imaging modality to assess skeletal maturity. This study aimed to chart the learning curves of paediatric radiologists when using an unfamiliar MRI grading system of skeletal maturity and to assess the clinical feasibility of implementing said system.
Materials and Methods
958 healthy paediatric volunteers were prospectively included in a dual-facility study. Each subject underwent a conventional MRI scan at 1.5 T. To perform the image reading, the participants were grouped into five subsets (subsets 1 to 5) of equal size (n∼192) in chronological order for scan acquisition. Two paediatric radiologists (R1–2) with different levels of MRI experience, both of whom were previously unfamiliar with the study’s MRI grading system, independently evaluated the subsets to assess skeletal maturity in five different growth plate locations. Congruent cases at blinded reading established the consensus reading. For discrepant cases, the consensus reading was obtained through an unblinded reading by a third paediatric radiologist (R3), also unfamiliar with the MRI grading system. Further, R1 performed a second blinded image reading for all included subjects with a memory wash-out of 180 days. Weighted Cohen’s kappa was used to assess interreader reliability (R1 vs consensus; R2 vs consensus) at non-cumulative and cumulative time points, as well as interreader (R1 vs R2) and intrareader (R1 vs R1) reliability at non-cumulative time points.
Results
Mean weighted Cohen’s kappa values for each pair of blinded readers compared to consensus reading (interreader reliability, R1–2 vs consensus) were ≥0.85, showing a strong to almost perfect interreader agreement at both non-cumulative and cumulative time points and in all growth plate locations. Weighted Cohen’s kappa values for interreader (R1 vs R2) and intrareader reliability (R1 vs R1) were ≥0.72 at non-cumulative time points, with values ≥ 0.82 at subset 5.
Conclusions
Paediatric radiologists’ clinical confidence when introduced to a new MRI grading system for skeletal maturity was high from the outset of their learning curve, despite the radiologists’ varying levels of work experience with MRI assessment. The MRI grading system for skeletal maturity investigated in this study is a robust clinical method when used by paediatric radiologists and can be used in clinical practice.
Advances in knowledge
Radiologists with fellowship training in paediatric radiology experienced no learning curve progress when introduced to a new MRI grading system for skeletal maturity and achieved desirable agreement from the first time point of the learning curve. The robustness of the investigated MRI grading system was not affected by the earlier different levels of MRI experience among the readers.
A major question in developmental and regenerative biology is how organ size and architecture are controlled by progenitor cells. While limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse fetal cartilage is repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states is delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death is over, chondroprogenitor differentiation is accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlates with, and is necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies.
Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin‐dependent kinase inhibitor p21. The activation of the TGF‐β/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF‐β/SMAD2/3 pathway.
Background
Previous studies on the effects of microplastics (MPs) on bone in early development are limited. This study aimed to investigate the adverse effects of MPs on bone in young rats and the potential mechanism.
Methods
Three‐week‐old female rats were orally administered MPs for 28 days, and endoplasmic reticulum (ER) stress inhibitor salubrinal (SAL) and ER stress agonist tunicamycin (TM) were added to evaluate the effect of ER stress on toxicity of MPs. The indicators of growth and plasma markers of bone turnover were evaluated. Tibias were analyzed using micro‐computed tomography (micro‐CT). Histomorphological staining of growth plates was performed, and related gene expression of growth plate chondrocytes was tested.
Results
After exposure of MPs, the rats had decreased growth, shortened tibial length, and altered blood calcium and phosphorus metabolism. Trabecular bone was sparse according to micro‐CT inspection. In the growth plate, the thickness of proliferative zone substantial reduced while the thickness of hypertrophic zone increased significantly, and the chondrocytes were scarce and irregularly arranged according to tibial histological staining. The transcription of the ER stress‐related genes BIP , PERK , ATF4 , and CHOP dramatically increased, and the transcription factors involved in chondrocyte proliferation, differentiation, apoptosis, and matrix secretion were aberrant according to RT‐qPCR and western blotting. Moreover, the addition of TM showed higher percentage of chondrocyte death. Administration of SAL alleviated all of the MPs‐induced symptoms.
Conclusion
These results indicated that MPs could induce growth retardation and longitudinal bone damage in early development. The toxicity of MPs may attribute to induced ER stress and impaired essential processes of the endochondral ossification after MPs exposure.
Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA) as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labelling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow towards the TCA cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes - a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.
Osteogenic differentiation is important for fracture healing. Microfibrial‐associated glycoprotein 2 (MAGP2) is found to function as a proangiogenic regulator in bone formation; however, its role in osteogenic differentiation during bone repair is not clear. Here, a mouse model of critical‐sized femur fracture was constructed, and the adenovirus expressing MAGP2 was delivered into the fracture site. Mice with MAGP2 overexpression exhibited increased bone mineral density and bone volume fraction (BV/TV) at Day 14 postfracture. Within 7 days postfracture, overexpression of MAGP2 increased collagen I and II expression at the fracture callus, with increasing chondrogenesis. MAGP2 inhibited collagen II level but elevated collagen I by 14 days following fracture, accompanied by increased endochondral bone formation. In mouse osteoblast precursor MC3T3‐E1 cells, MAGP2 treatment elevated the expression of osteoblastic factors (osterix, BGLAP and collagen I) and enhanced ALP activity and mineralization through activating β‐catenin signaling after osteogenic induction. Besides, MAGP2 could interact with lipoprotein receptor‐related protein 5 (LRP5) and upregulated its expression. Promotion of osteogenic differentiation and β‐catenin activation mediated by MAGP2 was partially reversed by LRP5 knockdown. Interestingly, β‐catenin/transcription factor 4 (TCF4) increased MAGP2 expression probably by binding to MAGP2 promoter. These findings suggest that MAGP2 may interact with β‐catenin/TCF4 to enhance β‐catenin/TCF4's function and activate LRP5‐activated β‐catenin signaling pathway, thus promoting osteogenic differentiation for fracture repair. mRNA sequencing identified the potential targets of MAGP2, providing novel insights into MAGP2 function and the directions for future research.
