Mineral Content and Extracellular Matrix Protein Expression in Mouse Growth Plates During Epiphyseal Fusion: An Observational Study

To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice. Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses. ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage. Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

Transforming growth factor alpha controls the transition from hypertrophic cartilage to bone during endochondral bone growth

To characterize the modifications of growth plate in individuals with growth impairment secondary to chronic renal failure, young rats were made uremic by subtotal nephrectomy (NX) and, after 14 d, their tibial growth plates were studied and compared with those of sham-operated rats fed ad libitum (SAL) or pair-fed with NX (SPF). NX rats were growth retarded and severely uremic. Growth plate height (mean +/- SD) was much greater (P<0.05) in NX (868.4+/-85.4 microm) than SAL (570.1+/-93.5 microm) and SPF (551.9+/-99.7 microm) rats as a result of a higher (P<0.05) hypertrophic zone (661.0+/-89.7 versus 362.8+/-71.6 and 353.0+/-93.9 microm, respectively). The increased size of the growth plate was associated with a greater number of chondrocytes and modifications in their structure, particularly in the hypertrophic zone adjacent to bone. In this zone, chondrocytes of NX animals were significantly (P<0.05) smaller (12080.4+/-1158.3 microm3) and shorter (34.1+/-2.5 microm) than those of SAL (16302.8+/-1483.4 microm3 and 37.8+/-2.0 microm) and SPF (14465.8+/-1521.0 microm3 and 36.3+/-1.8 microm). The interface between the growth plate cartilage and the metaphyseal bone appeared markedly irregular in NX rats. Kinetics of chondrocytes was also modified (P<0.05) in the NX rats, which had lower cell turnover per column per day (5.4+/-0.9), longer duration of hypertrophic phase (89.0+/-15.2 h), and reduced cellular advance velocity (7.4+/-2.2 microm/h) compared with SAL (8.0+/-1.6, 32.1+/-6.7 h, and 11.3+/-2.7 microm/h) and SPF (7.2+/-1.1, 34.8+/-5.1 h, and 10.1+/-2.5 microm/h). Cell proliferation was no different among the three groups. Because the growth plates of SPF and SAL rats were substantially not different, modifications observed in the NX rats cannot be attributed to the nutritional deficit associated with renal failure. These findings indicate that chronic renal failure depresses both the activity of the growth plate cartilage by altering chondrocyte hypertrophy and the replacement of cartilage by bone at the metaphyseal end. The two processes are differentially depressed since cartilage resorption is more severely lowered than cartilage enlargement and this leads to an accumulation of cartilage at the hypertrophic zone.

Immunofluorescent localization of estrogen receptor-α in growth plates of rabbits, but not in rats, at sexual maturity

The effects of ovariectomy on growth and estrogen receptor (ER) expression level in the epiphyseal growth plate in mice have been estimated by histomorphometry and immunohistochemistry. Twelve female ddY mice, 8-9 weeks of age, were subjected to bilateral ovariectomy and 12 others were sham operated. They were then killed 8 weeks later. Ovariectomy significantly increased the total thickness of the distal femoral and proximal tibial growth plate cartilage. Ovariectomy caused a 1.4-fold increase in the thickness of the proliferative layer in the distal growth plate of the femur and a 1.3-fold increase in the thickness of the proliferative layer in the proximal growth plate of the tibia. ERalpha and ERbeta immunoreactivity was detected in chondrocytes of the growth plate and the expression level of ERs in epiphyseal plates was increased in ovariectomized mice compared with controls. These data suggest that ERalpha and ERbeta are coexpressed in the growth plates of the mice and that the cartilage growth and the level of expression of ERs in these tissues are hormonally regulated.

