Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis.
Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues. (+info)
Expression of tissue transglutaminase in the developing chicken limb is associated both with apoptosis and endochondral ossification.
The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction. (+info)
Assessment of bone mineral density in adults with a history of juvenile chronic arthritis: a cross-sectional long-term followup study.
OBJECTIVE: To assess bone mineral density (BMD) and bone turnover in adults with a history of juvenile chronic arthritis (JCA) or persistent JCA, and to identify predictors of reduced BMD. METHODS: Sixty-five white patients (mean age 32.2 years) with a history of JCA and 65 age-, sex-, height-, and weight-matched healthy control subjects participated in the study. Densitometry of the left hip and the lumbar spine was performed, and osteocalcin (bone formation marker) and crosslinks (bone resorption marker) were measured. In addition, bone-related clinical parameters were assessed in the JCA group. RESULTS: BMD in the hip and lumbar spine was significantly lower in the JCA group than in the controls. Levels of osteocalcin and crosslinks were significantly increased in the JCA group. According to WHO definitions, significantly more subjects in the JCA group had "osteopenia" and "osteoporosis" than would be expected in a normal population sample. Active disease at the time of the study (1996-1997), baseline erosions evaluated in 1979, Steinbrocker functional class in 1996-1997, polyarticular course of JCA, and history of systemic steroid treatment for more than 1 year were significantly associated with reduced BMD. In linear regression analysis including both the JCA and control groups, presence of JCA proved to be the factor most strongly associated with reduced BMD, explaining approximately 20% of its variation. CONCLUSION: Reduced BMD and evidence of increased bone turnover suggest that JCA patients may be at risk of developing premature osteoporosis and associated fractures later in life. The data are consistent with the concept that BMD in JCA is determined by many factors. (+info)
Bone histology in patients with nephrotic syndrome and normal renal function.
BACKGROUND: The prevalence of metabolic bone disease in patients with nephrotic syndrome (NS) at normal level of renal function remains uncertain. METHODS: To address this issue, we studied 30 patients (20 men and 10 women, mean age 27.3 +/- 11.7 years) with NS who had normal renal function (mean creatinine clearance 103 +/- 4 ml/min). We evaluated their serum calcium, phosphorus, alkaline phosphatase, immunoreactive parathyroid hormone (iPTH), vitamin D metabolites, urinary calcium, and skeletal survey. The extent of bone mineralization was analyzed by histomorphometric analysis of iliac crest bone biopsy specimens in all patients. The findings on bone histology were correlated with biochemical parameters. RESULTS: The mean duration of NS was 35.5 +/- 26.9 months, with a protein excretion of 7.3 +/- 3.2 g/24 hr and a serum albumin of 2.2 +/- 0.8 g/dl. Total serum calcium was 7.8 +/- 0.8 mg/dl, whereas ionized calcium was 5.7 +/- 0.7 mg/dl, phosphorus 3.2 +/- 1.2 mg/dl, and alkaline phosphatase 149 +/- 48.6 U/liter. Serum iPTH levels were normal in all except two patients. The mean serum 25-hydroxyvitamin D [25(OH)D] level was 3.9 +/- 1.2 ng/ml (normal 15 to 30 ng/ml), whereas 1,25-dihydroxyvitamin D was 24 +/- 4.7 pg/ml (normal 16 to 65). There was an inverse correlation between serum levels of 25(OH)D and the magnitude of proteinuria (r = -0.42, P < 0.05). The mean 24-hour urinary calcium excretion was 82 +/- 21 mg/day. The skeletal survey was normal in all patients. Bone histology was normal in 33.3% of the patients, whereas 56.7% had isolated osteomalacia (OSM), and 10% had an increased bone resorption in association with defective mineralization. The severity of OSM measured by mineralization lag time correlated linearly with the duration (r = 0.94, P < 0.0001) and the amount (r = 0.97, P < 0.0001) of proteinuria. All patients with NS for more than three years had histological changes. Patients with OSM had lower 25(OH)D and serum albumin as compared with those with normal histology (P < 0.005). Bone mineralization had no significant correlation with serum iPTH, divalent ions, or vitamin D levels. CONCLUSIONS: OSM is a frequent finding in adult patients with NS, even at a normal level of renal function. Its severity correlates with the amount and duration of proteinuria. (+info)
Evidence for the promotion of bone mineralization by 1alpha,25-dihydroxycholecalciferol in the rat unrelated to the correction of deficiencies in serum calcium and phosphorus.
Concurrent administration of 1alpha,25-dihydroxycholecalciferol [1alpha,25-(OH)2-CC] to intact and thyroparathyroidectomized rats treated with ethane-1-hydroxy-1,1-diphosphonate (EHDP) prevented or reversed the EHDP-induced inhibition of bone mineralization as measured by changes in epiphyseal plate width and ash content of bone. An analog, 1alpha-droxycholecalciferol, was also effective. Recovery of bone after EHDP treatment was also significantly improved by administration of 1alpha,25-(OH)2-CC as evidenced by enhanced uptake of 45Ca by epiphyseal plates and decreased plate widths. Cholecalciferol (CC), ergocalciferol, dihydrotachysterol2, 5,6-trans-CC, 25-OH-CC, 5,6-Trans-25-OH-CC, and 1alpha24R,25-(OH)3-CC also blocked EHDP-induced epiphyseal plate widening, but required high, pharmacological dose levels. 24R,25- (OH)2-CC was inactive at doses up to 10 microgram/day. Since EHDP-treated rats are not deficient in calcium or phosphate, these data suggest that 1alpha,25-dihydroxycholecalciferol promoted bone mineralization independently of effects upon the intestinal absorption of calcium and phosphate. (+info)
Hard fallow deer antler: a living bone till antler casting?
Deer antlers are the only mammalian bone structures which regenerate completely every year. Once developed, antlers are cleaned of the velvet-like skin. Presently it is believed that due to velvet shedding the blood supply is interrupted in the solidifying antler bone. Histological examinations were made on different parts of fallow deer antlers investigated from the time of velvet shedding till the antler casting. The present study on hard (polished) antlers revealed living bone with regions presenting living osteocytes, active osteoblasts, osteoid seams and even early stages of trabecular microcallus formation, thus indicating to a continuous bone remodeling. A well developed vascular system was found despite the presence of hard antler bone. The pedicle bone exhibits a rich supply of capillaries and vessels connected to the spongy core of the main branch and the compact bone as well. There is evidence that hard fallow deer antlers possess a functioning vascular system that "keeps the antler moist" resulting in a high impact resistance when fights are most frequent. As late as 3 weeks prior to antler casting a large number of living cells were discovered within the antler core. As we have no doubt that parts of the polished fallow deer antler represent a living bone, we have concluded that a sufficient blood supply of the antler core is maintained almost till the time of antler casting by vessels passing through the antler base. (+info)
Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture. (+info)
Histochemical and immunohistochemical analysis of the mechanism of calcification of Meckel's cartilage during mandible development in rodents.
It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification. (+info)