Hypertrophic Growth Plate Chondrocytes Research Articles

A conserved regulation of cell expansion underlies notochord mechanics, spine morphogenesis, and endochondral bone lengthening.

Cell size is a key contributor to tissue morphogenesis 1 . As a notable example, growth plate hypertrophic chondrocytes use cellular biogenesis and disproportionate fluid uptake to expand 10-20 times in size to drive lengthening of endochondral bone 2,3 . Similarly, notochordal cells expand to one of the largest cell types in the developing embryo to drive axial extension 4-6 . In zebrafish, the notochord vacuolated cells undergo vacuole fusion to form a single large, fluid-filled vacuole that fills the cytoplasmic space and contributes to vacuolated cell expansion 7 . When this process goes awry, the notochord lacks sufficient hydrostatic pressure to support vertebral bone deposition resulting in adult spines with misshapen vertebral bones and scoliosis 8 . However, it remains unclear whether endochondral bone and the notochord share common genetic and cellular mechanisms for regulating cell and tissue expansion. Here, we demonstrate that the 5'-inositol phosphatase gene, inppl1a , regulates notochord expansion, spine morphogenesis, and endochondral bone lengthening in zebrafish. Furthermore, we show that inppl1a regulates notochord expansion independent of vacuole fusion, thereby genetically decoupling these processes. We demonstrate that inppl1a -dependent notochord expansion is essential to establish normal mechanical properties of the notochord to facilitate the development of a straight spine. Finally, we find that inppl1a is also important for endochondral bone lengthening in fish, as has been shown in the human INPPL1 -related endochondral bone disorder, Opsismodysplasia 9 . Overall, this work reveals a conserved mechanism of cell size regulation that influences disparate tissues critical for skeletal development and short-stature disorders.

Generation of an Ihh-mKate2-Dre knock-in mouse line.

Indian hedgehog (Ihh), a member of the Hh family, plays important roles in vertebrate development and homeostasis. To improve our understanding of the function of Ihh-expressing cells and their progeny as well, we generate an Ihh-mKate2tomm20 -Dre knock-in mouse line that can label Ihh-positive cells with a fluorescence protein mKate2 and trace Ihh-positive cells and their progeny via Dre-mediated recombination. Consistent with previous reports, we verified Ihh expression in hypertrophic chondrocytes of growth plate and granulosa cells of ovarian follicles by mKate2 immunostaining, and meanwhile confirmed Dre activity in these cells via a Dre reporter mouse line Rosa26-confetti2. We also found, for the first time, that Ihh can mark some cell types, including retinal ganglion cells, Purkinje cells, and gallbladder epithelial cells. Taken together, the Ihh-mKate2tomm20 -Dre mouse is a genetic tool for examining the precise expression profile of Ihh and tracing Ihh-expressing cells and their progeny.

Reconstitution of the host holobiont in germ-free born male rats acutely increases bone growth and affects marrow cellular content.

Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiological interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-wk-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, whereas the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short-chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyrate. Furthermore, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders.

Specificity for the Epiphyseal Round Cell Layer is Significantly Associated With Height GWAS​