Chondrocytes in growth plates are responsible for longitudinal growth in long bones during endochondral ossification. Discoidin domain receptor 1 (Ddr1) is expressed in chondrocytes, but the molecular mechanisms by which DDR1 regulates chondrocyte behaviors during the endochondral ossification process remain undefined. To elucidate Ddr1-mediate chondrocyte functions, we generated chondrocyte-specific Ddr1 knockout (CKOΔDdr1) mice in this study. The CKOΔDdr1 mice showed delayed development of the secondary ossification center and increased growth plate length in the hind limbs. In the tibial growth plate in CKOΔDdr1 mice, chondrocyte proliferation was reduced in the proliferation zone, and remarkable downregulation of Ihh, MMP13, and Col-X expression in chondrocytes resulted in decreased terminal differentiation in the hypertrophic zone. Furthermore, apoptotic chondrocytes were reduced in the growth plates of CKOΔDdr1 mice. We concluded that chondrocytes with Ddr1 knockout exhibit decreased proliferation, terminal differentiation, and apoptosis in growth plates, which delays endochondral ossification and results in short stature. We also demonstrated that Ddr1 regulates the Ihh/Gli1/Gli2/Col-X pathway to regulate chondrocyte terminal differentiation. These results indicate that Ddr1 is required for chondrocytes to regulate endochondral ossification in skeletal development.

Estrogen regulates skeletal growth and promotes epiphyseal fusion. To explore the mechanisms underlying these effects we investigated the expression of estrogen receptor-alpha (ERalpha) and -beta (ERbeta) in rat and rabbit growth plates during postnatal development, using immunohistochemistry. Immunoreactivity for ERalpha and ERbeta was observed in resting zone and proliferative zone chondrocytes at all ages studied for both rat (7, 14, 28 and 70 days of age) and rabbit (1, 7, 28 and 120 days of age). In the rat distal humerus and the rabbit proximal tibia, expression of both receptors in the hypertrophic zone was minimal at early ages, increasing only at the last time point prior to epiphyseal fusion. Expression was rarely seen in the hypertrophic zone of the rat proximal tibia, a growth plate that does not fuse until late in life. Therefore, we conclude that ERalpha and ERbeta are both expressed in the mammalian growth plate. The temporal and anatomical pattern suggests that ER expression in the hypertrophic zone in particular may play a role in epiphyseal fusion.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of parathyroid hormone-related protein (PTHrP) but increased Indian Hedgehog (IHH) signaling throughout the growth plate. Attenuation of smoothened-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

BackgroundEndochondral ossification, the process through which long bones are formed, involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. In a previous publication we showed that pharmacological inhibition of the PI3K signaling pathway results in reduced endochondral bone growth, and in particular, shortening of the hypertrophic zone in a tibia organ culture system. In this current study we aimed to investigate targets of the PI3K signaling pathway in hypertrophic chondrocytes.Methodology/Principal FindingsThrough the intersection of two different microarray analyses methods (classical single gene analysis and GSEA) and two different chondrocyte differentiation systems (primary chondrocytes treated with a pharmacological inhibitor of PI3K and microdissected growth plates), we were able to identify a high number of genes grouped in GSEA functional categories regulated by the PI3K signaling pathway. Genes such as Phlda2 and F13a1 were down-regulated upon PI3K inhibition and showed increased expression in the hypertrophic zone compared to the proliferative/resting zone of the growth plate. In contrast, other genes including Nr4a1 and Adamts5 were up-regulated upon PI3K inhibition and showed reduced expression in the hypertrophic zone. Regulation of these genes by PI3K signaling was confirmed by quantitative RT-PCR. We focused on F13a1 as an interesting target because of its known role in chondrocyte hypertrophy and osteoarthritis. Mouse E15.5 tibiae cultured with LY294002 (PI3K inhibitor) for 6 days showed decreased expression of factor XIIIa in the hypertrophic zone compared to control cultures.Conclusions/SignificanceDiscovering targets of signaling pathways in hypertrophic chondrocytes could lead to targeted therapy in osteoarthritis and a better understanding of the cartilage environment for tissue engineering.