The poultry skeletal system serves multiple functions, not only providing structural integrity but also maintaining the balance of essential minerals such as calcium and phosphorus. However, in recent years, the consideration of skeletal traits has been overlooked in the selective breeding of broilers, resulting in an inadequate adaptation of the skeletal system to cope with the rapid increase in body weight. Consequently, this leads to lameness and bone diseases such as tibial dyschondroplasia (TD), which significantly impact the production performance of broilers. Accumulating evidence has shown that microRNAs (miRNA) play a crucial role in the differentiation, formation, and disease of cartilage. However, the miRNA-mediated molecular mechanism underlying chicken TD formation is still poorly understood. The objective of this study was to investigate the biological function and regulatory mechanism of miRNA in chicken TD formation. Based on transcriptome sequencing of tibial cartilage in the healthy group and TD group, miR-206a-3p was found to be highly expressed in TD cartilage. The function of miR-206a-3p was explored through the transfection test of miR-206a-3p mimics and miR-206a-3p inhibitor. In this study, we utilized qRT-PCR, CCK-8, EdU, western blot, and flow cytometry to detect the proliferation, differentiation, and apoptosis of chondrocytes. The results revealed that miR-206a-3p suppressed the proliferation and differentiation of TD chondrocytes while promoting their programmed cell death. Furthermore, through biosynthesis and dual luciferase assays, it was determined that BMP6 was the direct target gene of miR-206a-3p. This finding was further supported by rescue experiments which confirmed the involvement of BMP6 in the regulatory pathway governed by miR-206a-3p. Our results suggest that miR-206a-3p can inhibits the proliferation and differentiation promote apoptosis through the target gene BMP-6 and suppressing the Smad2/3 signaling pathway in chicken TD chondrocytes.
Spatial and temporal regulation of chondrocyte maturation in the growth plate drives growth of many bones. One essential event to generate the ordered cell array characterizing growth plate cartilage is the formation of chondrocyte columns in the proliferative zone via 90-degree rotation of daughter cells to align with the long axis of the bone. Previous studies have suggested crucial roles for cadherins and integrin β1 in column formation. The purpose of this study was to determine the relative contributions of cadherin- and integrin-mediated cell adhesion in column formation. Here we present new mechanistic insights generated by application of live time-lapse confocal microscopy of cranial base explant cultures, robust genetic mouse models, and new quantitative methods to analyze cell behavior. We show that conditional deletion of either the cell-cell adhesion molecule Cdh2 or the cell-matrix adhesion molecule Itgb1 disrupts column formation. Compound mutants were used to determine a potential reciprocal regulatory interaction between the two adhesion surfaces and identified that defective chondrocyte rotation in a N-cadherin mutant was restored by a heterozygous loss of integrin β1. Our results support a model for which integrin β1, and not N-cadherin, drives chondrocyte rotation and for which N-cadherin is a potential negative regulator of integrin β1 function.
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During developmental processes, cells frequently condense along a preferred
axis, creating columnar arrangements—a pivotal step in shaping elongating tissue
structures and facilitating gradual building of tissue complexity. Despite advances
in biofabrication technology that has allowed researchers to recreate these axial
arrangements in vitro in 3D culture, maintaining these patterns for periods of
cultivation beyond 7 days has proven challenging, given cells' tendency to exhibit
random migratory patterns. In this study, we introduce EXPECT (EXtrusion
Patterned Embedded ConstruCTs), a thermosensitive hydrogel based on poly(Nisopropylacrylamide)
designed with specific rheological properties enabling the
creation of embedded, macroscopic, cell-laden channels within the hydrogel using
3D printing. EXPECT, coupled with mild temperature changes at regular
intervals, suppressed the random migratory tendencies of mesenchymal stromal
cells (MSCs), guiding the cells to laterally intercalate and aggregate
longitudinally. This resulted in the formation of continuous stacked arrangements
of MSCs sustained over 36 days of culture. Additionally, EXPECT led to the
elongation of initially spaced MSC spheroids toward each other, culminating in their fusion into narrowed, columnar assemblies. Our study presents a versatile
and readily applicable approach for orchestrating and maintaining cell
communication and movements along a preferred axis outside the developmental
niche. By addressing a key limitation in current in vitro 3D culture systems and
inducing cell movements reminiscent of both convergent extension and directed
chemotaxis, we present a novel tool for studying various facets of development,
disease, and repair.
Background
Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease characterized by malformation of the bilateral great toes and progressive heterotopic ossification. The clinical features of FOP occur due to dysfunction of the bone morphogenetic protein (BMP) signaling pathway induced by the mutant activin A type I receptor/activin‐like kinase‐2 (ACVR1/ALK2) which contributes to the clinical features in FOP. Dysregulation of the BMP signaling pathway causes the development of osteochondroma. Poor awareness of the association between FOP and osteochondromas always results in misdiagnosis and unnecessary invasive operation.
Case Presentation
In this study, we present a case of classical FOP involving osteochondroma. An 18‐year‐old male adolescent, born with deformity of bilateral big toes, complained multiple masses on his back for 1 year. The mass initially emerged with a tough texture and did not cause pain. It was misdiagnosed as an osteochondroma. After two surgeries, the masses became hard and spread around the entire back region. Meanwhile, extensive heterotopic ossification was observed around the back, neck, hip, knee, ribs, and mandible during follow‐up. Osteochondromas were observed around the bilateral knees. No abnormalities were observed in the laboratory blood test results. Whole exome sequencing revealed missense mutation of ACVR1/ALK2 (c.617G > A; p.R206H) in the patient and confirmed the diagnosis of FOP.