Human height is a model polygenic trait with thousands of height-related SNPs identified in GWAS to date. An important determinant of height is the proliferation and hypertrophy of growth plate chondrocytes during childhood long bone elongation. Connecting the expression of specific genes that affect skeletal biology to associated variants in GWAS remains a difficult challenge. To connect the genetics of height and growth plate gene expression, we studied the relationship between gene expression in the murine growth plate and common-variant associations from GWAS of height. To obtain gene expression data from the growth plate, we dissected three layers of murine tibial growth plates, extracted RNA from each layer, and measured expression using the Affymetrix GeneChip 430 3.0. For each gene, we derived a specificity score for each growth plate layer, and SNP-level p-values from a published GWAS of height (N~700000) were combined into gene-level p-values using MAGMA. We then used MAGMA to test for association between specificity of expression for each growth plate layer and the GWAS gene level p-values for height. We found that specificity for the round cell layer is significantly associated with height GWAS p-values (p = 8.5x10-9). This association remains when we condition on each of the other cell layers and on membership in a set of genes from OMIM that cause skeletal growth disorders (3.3x10-8 < p < 4.1x10-6). We replicated this result in a RNA-seq dataset of maturing chondrocytes sampled at three time points during development in vitro (days 3, 5, and 10): we found that z-scores for expression in the earliest two days of development are significantly associated with gene-level p-values from height GWAS (pDay3 = 1.2x10-21 and pDay5 = 2.0x10-20) and that this association remains after conditioning on the other timepoints and on the OMIM gene set (3.1x10-20 < pDay3 < 8.3x10-5; 3.7x10-19 < pDay5 < 0.002). We then performed pathway analysis of genes that are both highly specific to the round layer and highly significant in GWAS using Enrichr. Together, our results suggest that genes expressed in early chondrocyte development (the round cell layer) are particularly relevant to the contribution of growth plate-expressed genes to height. This conclusion both sheds light on the regulation of human skeletal growth and also helps prioritize relevant genes implicated from the height GWAS in skeletal biology.

Gut Microbiota Accelerates Bone Growth and Marrow Adiposity of the Adolescent Gnotobiotic Rat

Skeletal growth and bone homeostasis are significant holobiome components strongly tied to the gut microbiota. We aimed to explore the requirement of gut microbiome for skeletal maturation in an adolescent organism. For our approach, we co-housed young (7-week-old) germ-free (GF) rats with conventional rats of the same age, sex (male), and strain (Sprague Dawley) for 10 days to naturally conventionalize GF (GFC) rats with the gut microbiota by coprophagy. Bone morphometry by micro-CT scanning and histological staining revealed accelerated longitudinal and radial bone growth in GFC rats when compared to GF rats. Changes in bone mass and structure for GFC rats encompassed an increase of bone accrual in cortical and trabecular compartments, greater tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. Analysis of bone marrow at the tibia midshaft depicted more adipocyte number, greater adipocyte density, and altered cell size distribution to favor small adipocytes, but reduced megakaryocytes, in GFC rats. Interestingly, putative effector genes responsible for bone mass accumulation were found to be comparable between GF and GFC rats, which shifted our focus onto systemic host and microbial regulatory factors. Assessment of serum markers indicated that the active bone formation could be due to elevated levels of 25-hydroxy vitamin D and alkaline phosphatase. Importantly, 16S ribosomal RNA gene sequencing demonstrated that the prominent bacterial colonizers were Clostridiaceae and Clostridium members, which are potent short chain fatty acid (SCFA) producers. 1H-NMR metabolomics in the cecal contents showed a dramatic increase of SCFA, specifically butyrate, following bacterial colonization. In relation with the literature, our observation of increased hepatic insulin-like growth factor-1 (IGF-1) expression and systemic IGF-1 levels suggest that butyrate promotes bone growth through the somatotropic axis. Collectively, our study demonstrates that gut microbiota can deliver signals to the skeleton for accelerating bone expansion and accumulating marrow fat.

New Developments in the Treatment of X-Linked Hypophosphataemia: Implications for Clinical Management.

X-linked hypophosphataemia (XLH) is due to mutations in phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and represents the most common heritable form of rickets. In this condition, the hormone fibroblast growth factor 23 (FGF23) is produced in excessive amounts for still unknown reasons, and causes renal phosphate wasting and suppression of 1,25-dihydroxyvitamin D, leading to low serum phosphate concentrations. Prolonged hypophosphataemia decreases apoptosis of hypertrophic chondrocytes in growth plates (causing rickets) and decreases mineralisation of existing bone (causing osteomalacia). In contrast to historical conventional treatment with oral phosphate supplements and active vitamin D for the last 50 years, the new anti-FGF23 antibody treatment (burosumab) targets the primary pathology by blocking FGF23, thereby restoring phosphate homeostasis. In this review, we describe the changes in treatment monitoring, treatment targets and long-term treatment goals, including future opportunities and challenges in the treatment of XLH in children.