Disclosure: K. Hansdah: None. A. Rama-Krishnan: None. K. Tailor: None. J. Lui: None. J. Baron: None.Introduction: During malnutrition, growth plate chondrogenesis is impaired, which results in a decreased bone length and therefore a decrease in overall body size. Objective: To explore the molecular mechanisms by which caloric restriction regulates growth plate chondrogenesis. Methods: The study investigated the effect of maternal food restriction during lactation on offspring in C57BL/6 mice. Beginning at 3 days after parturition, lactating dams were fed either ad-libitum (AL) or calorie-restricted (CR) with 60% of the food consumed by the AL group. After 9 days of caloric restriction, offspring were euthanized for measurements of body and tibial length, and histological analysis of the growth plate. We performed Visium HD spatial gene expression analysis to compare gene expressions in specific regions of growth plate. Results: CR mice exhibited lower body weight, body length, and tibial length compared to the AL group (all p less than 0.01). Histological analysis showed a significant decrease in heights of the growth plate, proliferative and hypertrophic zone in CR mice compared to AL mice (all p less than 0.01). Cell proliferation (by EdU incorporation) in the proliferative zone of growth plate was significantly (p=0.03) decreased in CR group compared to AL group. We also detected significantly (p less than 0.0001) lower levels of serum IGF-1 protein in CR group compared to AL (by ELISA). Spatial transcriptomic profiling revealed markedly upregulated expression of anti-apoptotic gene Bcl2 in the resting zone chondrocytes, which could enhance the survival of chondroprogenitor cells. Conversely, Gnai1 and Gna11, both of which are implicated in PTHrP and G-protein coupled receptor signaling that are important in the resting zone, were downregulated due to caloric restriction. In the proliferative zone we found that ITGA11, a gene associated with cell proliferation, was significantly downregulated in caloric restricted mice. Similarly, we found decreased expression of ADAMST4, which is involved in the degradation of aggrecan and matrix turnover, suggesting a slower rate of cartilage remodeling in proliferative zone under caloric restriction. In the hypertrophic zone, expression of Gdf10 and Prelp, which are markers typically associated with the proliferative zone, were upregulated suggesting delayed hypertrophy in caloric restricted mice. In contrast, we found downregulation of IGF1R, suggesting decreased IGF-1 signaling in the hypertrophic zone during caloric restriction. Conclusions: Our findings provide molecular insights into dysregulated growth plate chondrogenesis and cell survival that may help explain the etiology of short stature due to malnutrition.Presentation: Saturday, July 12, 2025

Epiphyseal plate cartilage, epiphyseal cartilage, synchondroseal cartilage and mandibular condylar cartilage were studied morphologically and histochemically in 14 days old rats. Ordinary decalcified paraffin sections were stained with hematoxylin & eosin, van Giesons connective tissue stain, or toluidine blue, and used for morphological studies of the different cartilaginous structures. Undecalcified cryostat sections were used for demonstration of acid and alkaline phosphatase. The enzyme activity was tested for at regular intervals during incubation from 15 sec to 120 min. The morphologic study revealed that a marked similarity of construction exists between epiphyseal plate cartilage and synchrondroseal cartilage. The construction of epiphyseal and condylar cartilage differ from that of the other two structures and also differ mutually. With small variations the reaction for both alkaline and acid phosphatase was found to be identical in the zones of erosion, hypertrophy and maturation of the four structures. Intercellularly, acid phosphatase is present in all zones in the synchondroseal and the epiphyseal plate cartilage, while in the epiphyseal and condylar cartilages it is only present in the zones of erosion, hypertrophy and maturation. The identical reaction for acid phosphatase in the epiphyseal and the condylar cartilage is thought, in all likelihood, to be accidental. When kinetic conditions are taken into account, epiphyseal cartilage seems to react like epiphyseal plate and synchondroseal cartilage, while the condylar cartilage takes up an exceptional position among growth cartilages.

Misconceptions (1): epiphyseal fusion causes cessation of growth