Conclusion
In summary, classical FOP always behaves as a bilateral deformity of the big toes, as well as progressive ectopic ossification and osteochondromas in the distal femur and proximal tibia. An understanding of the association between osteochondromas and FOP aids in diagnosis and avoids unnecessary invasive management in patients.
Endosteal stem cells are a subclass of bone marrow skeletal stem cell populations that are particularly important for rapid bone formation occurring in growth and regeneration. These stem cells are strategically located near the bone surface in a specialized microenvironment of the endosteal niche. These stem cells are abundant in young stages but eventually depleted and replaced by other stem cell types residing in a non‐endosteal perisinusoidal niche. Single‐cell molecular profiling and in vivo cell lineage analyses play key roles in discovering endosteal stem cells. Importantly, endosteal stem cells can transform into bone tumor‐making cells when deleterious mutations occur in tumor suppressor genes. The emerging hypothesis is that osteoblast‐chondrocyte transitional identities confer a special subset of endosteal stromal cells with stem cell‐like properties, which may make them susceptible for tumorigenic transformation. Endosteal stem cells are likely to represent an important therapeutic target of bone diseases caused by aberrant bone formation.
Social insects often display extreme variation in body size and morphology within the same colony. In many species, adult morphology is socially regulated by workers during larval development. While larval nutrition may play a role in this regulation, it is often difficult to identify precisely what larvae receive from rearing workers, especially when larvae are fed through social regurgitation. Across insects, juvenile hormone is a major regulator of development. In the ant Camponotus floridanus, this hormone is present in the socially regurgitated fluid of workers. We investigated the role the social transfer of juvenile hormone in the social regulation of development. To do this, we administered an artificial regurgitate to larvae through a newly developed handfeeding method that was or was not supplemented with juvenile hormone. Orally administered juvenile hormone increased the nutritional needs of larvae, allowing them to reach a larger size at pupation. Instead of causing them to grow faster, the juvenile hormone treatment extended larval developmental time, allowing them to accumulate resources over a longer period. Handfeeding ant larvae with juvenile hormone resulted in larger adult workers after metamorphosis, suggesting a role for socially transferred juvenile hormone in the colony-level regulation of worker size over colony maturation.
The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-indian hedgehog (Ihh) feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and trace the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with Patched-1 (Ptch1) floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large concentric clonally expanded cell populations within the resting zone ('patched roses') and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell-descendants migrated away from the growth plate and eventually transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.
Cicatricial tracheal stenosis is a fairly common complication that occurs after tracheal intubation or tracheostomy. However, critical tracheal stenosis is a rare case, sometimes not associated with trauma, and is probably due to the peculiarities of the patient’s immune response during the development of stenosis. In this regard, the study of the immune mechanisms of the development of inflammation in the upper respiratory tract is a very relevant and promising direction. This review is devoted to the analysis of the immunological mechanisms of tracheal stricture formation, and presents modern data on the immunopathogenesis of the disease.
Clarification of some pathogenetic mechanisms of the immune response during the formation of tracheal strictures of various origins can help in identifying laboratory markers as risk factors for tracheal stricture and timely prevention of such complications.
Osteoarthritis (OA) is a highly prevalent chronic joint disease and the leading cause of disability. Currently, no drugs are available to control joint damage or ease the associated pain. The wingless-type (WNT) signalling pathway is vital in OA progression. Excessive activation of the WNT signalling pathway is pertinent to OA progression and severity. Therefore, agonists and antagonists of the WNT pathway are considered potential drug candidates for OA treatment. For example, SM04690, a novel small molecule inhibitor of WNT signalling, has demonstrated its potential in a recent phase III clinical trial as a disease-modifying osteoarthritis drug (DMOAD). Therefore, targeting the WNT signalling pathway may be a distinctive approach to developing particular agents helpful in treating OA. This review aims to update the most recent progress in OA drug development by targeting the WNT pathway. In this, we introduce WNT pathways and their crosstalk with other signalling pathways in OA development and highlight the role of the WNT signalling pathway as a key regulator in OA development. Several articles have reviewed the Wnt pathway from different aspects. This candid review provides an introduction to WNT pathways and their crosstalk with other signalling pathways in OA development, highlighting the role of the WNT signalling pathway as a key regulator in OA development with the latest research. Particularly, we emphasise the state-of-the-art in targeting the WNT pathway as a promising therapeutic approach for OA and challenges in their development and the nanocarrier-based delivery of WNT modulators for treating OA.
Condensation is the pivotal stage in the development of skeletal and other mesenchymal tissues. It occurs when a previously dispersed population of cells gathers together to differentiate into a single cell/tissue type such as cartilage, bone, muscle, tendon, kidney, and lung and is the earliest stage during organ formation when tissue-specific genes are upregulated. We present a synopsis of our current understanding of how condensations are initiated and grown, how their boundaries and sizes are set, how condensation ceases, and how overt differentiation begins. Extracellular matrix molecules, cell surface receptors and cell adhesion molecules, such as fibronectin, tenascin, syndecan, and N-CAM, initiate condensation formation and set condensation boundaries. Hox genes (Hoxd-11-13) and other transcription factors (CFKH-1, MFH-1, osf-2), modulate the proliferation of cells within condensations. Cell adhesion is ensured indirectly through Hox genes (Hoxa-2, Hoxd-13), and directly via cell adhesion molecules (N-CAM and N-cadherin). Subsequent growth of condensations is regulated by BMPs, which activate Pax-2, Hoxa-2 and Hoxd-11 among other genes. Growth of a condensation ceases when Noggin inhibits BMP signalling, setting the stage for transition to the next stage of skeletal development, namely overt cell differentiation. BioEssays 22:138–147, 2000. ©2000 John Wiley & Sons, Inc.
The parathyroid hormone-related peptide (PTHrP) gene was disrupted in murine embryonic stem cells by homologous recombination, and the null allele was introduced into the mouse germ line. Mice homozygous for the PTHrP null mutation died postnatally, probably from asphyxia, and exhibited widespread abnormalities of endochondral bone development. Histological examination revealed a diminution of chondrocyte proliferation, associated with premature maturation of chondrocytes and accelerated bone formation. Analysis of earlier developmental stages revealed that disturbance in cartilage growth preceded abnormal endochondral bone formation. There were no morphological abnormalities apparent in other tissues. These results provide direct evidence implicating PTHrP in normal skeletal development and serve to emphasize its potential involvement in human osteochondrodysplasias.
Fibroblast growth factor receptor 3 (Fgfr3) is a tyrosine kinase receptor expressed in developing bone, cochlea, brain and spinal cord. Achondroplasia, the most common genetic form of dwarfism, is caused by mutations in FGFR3. Here we show that mice homozygous for a targeted disruption of Fgfr3 exhibit skeletal and inner ear defects. Skeletal defects include kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae. Contrasts between the skeletal phenotype and achondroplasia suggest that activation of FGFR3 causes achondroplasia. Inner ear defects include failure of pillar cell differentiation and tunnel of Corti formation and result in profound deafness. Our results demonstrate that Fgfr3 is essential for normal endochondral ossification and inner ear development.
The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.
Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone-related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (-/-) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (-/-) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.
Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.
Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat alpha1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP-/-). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP-/- mice and allowed their prolonged survival. "Rescued" animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP-/- mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.
During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.
Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium.
Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis.
Mice carrying a targeted disruption of BmprIB were generated by homologous recombination in embryonic stem cells. BmprIB(-/-) mice are viable and, in spite of the widespread expression of BMPRIB throughout the developing skeleton, exhibit defects that are largely restricted to the appendicular skeleton. Using molecular markers, we show that the initial formation of the digital rays occurs normally in null mutants, but proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region are markedly reduced. Our results suggest that BMPRIB-mediated signaling is required for cell proliferation after commitment to the chondrogenic lineage. Analyses of BmprIB and Gdf5 single mutants, as well as BmprIB; Gdf5 double mutants suggests that GDF5 is a ligand for BMPRIB in vivo. BmprIB; Bmp7 double mutants were constructed in order to examine whether BMPRIB has overlapping functions with other type I BMP receptors. BmprIB; Bmp7 double mutants exhibit severe appendicular skeletal defects, suggesting that BMPRIB and BMP7 act in distinct, but overlapping pathways. These results also demonstrate that in the absence of BMPRIB, BMP7 plays an essential role in appendicular skeletal development. Therefore, rather than having a unique role, BMPRIB has broadly overlapping functions with other BMP receptors during skeletal development.
Indian hedgehog (Ihh) and Parathyroid Hormone-related Protein (PTHrP) play a critical role in the morphogenesis of the vertebrate skeleton. Targeted deletion of Ihh results in short-limbed dwarfism, with decreased chondrocyte proliferation and extensive hypertrophy, features shared by mutants in PTHrP and its receptor. Activation of Ihh signaling upregulates PTHrP at the articular surface and prevents chondrocyte hypertrophy in wild-type but not PTHrP null explants, suggesting that Ihh acts through PTHrP. To investigate the relationship between these factors during development of the appendicular skeleton, mice were produced with various combinations of an Ihh null mutation (Ihh(-/-)), a PTHrP null mutation (PTHrP(-/-)), and a constitutively active PTHrP/Parathyroid hormone Receptor expressed under the control of the Collagen II promoter (PTHrPR*). PTHrPR* rescues PTHrP(-/-) embryos, demonstrating this construct can completely compensate for PTHrP signalling. At 18.5 dpc, limb skeletons of Ihh, PTHrP compound mutants were identical to Ihh single mutants suggesting Ihh is necessary for PTHrP function. Expression of PTHrPR* in chondrocytes of Ihh(-/-) mice prevented premature chondrocyte hypertrophy but did not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation. These experiments demonstrate that the molecular mechanism that prevents chondrocyte hypertrophy is distinct from that which drives proliferation. Ihh positively regulates PTHrP, which is sufficient to prevent chondrocyte hypertrophy and maintain a normal domain of cells competent to undergo proliferation. In contrast, Ihh is necessary for normal chondrocyte proliferation in a pathway that can not be rescued by PTHrP signaling. This identifies Ihh as a coordinator of skeletal growth and morphogenesis, and refines the role of PTHrP in mediating a subset of Ihh's actions.
Vertebrate skeletogenesis requires a well-coordinated transition from chondrogenesis to osteogenesis. Hypertrophic chondrocytes in the growth plate play a pivotal role in this transition. Parathyroid hormone-related peptide (PTHrP), synthesized in the periarticular growth plate, regulates the site at which hypertrophy occurs. By comparing PTH/PTHrP receptor(-/-)/wild-type (PPR(-/-)/wild-type) chimeric mice with IHH(-/-);PPR(-/-)/wild-type chimeric and IHH(-/-)/wild-type chimeric mice, we provide in vivo evidence that Indian hedgehog (IHH), synthesized by prehypertrophic and hypertrophic chondrocytes, regulates the site of hypertrophic differentiation by signaling to the periarticular growth plate and also determines the site of bone collar formation in the adjacent perichondrium. By providing crucial local signals from prehypertrophic and hypertrophic chondrocytes to both chondrocytes and preosteoblasts, IHH couples chondrogenesis to osteogenesis in endochondral bone development.
Chondrocyte hypertrophy is a mandatory step during endochondral ossification. Cbfa1-deficient mice lack hypertrophic chondrocytes in some skeletal elements, indicating that Cbfa1 may control hypertrophic chondrocyte differentiation. To address this question we generated transgenic mice expressing Cbfa1 in nonhypertrophic chondrocytes (alpha1(II) Cbfa1). This continuous expression of Cbfa1 in nonhypertrophic chondrocytes induced chondrocyte hypertrophy and endochondral ossification in locations where it normally never occurs. To determine if this was caused by transdifferentiation of chondrocytes into osteoblasts or by a specific hypertrophic chondrocyte differentiation ability of Cbfa1, we used the alpha1(II) Cbfa1 transgene to restore Cbfa1 expression in mesenchymal condensations of the Cbfa1-deficient mice. The transgene restored chondrocyte hypertrophy and vascular invasion in the bones of the mutant mice but did not induce osteoblast differentiation. This rescue occurred cell-autonomously, as skeletal elements not expressing the transgene were not affected. Despite the absence of osteoblasts in the rescued animals there were multinucleated, TRAP-positive cells resorbing the hypertrophic cartilage matrix. These results identify Cbfa1 as a hypertrophic chondrocyte differentiation factor and provide a genetic argument for a common regulation of osteoblast and chondrocyte differentiation mediated by Cbfa1.
During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.
Fibroblast growth factor (FGF) signaling is involved in skeletal development of the vertebrate. Gain-of-function mutations of FGF receptors (FGFR) cause craniosynostosis, premature fusion of the skull, and dwarfism syndromes. Disruption of Fgfr3 results in prolonged growth of long bones and vertebrae. However, the role that FGFs actually play in skeletal development in the embryo remains unclear. Here we show that Fgf18 is expressed in and required for osteogenesis and chondrogenesis in the mouse embryo. Fgf18 is expressed in both osteogenic mesenchymal cells and differentiating osteoblasts during calvarial bone development. In addition, Fgf18 is expressed in the perichondrium and joints of developing long bones. In calvarial bone development of Fgf18-deficient mice generated by gene targeting, the progress of suture closure is delayed. Furthermore, proliferation of calvarial osteogenic mesenchymal cells is decreased, and terminal differentiation to calvarial osteoblasts is specifically delayed. Delay of osteogenic differentiation is also observed in the developing long bones of this mutant. Conversely, chondrocyte proliferation and the number of differentiated chondrocytes are increased. Therefore, FGF18 appears to regulate cell proliferation and differentiation positively in osteogenesis and negatively in chondrogenesis.
Over the last decade the identification of mutations in the receptors for fibroblast growth factors (FGFs) has de- fined essential roles for FGF signaling in both endochon- dral and intramembranous bone development. FGF sig- naling pathways are essential for the earliest stages of limb development and throughout skeletal develop- ment. In this review, we examine the role of FGF signal- ing in bone development and in human genetic diseases that affect bone development. We also explore what is presently known about how FGF signaling pathways in- teract with other major signaling pathways that regulate chondrogenesis and osteogenesis.
To examine whether the transcription factor Sox9 has an essential role during the sequential steps of chondrocyte differentiation, we have used the Cre/loxP recombination system to generate mouse embryos in which either Sox9 is missing from undifferentiated mesenchymal cells of limb buds or the Sox9 gene is inactivated after chondrogenic mesenchymal condensations. Inactivation of Sox9 in limb buds before mesenchymal condensations resulted in a complete absence of both cartilage and bone, but markers for the different axes of limb development showed a normal pattern of expression. Apoptotic domains within the developing limbs were expanded, suggesting that Sox9 suppresses apoptosis. Expression of Sox5 and Sox6, two other Sox genes involved in chondrogenesis, was no longer detected. Moreover, expression of Runx2, a transcription factor needed for osteoblast differentiation, was also abolished. Embryos, in which Sox9 was deleted after mesenchymal condensations, exhibited a severe generalized chondrodysplasia, similar to that in Sox5; Sox6 double-null mutant mice. Most cells were arrested as condensed mesenchymal cells and did not undergo overt differentiation into chondrocytes. Furthermore, chondrocyte proliferation was severely inhibited and joint formation was defective. Although Indian hedgehog, Patched1, parathyroid hormone-related peptide (Pthrp), and Pth/Pthrp receptor were expressed, their expression was down-regulated. Our experiments further suggested that Sox9 is also needed to prevent conversion of proliferating chondrocytes into hypertrophic chondrocytes. We conclude that Sox9 is required during sequential steps of the chondrocyte differentiation pathway.
Chondrocytes play critical roles at several stages of endochondral ossification. At the onset of skeletal development, in the areas of the skeleton that will undergo endochondral ossification, undifferentiated mesenchymal cells form condensations that have the shape of the future skeletal elements. Cells within these mesenchymal condensations differentiate into chondrocytes that express specific molecular markers, such as α1(II) collagen, whereas the remaining undifferentiated mesenchymal cells at the periphery of the condensations form the perichondrium (for review, see ref. 1). Once these cartilaginous models have formed, chondrocytes in their centers further differentiate into hypertrophic chondrocytes. At the time chondrocyte hypertrophy occurs, the perichondrial cells differentiate into osteoblasts to form, around the cartilaginous core, the bone collar (2).
The mouse short ear gene is required for normal growth and patterning of skeletal structures, and for repair of bone fractures in adults. We have carried out an extensive chromosome walk in the chromosome region that surrounds this locus. Here we show that the short ear region contains the gene for a TGFβ-related protein called bone morphogenetic protein 5 (Bmp-5). This gene is deleted or rearranged in several independent mutations at the short ear locus. Mice homozygous for large deletions of the Bmp-5 coding region are viable and fertile. Mutations at the short ear locus provide an important new tool for defining the normal functions of BMPs in mammals. The specific skeletal defects seen in short-eared animals, which occur against a background of otherwise normal skeletal structures, suggest that particular aspects of skeletal morphology may be determined by individual members of a family of signaling factors that can induce the formation of cartilage and bone in vivo.
The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH),
and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor.
The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most
PTH/PTHrP receptor (−/−) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (−/−) mice, perhaps because
of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their
bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the
PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.
Cbfa1, a transcription factor that belongs to the runt-domain gene family, plays an essential role in osteogenesis. Cbfa1-deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5–16.5. In Cbfa1-deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bonesialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation. Dev Dyn 1999;214:279–290. © 1999 Wiley-Liss, Inc.
The mouse short ear gene is required for normal growth and patterning of skeletal structures, and for repair of bone fractures in adults. We have carried out an extensive chromosome walk in the chromosome region that surrounds this locus. Here we show that the short ear region contains the gene for a TGF beta-related protein called bone morphogenetic protein 5 (Bmp-5). This gene is deleted or rearranged in several independent mutations at the short ear locus. Mice homozygous for large deletions of the Bmp-5 coding region are viable and fertile. Mutations at the short ear locus provide an important new tool for defining the normal functions of BMPs in mammals. The specific skeletal defects seen in short-eared animals, which occur against a background of otherwise normal skeletal structures, suggest that particular aspects of skeletal morphology may be determined by individual members of a family of signaling factors that can induce the formation of cartilage and bone in vivo.
Endochondral ossification is a major mode of bone that occurs as chondrocytes undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. We have identified a role for fibroblast growth factor receptor 3 (FGFR-3) in this process by disrupting the murine Fgfr-3 gene to produce severe and progressive bone dysplasia with enhanced and prolonged endochondral bone growth. This growth is accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. Thus, FGFR-3 appears to regulate endochondral ossification by an essentially negative mechanism, limiting rather than promoting osteogenesis. In light of these mouse results, certain human disorders, such as achondroplasia, can be interpreted as gain-of-function mutations that activate the fundamentally negative growth control exerted by the FGFR-3 kinase.
We have generated Cbfa1-deficient mice. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). These defects are also observed in a mouse Ccd mutant for this disease. The Cbfa1 gene was shown to be deleted in the Ccd mutation. Analysis of embryonic Cbfa1 expression using a lacZ reporter gene revealed strong expression at sites of bone formation prior to the earliest stages of ossification. Thus, the Cbfa1 gene is essential for osteoblast differentiation and bone formation, and the Cbfa1 heterozygous mouse is a paradigm for a human skeletal disorder.
A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. To elucidate the function of Cbfa1, we generated mice with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 died just after birth without breathing. Examination of their skeletal systems showed a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in the cartilage. Therefore, our data suggest that both intramembranous and endochondral ossification were completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice, and demonstrate that Cbfa1 plays an essential role in osteogenesis.
Noggin is a bone morphogenetic protein (BMP) antagonist expressed in Spemann's organizer. Murine Noggin is expressed in condensing cartilage and immature chondrocytes, as are many BMPs. In mice lacking Noggin, cartilage condensations initiated normally but developed hyperplasia, and initiation of joint development failed as measured by the expression of growth and differentiation factor-5. The maturation of cartilage and Hoxd expression were unaffected. Excess BMP activity in the absence of Noggin antagonism may enhance the recruitment of cells into cartilage, resulting in oversized growth plates; chondrocytes are also refractory to joint-inducing positional cues.
To define the contributions of changes in cell, matrix compartment, and fibrillar collagen volumes to longitudinal bone growth, we measured the differences in cell, pericellular/territorial matrix and interterritorial matrix volumes, and fibrillar collagen concentrations between the upper proliferative and lower hypertrophic zones of the proximal tibial physes of six miniature pigs. The mean numerical density of cells decreased from 110,000 cells/mm3 in the upper proliferative zone to 59,900 cells/mm3 in the lower hypertrophic zone. The mean cell volume increased nearly 5-fold (from 1,174 to 5,530 microm3), and the total matrix volume per cell increased 46% (from 8,040 to 11,760 microm3/cell) between the upper proliferative and lower hypertrophic zones. Both the pericellular/territorial matrix volume per cell and the interterritorial matrix volume per cell increased between the upper proliferative and lower hypertrophic zones; the pericellular/territorial matrix volume per cell increased 61% (from 4,580 to 7,390 microm3/cell), whereas the interterritorial matrix volume per cell increased 26% (from 3,460 to 4,370 microm3/cell). The total increase in mean cell volume of 4,356 microm3 exceeded the total increase in mean matrix volume per cell of 3,720 microm3; the total mean pericellular/territorial matrix volume per cell increased more than the total mean interterritorial matrix volume per cell (2,810 compared with 910 microm3/cell). Fibrillar collagen concentration was greater in the interterritorial matrix than in the pericellular/territorial matrix in both zones and increased in both matrix compartments between the upper proliferative and lower hypertrophic zones. The amount of fibrillar collagen per cell also increased in both matrix compartments between the upper proliferative and lower hypertrophic zones (from 1,720 to 3,100 microm3/cell in the pericellular/territorial matrix and from 1,490 to 2,230 microm3/cell in the interterritorial matrix; thus, the total amount of fibrillar collagen per cell increased from 3,210 to 5,530 microm3/cell). Growth rate was inversely related to the cell numerical density in the upper proliferative and lower hypertrophic zones and was directly related to interterritorial matrix volume per cell in the upper proliferative zone and to pericellular/territorial matrix volume per cell in the lower hypertrophic zone. These results show that cell enlargement contributes more to longitudinal bone growth than does increased matrix volume, that increased pericellular/territorial matrix volume makes a greater contribution to growth than does increased interterritorial matrix volume, and that the total amount of fibrillar collagen per cell increases between the upper proliferative and lower hypertrophic zones. The differences between the two matrix compartments in increase in volume, fibrillar collagen concentration, and amount of fibrillar collagen per cell strongly suggest that they differ not only in matrix organization but in rate of matrix accumulation and assembly and that these differences give the two compartments different roles in skeletal growth.
A functional skeletal system requires the coordinated development of many different tissue types, including cartilage, bones, joints, and tendons. Members of the Bone morphogenetic protein (BMP) family of secreted signaling molecules have been implicated as endogenous regulators of skeletal development. This is based on their expression during bone and joint formation, their ability to induce ectopic bone and cartilage, and the skeletal abnormalities present in animals with mutations in BMP family members. One member of this family, Growth/differentiation factor 5 (GDF5), is encoded by the mouse brachypodism locus. Mice with mutations in this gene show reductions in the length of bones in the limbs, altered formation of bones and joints in the sternum, and a reduction in the number of bones in the digits. The expression pattern of Gdf5 during normal development and the phenotypes seen in mice with single or double mutations in Gdf5 and Bmp5 suggested that Gdf5 has multiple functions in skeletogenesis, including roles in joint and cartilage development. To further understand the function of GDF5 in skeletal development, we assayed the response of developing chick and mouse limbs to recombinant GDF5 protein. The results from these assays, coupled with an analysis of the development of brachypodism digits, indicate that GDF5 is necessary and sufficient for both cartilage development and the restriction of joint formation to the appropriate location. Thus, GDF5 function in the digits demonstrates a link between cartilage development and joint development and is an important determinant of the pattern of bones and articulations in the digits.
Chondrogenesis results in the formation of cartilages, initial skeletal elements that can serve as templates for endochondral bone formation. Cartilage formation begins with the condensation of mesenchyme cells followed by their differentiation into chondrocytes. Although much is known about the terminal differentiation products that are expressed by chondrocytes, little is known about the factors that specify the chondrocyte lineage. SOX9 is a high-mobility-group (HMG) domain transcription factor that is expressed in chondrocytes and other tissues. In humans, SOX9 haploinsufficiency results in campomelic dysplasia, a lethal skeletal malformation syndrome, and XY sex reversal. During embryogenesis, Sox9 is expressed in all cartilage primordia and cartilages, coincident with the expression of the collagen alpha1(II) gene (Col2a1) . Sox9 is also expressed in other tissues, including the central nervous and urogenital systems. Sox9 binds to essential sequences in the Col2a1 and collagen alpha2(XI) gene (Col11a2) chondrocyte-specific enhancers and can activate these enhancers in non-chondrocytic cells. Here, Sox9 is identified as a regulator of the chondrocyte lineage. In mouse chimaeras, Sox9-/- cells are excluded from all cartilages but are present as a juxtaposed mesenchyme that does not express the chondrocyte-specific markers Col2a1, Col9a2, Col11a2 and Agc. This exclusion occurred cell autonomously at the condensing mesenchyme stage of chondrogenesis. Moreover, no cartilage developed in teratomas derived from Sox9-/- embryonic stem (ES) cells. Our results identify Sox9 as the first transcription factor that is essential for chondrocyte differentiation and cartilage formation.
The mechanisms that control cell proliferation and cell differentiation during morphogenesis of the endochondral skeleton of vertebrates are poorly understood. Indian hedgehog (Ihh) signaling from prehypertrophic chondrocytes has been implicated in the control of chondrocyte maturation by way of feedback control of a second secreted factor parathyroid hormone-related peptide (PTHrP) at the articular surfaces. Analysis of an Ihh null mutant suggests a more extensive role for Ihh in skeletal development. Mutants display markedly reduced chondrocyte proliferation, maturation of chondrocytes at inappropriate position, and a failure of osteoblast development in endochondral bones. Together, the results suggest a model in which Ihh coordinates diverse aspects of skeletal morphogenesis through PTHrP-dependent and independent processes.
In this study, we use a mouse insertional mutant to delineate gene activities that shape the distal limb skeleton. A recessive mutation that results in brachydactyly was found in a lineage of transgenic mice. Sequences flanking the transgene insertion site were cloned, mapped to chromosome 3, and used to identify the brachydactyly gene as the type IB bone morphogenetic protein receptor, BmprIB (ALK6). Expression analyses in wild-type mice revealed two major classes of BmprIB transcripts. Rather than representing unique coding RNAs generated by alternative splicing of a single pro-mRNA transcribed from one promoter, the distinct isoforms reflect evolution of two BmprIB promoters: one located distally, driving expression in the developing limb skeleton, and one situated proximally, initiating transcription in neural epithelium. The distal promoter is deleted in the insertional mutant, resulting in a regulatory allele (BmprIB(Tg)) lacking cis-sequences necessary for limb BmprIB expression. Mutants fail to generate digit cartilage, indicating that BMPRIB is the physiologic transducer for the formation of digit cartilage from the skeletal blastema. Expansion of BmprIB expression into the limb through acquisition of these distal cis-regulatory sequences appears, therefore, to be an important genetic component driving morphological diversity in distal extremities. GDF5 is a BMP-related signal, which is also required for proper digit formation. Analyses incorporating both Gdf5 and BmprIB(Tg) alleles revealed that BMPRIB regulates chondrogenesis and segmentation through both GDF5-dependent and -independent processes, and that, reciprocally, GDF5 acts through both IB and other type I receptors. Together, these findings provide in vivo support for the concept of combinatorial BMP signaling, in which distinct outcomes result both from a single receptor being triggered by different ligands and from a single ligand binding to different receptors.