New morphological evidence of the 'fate' of growth plate hypertrophic chondrocytes in the general context of endochondral ossification.

The 'fate' of growth plate hypertrophic chondrocytes has been long debated with two opposing theories: cell apoptosis or survival with transformation into osteogenic cells. This study was carried out on the proximal tibial growth plate of rabbits using light microscopy, scanning and transmission electron microscopy. We focused particularly on the orientation of the specimens included in order to define the mineral deposition and the vascular invasion lines and obtain histological and ultrastructural images at the corresponding height of the plate. Chondrocyte morphology transformation through the maturation process (characterized by vesicles and then large cytoplasmic lacunae before condensation, fragmentation and disappearance of the nuclear chromatin) did not correspond to that observed in the 'in vitro' apoptosis models. These findings rather suggested the passage of free water from the cartilage matrix into a still live cell (swelling). The level of these changes suggested a close relationship with the mineral deposition line. Furthermore, the study provided evidence that the metaphyseal capillaries could advance inside the columns of stacked hypertrophic chondrocytes (delimited by the intercolumnar septa) without the need for calcified matrix resorption because the thin transverse septa between the stacked chondrocyte (below the mineral deposition line) were not calcified. The zonal distribution of cell types (hypertrophic chondrocytes, osteoblasts, osteoclasts and macrophages) did not reveal osteoclasts or chondroclasts at this level. Morphological and morphometric analysis recorded globular masses of an amorphous, necrotic material in a zone 0-70μm below the vascular invasion line occasionally surrounded by a membrane (indicated as 'hypertrophic chondrocyte ghosts'). These masses and the same material not bound by a membrane were surrounded by a large number of macrophages and other blood cell precursors, suggesting this could be the cause of macrophage recall and activation. The most recent hypotheses based on genetic and lineage tracing studies stating that hypertrophic chondrocytes can survive and transform into osteoblasts and osteocytes (trans-differentiation) were not confirmed by the ultrastructural morphology or by the zonal comparative counting and distribution of cell types below the vascular invasion line.

SAT-283 A Mutation In Ccdc53 Affects Pth1r Trafficking And Causes Disproportionate Short Stature With Noonan-like Facies

In many children with short stature, the genetic etiology remains unknown. Short stature due to defects in trafficking of membrane-associated proteins, such as receptors, has not been reported. A mother and two daughters presented with disproportionate short stature and Noonan-like facies. Exome sequencing, confirmed by Sanger sequencing, identified a de novo dominant mutation (L69F) in CCDC53. CCDC53 normally binds to WASH1 within a protein complex termed the WASH complex. To study the effect of the mutation on this interaction, CCDC53 was first ablated in HEK293 cells (CCDC53KO-HEK) by CRISPER-Cas9 and then wild-type (WT) or mutant (mut) CCDC53 or empty vector (EV) was expressed in these cells. The identified mutation impaired CCDC53 interaction with WASH1 as assessed by co-immunoprecipitation. CCDC53 and the WASH complex are involved in trafficking of cell-surface proteins that have undergone endocytosis, including G-coupled protein receptors that contain a PDZ motif, such as PTH1R. We therefore hypothesized that the mutation would affect PTH1R endocytic trafficking. CCDC53KO-HEK cells were transfected with vectors to express PTH1R and either EV, WT or mut CCDC53 and treated with PTH, after which endosomes were extracted and PTH1R was detected by western blot. Compared to WT, EV and mut CCDC53 showed decreased PTH1R localization in endosomes. In this system, the mutation also decreased generation of cAMP by extracellular PTH (P=0.001 vs WT), indicating that the mutation interferes with PTH1R signal transduction. PTH1R acts as the receptor for PTHrP and prevents premature hypertrophy of growth plate chondrocytes. We therefore hypothesized that the CCDC53 mutation would allow premature hypertrophy of growth plate chondrocytes. Primary rat epiphyseal chondrocytes were transfected with siRNA against Ccdc53 and with vectors to express either WT or mut Ccdc53. Compared to WT, mut Ccdc53 led to increased expression of hypertrophy-associated genes: Col10a1 (P=0.001), Ihh (P=0.031), and Mmp13 (P=0.003), consistent with the hypothesized effect on hypertrophic differentiation. In conclusion, a mutation in CCDC53 was identified in a family with disproportionate short stature and Noonan-like facies. The mutation interfered with the ability of CCDC53 to participate in the WASH complex, affecting endosomal localization of PTH1R, PTH1R signal transduction, and chondrocyte hypertrophy. Because the timing of growth plate chondrocyte hypertrophy is critical for linear growth, the findings provide a potential explanation for the short stature in this disorder. More generally, the findings also indicate that endocytic trafficking of PTH1R by the WASH complex plays an important role in the regulation of human linear growth.

