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PubMed1999

Localization of Smads, the TGF-beta family intracellular signaling components during endochondral ossification.

Members of the transforming growth factor-beta (TGF-beta) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-beta signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-beta superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-beta1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-beta1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-beta/activin-restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-beta superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.

Activin Receptors, Type I↗
PubMed2001

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

Animals↗
PubMed1985

Calcium metabolism in paravertebral ligamentous ossification.

Twenty-eight patients with paravertebral ligamentous ossification (PVLO) and 11 control subjects were studied in an attempt to examine possible involvement of disturbances in mineral metabolism in the development of PVLO. No significant difference in baseline serum calcium and fasting urinary calcium excretion was found between patients with PVLO and controls. Patients with PVLO showed lower serum inorganic phosphate (Pi) and tubular reabsorptive capacity for Pi (TmP/GFR) than controls. Basal nephrogenous cyclic 3',5'-AMP (NcAMP) was elevated in some patients with PVLO. A significant inverse relationship was found between basal TmP/GFR and NcAMP in patients with PVLO (r = -0.50; P less than 0.01). Compared with controls, patients with PVLO had significantly lower calciuric responses to an oral calcium load (P less than 0.05), suggesting decreased intestinal calcium absorption. Furthermore, a significant inverse correlation was found between basal NcAMP and the calciuric response in patients with PVLO (r = -0.42; P less than 0.05). Serum 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)2D) levels were not significantly different between the two groups. The size of ossified areas in paravertebral ligaments estimated from spinal radiograms inversely correlated with the calciuric response to the oral calcium load (r = -0.50; P less than 0.01). These data demonstrate that the development of PVLO is associated with decreased intestinal calcium absorption in the face of normal serum 1,25(OH)2D level. Thus, together with previous observations showing a high incidence of PVLO in patients with hypoparathyroidism or familial hypophosphataemic rickets/osteomalacia, the present results suggest that the defect in the action of 1,25(OH)2D may underlie the development of PVLO.

Calcifediol↗
PubMed1990

Matrix vesicles as a marker of endochondral ossification.

Endochondral ossification in bone development and repair, and in induced bone formation in mesenchymal tissues, involves recruitment of mesenchymal cells, their differentiation into chondrocytes, and calcification of the cartilagenous matrix. Stimulation of proteoglycan synthesis is used as a biochemical marker of chondrogenesis, however it does not distinguish among chondrogenic phenotypes. Chondrocytes derived from the resting zone and adjacent growth zone cartilage of the costochondral junction of young rats, produce matrix vesicles in culture which are enriched in alkaline phosphatase specific activity with respect to the plasma membrane. Matrix vesicles isolated from cultures of neonatal rat muscle mesenchymal cells are not enriched in this enzyme activity. Alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is stimulated by 1,25(OH)2D3; enzyme in matrix vesicles produced by resting zone chondrocytes is stimulated by 24,25(OH)2D3; enzyme in matrix vesicles isolated from mesenchymal cell cultures is responsive to neither metabolite. Matrix vesicle phospholipase A2 is stimulated by 1,25(OH)2D3 in growth zone chondrocytes cultures; inhibited by 24,25(OH)2D3 in resting zone chondrocyte cultures; and is unaffected by either metabolite in mesenchymal cell cultures. These observations suggest that matrix vesicle production, as defined by alkaline phosphatase enrichment, and responsiveness of matrix vesicle enzymes to vitamin D metabolites, can be used as markers of phenotypic maturation during chondrogenesis in vivo and in vitro.

24,25-Dihydroxyvitamin D 3↗
PubMed1996

The role of type X collagen in endochondral ossification as deduced by Fourier transform infrared microscopy analysis.

Type X collagen has been implicated in the morphogenetic events of endochondral ossification (EO), including the calcification of hypertrophic cartilage and trabeculae prior to their replacement by bone and marrow. Recently, transgenic mice which expressed a truncated collagen X protein were reported to exhibit morphologic alterations in all tissues arising through EO. