Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium.

Epiphyseal chondrocytes cultured in a medium containing 10% serum may be maintained as three dimensional aggregates and differentiate terminally into hypertrophic cells. There is an attendant expression of genes encoding type X collagen and high levels of alkaline phosphatase activity. Manipulation of the serum concentration to optimal levels of 0.1 or 0.01% in this chondrocyte pellet culture system results in formation of features of developing cartilage architecture which have been observed exclusively in growth cartilage in vivo. Cells are arranged in columns radiating out from the center of the tissue, and can be divided into distinct zones corresponding to the recognized stages of chondrocyte differentiation. Elimination of the optimal serum concentration in a chemically defined medium containing insulin eliminates the events of terminal differentiation of defined cartilage architecture. Chondrocytes continue to enlarge into hypertrophic cells and synthesize type X collagen mRNA and protein, but in the absence of the optimal serum concentration, alkaline phosphatase activity does not increase and the cells retain a random orientation. Addition of thyroxine to the chemically defined medium containing insulin and growth hormone results in dose-dependent increases in both type X collagen synthesis and alkaline phosphatase activity, and reproduces the optimal serum-induced morphogenesis of chondrocytes into a columnar pattern. These experiments demonstrate the critical role of thyroxine in cartilage morphogenesis

A case of familial isolated hemihyperplasia

BACKGROUND: Hemihyperplasia (hemihypertrophy) is defined as asymmetric body overgrowth of one or more body parts. Hemihyperplasia can be isolated or be part of well-defined syndromes such as in the case of Beckwith-Wiedemann syndrome (BWS). Isolated hemihyperplasia is usually sporadic, but a number of familial occurrences have been described. CASE PRESENTATION: We describe a Tunisian family in which three maternal cousins and their maternal grandfather present with isolated hemihyperplasia. CONCLUSIONS: The etiology of isolated hemihyperplasia is unknown although in BWS, genomic imprinting has been shown to play a role in the asymmetric overgrowth. Given the similarity between these two conditions, it is possible that both may share a common pathogenesis. We also discuss the possible genetic mechanisms leading to the production of hemihyperplasia in this family

Peripheral chondrosarcoma progression is associated with increased type X collagen and vascularisation

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours

Loss of ATRX in Chondrocytes Has Minimal Effects on Skeletal Development

BACKGROUND:Mutations in the human ATRX gene cause developmental defects, including skeletal deformities and dwarfism. ATRX encodes a chromatin remodeling protein, however the role of ATRX in skeletal development is currently unknown. METHODOLOGY/PRINCIPAL FINDINGS:We induced Atrx deletion in mouse cartilage using the Cre-loxP system, with Cre expression driven by the collagen II (Col2a1) promoter. Growth rate, body size and weight, and long bone length did not differ in Atrx(Col2cre) mice compared to control littermates. Histological analyses of the growth plate did not reveal any differences between control and mutant mice. Expression patterns of Sox9, a transcription factor required for cartilage morphogenesis, and p57, a marker of cell cycle arrest and hypertrophic chondrocyte differentiation, was unaffected. However, loss of ATRX in cartilage led to a delay in the ossification of the hips in some mice. We also observed hindlimb polydactily in one out of 61 mutants. CONCLUSIONS/SIGNIFICANCE:These findings indicate that ATRX is not directly required for development or growth of cartilage in the mouse, suggesting that the short stature in ATR-X patients is caused by defects in cartilage-extrinsic mechanisms

Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

BACKGROUND: Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias) – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. METHODS: Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. RESULTS: We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor). CONCLUSION: Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors

Histomorphological study of the spinal growth plates from the convex side and the concave side in adolescent idiopathic scoliosis

Asymmetrical growth of the vertebrae has been implicated as one possible etiologic factor in the pathogenesis of adolescent idiopathic scoliosis. The longitudinal vertebral growth derives from the endochondral ossification of the vertebral growth plate. In the present study, the growth plates from the convex and concave side of the vertebrae were characterized by the method of histology and immunohistochemistry to evaluate the growth activity, cell proliferation, and apoptosis. Normal zoned architectures were observed in the convex side of the growth plate and disorganized architectures in the concave side. The histological grades were significantly different between the convex and the concave side of the growth plate in the apex vertebrae (P < 0.05). The histological difference was also found significant statistically between end vertebrae and apex vertebrae in the concave side of vertebral growth plates (P < 0.05). The proliferative potential indexes and apoptosis indexes of chondrocytes in the proliferative and hypertrophic zone in the convex side were significantly higher than that in the concave side in the apex vertebral growth plate (P < 0.05). There was a significant difference of the proliferative potential index (proliferating cell nuclear antigen, PCNA index) between convex side and concave side at the upper end vertebra (P < 0.05). The difference of the proliferative potential index and apoptosis index were found significant statistically in the concave side of the vertebral growth plate between end vertebrae and apex vertebrae (P < 0.05). The same result was also found for the apoptosis index (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling assay, TUNEL index) in the convex side of vertebral growth plate between end vertebrae and apex vertebrae (P < 0.05). Some correlation were found between radiographic measurements and proliferation and apoptosis indexes. The difference in histological grades and cellular activity between the convex and concave side indicated that the bilateral growth plate of the vertebrae in AIS patients have different growth kinetics which may affect the curve progression

Long Term Cyclic Pamidronate Reduces Bone Growth by Inhibiting Osteoclast Mediated Cartilage-to-Bone Turnover in the Mouse

Bisphosphonates, used to treat diseases exhibiting increased osteoclast activity, reduce longitudinal bone growth through an as yet undefined mechanism. Pamidronate, an aminobisphosphonate, was given weekly to mice at 0, 1.25, or 2.50 mg/kg/wk beginning at 4 weeks of age. At 12 weeks of age, humeral length, growth plate area, regional chondrocyte cell numbers, chondrocyte apoptosis, TRAP stained osteoclast number, and osteoclast function assessed by cathepsin K immunohistochemistry were quantified. Humeral length was decreased in pamidronate treated mice compared to vehicle control mice, and correlated with greater growth plate areas reflecting greater proliferative and hypertrophic chondrocyte cell numbers with fewer hypertrophic cells undergoing apoptosis. Pamidronate treatment increased TRAP stained osteoclast numbers yet decreased cathepsin K indicating that pamidronate repressed osteoclast maturation and function. The data suggest that long term cyclic pamidronate treatment impairs bone growth by inhibition of osteoclast maturation thereby reducing cartilage-to-bone turnover within the growth plate

Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation

Platelet-­rich plasma (PRP) is used to stimulate the repair of acute and chronic cartilage damage even though there is no definitive evidence of how this is achieved. Chondrocytes in injured and diseased situations frequently re­ express phenotypic biomarkers of immature cartilage so tissue maturation is a potential pathway for restoration of normal structure and function. We used an in vitro model of growth factor­induced maturation to perform a comparative study in order to determine whether PRP can also induce this specific form of remodeling that is characterised by increased cellular proliferation and tissue stiffness. Gene expression patterns specific for maturation were mimicked in PRP treated cartilage, with chondromodulin, collagen types II/X downregulated, deiodinase II and netrin­1 upregulated. PRP increased cartilage surface cell density 1.5­fold (P &#60; 0.05), confirmed by bromodeoxyuridine incorporation and proportionate increases in proliferating cell nuclear antigen gene expression. Atomic force microscopy analysis of PRP and growth factor treated cartilage gave a 5­fold increase in stiffness correlating with a 10­fold upregulation of lysyl oxidase like­1 gene expression (P &#60; 0.001). These data show PRP induces key aspects of post­natal maturation in immature cartilage and provides the basis to evaluate a new biological rationale for its activity when used clinically to initiate joint repair