Endoplasmic reticulum stress is induced in growth plate hypertrophic chondrocytes in G610C mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a hereditary bone disorder most commonly caused by autosomal dominant mutations in genes encoding type I collagen. In addition to bone fragility, patients suffer from impaired longitudinal bone growth. It has been demonstrated that in OI, an accumulation of mutated type I collagen in the endoplasmic reticulum (ER) induces ER stress in osteoblasts, causing osteoblast dysfunction leading to bone fragility. We hypothesize that ER stress is also induced in the growth plate where bone growth is initiated, and examined a mouse model of dominant OI that carries a G610C mutation in the procollagen α2 chain. The results demonstrated that G610C OI mice had significantly shorter long bones with growth plate abnormalities including elongated total height and hypertrophic zone. Moreover, we found that mature hypertrophic chondrocytes expressed type I collagen and ER dilation was more pronounced compared to wild type littermates. The results from in vitro chondrocyte cultures demonstrated that the maturation of G610C OI hypertrophic chondrocytes was significantly suppressed and ER stress related genes were upregulated. Given that the alteration of hypertrophic chondrocyte activity often causes dwarfism, our findings suggest that hypertrophic chondrocyte dysfunction induced by ER stress may be an underlying cause of growth deficiency in G610C OI mice.

Identification of distinctive DNA methylation patterns correlated with differential gene expression in murine hypertrophic chondrocytes in vitro

Purpose: Osteoarthritis (OA) is joint disorder in which the articular cartilage is permanently damaged. Articular chondrocytes undergo profound phenotypic changes in OA, part of which mimic developmental steps observed in hypertrophic growth plate chondrocytes, including increased Runx2, Mmp13 and collagen type X expression. Hypertrophy is a normal step during endochondral ossification processes, which are required for normal chondrogenesis, long bone growth and ossification. Thus, to better understand the molecular mechanisms that trigger a hypertrophy-like reprogramming of articular chondocytes in osteoarthritis, we require a better understanding of gene regulation during hypertrophy. DNA methylation is one of the principal mechanisms by which cells maintain dominant and stable phenotypes, and altered DNA methylation is associated with abnormal gene expression in different pathologies, including OA. This study aims to identify genes that are differentially expressed during hypertrophic differentiation in vitro, and that are correlated with changes in DNA methylation patterns. Methods: Chondrocytes were isolated from articular cartilage obtained from 5 to 6 days old C57BL/6J mice. We used 3D/pellet culture systems to drive chondrocyte hypertrophy in vitro. Pellets were harvested at 7 and 21 days for RNA or DNA isolation. Total RNA was used for RNA sequencing, and DNA was used for Reduced Representation Oxidative Bisulfite Sequencing (RRoxBS) at the Epigenomics Core at Weill Cornell Medicine (EC-WCM). After sequencing, the RNAseq reads were processed using a dedicated RNAseq pipeline, and the expression of selected genes was further validated using SYBR-green-based real-time PCR analyses (RTqPCR). For methylation profiling, libraries were made using the ERRBS protocol. Per sample, 50-60 million ERRBS reads were aligned and processed using the EC-WCM’s in-house bioinformatics pipeline to yield methylation values for each CpG. Oxidative bisulfite (oxBS) technology was applied to distinguish between 5mC and 5hmC. Differential DNA methylation was analyzed using Methylkit and eDMR, and subsequent assessment and annotation of differentially methylated regions was done using R Core Team. Results: Hypertrophic differentiation in vitro was confirmed by RTqPCR analyses, which showed upregulation of Mmp13, collagen X and Runx2 and down-regulation of Sox9 and collagen II. RNAseq analyses uncovered more than 3000 differentially expressed genes between 7 (non hypertrophic) and 21 (hypertrophic) days (p less than 0.01). Among these genes, we identified novel targets with previously unreported roles in chondrocyte hypertrophy. Analysis of RRoxBS sequencing data also revealed changes in DNA methylation patterns accompanying hypertrophic differentiation and differential expression of hypertophic-related genes, highlighting the contribution of DNA methylation to gene expression during chondrocyte hypertrophy. Conclusions: Our results suggest that DNA methylation significantly contributes to changes in phenotype and gene expression during chondrocyte hypertrophy. In addition, we found a number of differentially expressed genes with previously unreported roles in hypertrophic chondrocytes, and with potential relevance to developmental and pathological processes. Ongoing studies are aimed to verify the results obtained in vitro using in vivo systems of chondrocyte hypertrophy during developmental processes (i.e., growth plate chondrocytes) and of hypertrophic-like conversion during OA, using surgical murine models of OA and human samples obtained from OA patients. We believe that our novel integrative approaches have the potential of uncovering new targets for therapeutic intervention.