Recent experiments have established that Sox9 is required for chondrocyte differentiation. Here, we show that fibroblast growth factors (FGFs) markedly enhance Sox9 expression in mouse primary chondrocytes as well as in C3H10T1/2 cells that express low levels of Sox9. FGFs also strongly increase the activity of a Sox9-dependent chondrocyte-specific enhancer in the gene for collagen type II. Transient transfection experiments using constructs encoding FGF receptors strongly suggested that all FGF receptors, FGFR1-R4, can transduce signals that lead to the increase in Sox9 expression. The increase in Sox9 levels induced by FGF2 was inhibited by a specific mitogen-activated protein kinase kinase (MAPKK)/mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 in primary chondrocytes. In addition, coexpression of a dual-specificity phosphatase, CL100/MKP-1, that is able to dephosphorylate and inactivate mitogen-activated protein kinases (MAPKs) inhibited the FGF2-induced increase in activity of the Sox9-dependent enhancer. Furthermore, coexpression of a constitutively active mutant of MEK1 increased the activity of the Sox9-dependent enhancer in primary chondrocytes and C3H10T1/2 cells, mimicking the effects of FGFs. These results indicate that expression of the gene for the master chondrogenic factor Sox9 is stimulated by FGFs in chondrocytes as well as in undifferentiated mesenchymal cells and strongly suggest that this regulation is mediated by the MAPK pathway. Because Sox9 is essential for chondrocyte differentiation, we propose that FGFs and the MAPK pathway play an important role in chondrogenesis.
Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes.
L-Sox5 and Sox6 are highly identical Sry-related transcription factors coexpressed in cartilage. Whereas Sox5 and Sox6 single null mice are born with mild skeletal abnormalities, Sox5; Sox6 double null fetuses die with a severe, generalized chondrodysplasia. In these double mutants, chondroblasts poorly differentiate. They express the genes for all essential cartilage extracellular matrix components at low or undetectable levels and initiate proliferation after a long delay. All cartilages are thus extracellular matrix deficient and remain rudimentary. While chondroblasts in the center of cartilages ultimately activate prehypertrophic chondrocyte markers, epiphyseal chondroblasts ectopically activate hypertrophic chondrocyte markers. Thick intramembranous bone collars develop, but the formation of cartilage growth plates and endochondral bones is disrupted. L-Sox5 and Sox6 are thus redundant, potent enhancers of chondroblast functions, thereby essential for endochondral skeleton formation.
During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. To analyze the relationship between the two signaling pathways, we used an organ culture system for limb explants of mouse and chick embryos. We manipulated chondrocyte differentiation by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice did not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, could not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. We further investigated the role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes and identified three functions of BMP signaling in this process. First we found that maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel. We further identified a role for BMP signaling in modulating the expression of IHH: Finally, the application of Noggin to mouse limb explants resulted in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This role of BMP signaling is independent of the Ihh/PTHrP pathway.
Indian hedgehog (Ihh), one of the three mammalian hedgehog (Hh) proteins, coordinates proliferation and differentiation of chondrocytes during endochondral bone development. Smoothened (Smo) is a transmembrane protein that transduces all Hh signals. In order to discern the direct versus indirect roles of Ihh in cartilage development, we have used the Cre-loxP approach to remove Smo activity specifically in chondrocytes. Animals generated by this means develop shorter long bones when compared to wild-type littermates. In contrast to Ihh mutants (Ihh(n)/Ihh(n)), chondrocyte differentiation proceeds normally. However, like Ihh(n)/Ihh(n) mice, proliferation of chondrocytes is reduced by about 50%, supporting a direct role for Ihh in the regulation of chondrocyte proliferation. Moreover, by overexpressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, we demonstrate that activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Finally, expression of cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least in part by modulating the transcription of cyclin D1. Taken together, the present study establishes Ihh as a key mitogen in the endochondral skeleton.
Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. The ligands interacting with FGF receptors (FGFRs) in developing bone have remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are not known. Here we show that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibited expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone.
Mutations in fibroblast growth factor (FGF) receptor 3 lead to the human dwarfism syndrome achondroplasia. Using a limb culture system, we have analyzed the role of FGF signaling and its interaction with the Ihh/Pthlh and BMP pathways in regulating chondrocyte differentiation. In contrast to previous suggestions, we demonstrate that FGF signaling accelerates both the onset and the pace of hypertrophic differentiation. We furthermore found that FGF and BMP signaling act in an antagonistic relationship regulating chondrocyte proliferation, Ihh expression, and the process of hypertrophic differentiation. Importantly, BMP signaling rescues the reduced domains of proliferating and hypertrophic chondrocytes in a mouse model for achondroplasia. We propose a model in which the balance of BMP and FGF signaling adjusts the pace of the differentiation process to the proliferation rate.
Lethal skeletal dysplasia from targeted disruption of the parathyroid hormonerelated peptide gene 6. Lanske, B. et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth
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Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3
- J S Colvin
- B A Bohne
- G W Harding
- D G Mcewen
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- V Rosen