Activation of mTORC1 in chondrocytes does not affect proliferation or differentiation, but causes the resting zone of the growth plate to become disordered

There are several pitfalls associated with research based on transgenic mice. Here, we describe our interpretation and analysis of mTORC1 activation in growth plate chondrocytes and compare these to a recent publication (Yan et al., Nature Communications 2016, 7:11151). Both laboratories employed TSC1-floxed mice crossed with collagen type 2-driven Cre (Col2-Cre), but drew substantially different conclusions. It was reported that activation of mechanistic target of rapamycin complex 1 (mTORC1) via Tsc1 ablation promotes the hypertrophy of growth plate chondrocytes, whereas we observe only disorganization in the resting zone, with no effect on chondrocyte hypertrophy or proliferation. Here, we present our data and discuss the differences in comparison to the earlier phenotypic characterization of TSC1 ablation in cartilage. Importantly, we detect Col2-Cre activity in non-cartilaginous tissues (including the brain) and discuss it in relation to other studies reporting non-cartilaginous expression of collagen alpha(1) II. Altogether, we conclude that mouse phenotypes following genetic ablation using Col2-Cre should be interpreted with care. We also conclude that activation of mTORC1 by TSC1 ablation in postnatal chondrocytes with inducible Col2-Cre (Col2-CreERt) leads to disorganization of the resting zone but causes no changes in chondrocyte proliferation or differentiation.

SHP2 Regulates the Osteogenic Fate of Growth Plate Hypertrophic Chondrocytes

Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using “Cre-loxP”-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.

Mesoderm-specific Stat3 deletion affects expression of Sox9 yielding Sox9-dependent phenotypes.

To date, mutations within the coding region and translocations around the SOX9 gene both constitute the majority of genetic lesions underpinning human campomelic dysplasia (CD). While pathological coding-region mutations typically result in a non-functional SOX9 protein, little is known about what mechanism(s) controls normal SOX9 expression, and subsequently, which signaling pathways may be interrupted by alterations occurring around the SOX9 gene. Here, we report the identification of Stat3 as a key modulator of Sox9 expression in nascent cartilage and developing chondrocytes. Stat3 expression is predominant in tissues of mesodermal origin, and its conditional ablation using mesoderm-specific TCre, in vivo, causes dwarfism and skeletal defects characteristic of CD. Specifically, Stat3 loss results in the expansion of growth plate hypertrophic chondrocytes and deregulation of normal endochondral ossification in all bones examined. Conditional deletion of Stat3 with a Sox9Cre driver produces palate and tracheal irregularities similar to those described in Sox9+/- mice. Furthermore, mesodermal deletion of Stat3 causes global embryonic down regulation of Sox9 expression and function in vivo. Mechanistic experiments ex vivo suggest Stat3 can directly activate the expression of Sox9 by binding to its proximal promoter following activation. These findings illuminate a novel role for Stat3 in chondrocytes during skeletal development through modulation of a critical factor, Sox9. Importantly, they further provide the first evidence for the modulation of a gene product other than Sox9 itself which is capable of modeling pathological aspects of CD and underscore a potentially valuable therapeutic target for patients with the disorder.

CCAAT/enhancer binding protein β (C/EBPβ) regulates the transcription of growth arrest and DNA damage-inducible protein 45 β (GADD45β) in articular chondrocytes

Osteoarthritis (OA) is a whole joint disease characterized by cartilage degradation, which causes pain and disability in older adults. Our previous work showed that growth arrest and DNA damage-inducible protein 45 β (GADD45β) is upregulated in chondrocyte clusters in OA cartilage, especially in the early stage of this disease. CCAAT/enhancer binding protein β (C/EBPβ) is expressed in the hypertrophic growth plate chondrocytes and functions in synergy with GADD45β. Here, the presence and localization of these proteins was assessed by immunohistochemistry using articular cartilage from OA patients, revealing colocalization of C/EBPβ and GADD45β in OA chondrocytes. GADD45β promoter analysis was performed to determine whether C/EBPβ directly regulates GADD45β transcription. Furthermore, we analyzed the effect of C/EBPβ on Gadd45β gene regulation in articular chondrocytes in vivo and in vitro. Immunohistochemical analysis of C/ebpβ-haploinsufficient mice (C/ebpβ+/−) cartilage showed that C/ebpβ haploinsufficiency led to reduced Gadd45β gene expression in these cells. In vitro, we evaluated the effects of conditional C/EBPβ overexpression driven by the cartilage oligomeric matrix protein (Comp) promoter in mComp-tTA;pTRE-Tight-BI-DsRed-mC/ebpβ transgenic mice. C/EBPβ overexpression significantly stimulated Gadd45β gene expression in articular chondrocytes. Taken together, our data demonstrate that C/EBPβ plays a central role in controlling Gadd45β gene expression in these cells.

Losartan increases bone mass and accelerates chondrocyte hypertrophy in developing skeleton

Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development.

Fate of growth plate hypertrophic chondrocytes: Death or lineage extension?

The vertebrate growth plate is an essential tissue that mediates and controls bone growth. It forms through a multistep differentiation process in which chondrocytes differentiate, proliferate, stop dividing and undergo hypertrophy, which entails a 20-fold increase in size. Hypertrophic chondrocytes are specialized cells considered to be the end state of the chondrocyte differentiation pathway, and are essential for bone growth. They are characterized by expression of type X collagen encoded by the Col10a1 gene, and synthesis of a calcified cartilage matrix. Whether hypertrophy marks a transition preceding osteogenesis, or it is the terminal differentiation stage of chondrocytes with cell death as the ultimate fate has been the subject of debate for over a century. In this review, we revisit this debate in the light of new findings arising from genetic-mediated lineage tracing studies showing that hypertrophic chondrocytes can survive at the chondro-osseous junction and further make the transition to become osteoblasts and osteocytes. The contribution of chondrocytes to the osteoblast lineage has important implications in bone development, disease and repair.

Insulin exerts direct, IGF-1 independent actions in growth plate chondrocytes.

Insufficient insulin production or action in diabetic states is associated with growth retardation and impaired bone healing, while the underling mechanisms are unknown. In this study, we sought to define the role of insulin signaling in the growth plate. Insulin treatment of embryonic metatarsal bones from wild-type mice increased chondrocyte proliferation. Mice lacking insulin receptor (IR) selectively in chondrocytes (CartIR−/−) had no discernable differences in total femoral length compared to control littermates. However, CartIR−/− mice exhibited an increase in chondrocyte numbers in the growth plate than that of the controls. Chondrocytes lacking IR had elevated insulin-like growth factor (IGF)-1R mRNA and protein levels. Subsequently, IGF-1 induced phosphorylation of Akt and ERK was enhanced, while this action was eliminated when the cells were treated with IGF-1R inhibitor Picropodophyllin. Deletion of the IR impaired chondrogenic differentiation, and the effect could not be restored by treatment of insulin, but partially rescued by IGF-1 treatment. Intriguingly, the size of hypertrophic chondrocytes was smaller in CartIR−/− mice when compared with that of the control littermates, which was associated with upregulation of tuberous sclerosis complex 2 (TSC2). These results suggest that deletion of the IR in chondrocytes sensitizes IGF-1R signaling and action, IR and IGF-1R coordinate to regulate the proliferation, differentiation and hypertrophy of growth plate chondrocytes.

The role of smoothened for the osteophyte formation in mouse osteoarthritis

Objective: Osteoarthritis (OA) shows a progressive degeneration of articular cartilage and osteophyte formation. Endochondral ossification is considered an important pathophysiological process for the osteophyte formation. Chondrocyte hypertrophy is also an essential process required for endochondral bone formation. It is reported that the expression of Indian hedgehog (Ihh) in pre- and early hypertrophic chondrocytes of growth plate and osteophytes, and a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy. Therefore, in this study, we investigated whether the deletion of smoothened (Smo), an essential transducer of Hedgehog signaling, affected osteophyte formation in mouse osteoarthritis. Methods: To investigate the role of Smo in osteophyte formation, we crossed Smo/flox mice, which express a null allele of Smo upon exposure to Cre recombinase, with RosaCreER(T) mice to generate Smo conditional knockout (CKO) mice upon administration of Tamoxifen. CKO mice and Cre-negative control mice were administrated Tamoxifen on 3, 5 weeks after birth and 3days, 1day before surgery, resulting in downregulated Hedgehog signaling. OA was surgically induced in 8-week-old CKO mice and Cre-negative control mice using the anterior cruciate ligament transection model. 1week after surgery, mice were caged in climbing cage to accelerate degenerative change. Mice were sacrificed at 4, 6, 8 weeks after surgery. Histomorphometric and immunohistochemical (IHC) assessment was performed. Result: The immature osteophyte-like lesion composed of chondrocytes and cartilage was observed at the posterior margins of tibia in both CKO mice and Cre-negative control mice in 4 weeks post-surgery. However, the maturity of osteophytes was suppressed in CKO mice compared to Cre-negative control mice at 6 and 8 weeks post-surgery. In contrast, Cre-negative control mice showed mature ossifying lesion and hypertrophic chondrocytes in osteophytes, and had a larger and more mature osteophytes at 6 and 8 weeks post-surgery by histomorphometric analysis. Furthermore, it is revealed that lesser expression of type X collagen in osteophytes of CKO mice. These data suggests that the deletion of Smo delayed the formation of osteophytes and the suppression of the endochondral ossification process with blocking the hypertrophy of chondrocytes and type X collagen expression. Conclusions: Smo plays a crucial role of osteophyte formation in mouse osteoarthritis, especially, the maturity of osteophytes through endochondral ossification process.

Thyroid Hormone-Induced Hypertrophy in Mesenchymal Stem Cell Chondrogenesis Is Mediated by Bone Morphogenetic Protein-4

Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair.

Specification of chondrocytes and cartilage tissues from embryonic stem cells

Osteoarthritis primarily affects the articular cartilage of synovial joints. Cell and/or cartilage replacement is a promising therapy, provided there is access to appropriate tissue and sufficient numbers of articular chondrocytes. Embryonic stem cells (ESCs) represent a potentially unlimited source of chondrocytes and tissues as they can generate a broad spectrum of cell types under appropriate conditions in vitro. Here, we demonstrate that mouse ESC-derived chondrogenic mesoderm arises from a Flk-1(-)/Pdgfrα(+) (F(-)P(+)) population that emerges in a defined temporal pattern following the development of an early cardiogenic F(-)P(+) population. Specification of the late-arising F(-)P(+) population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo.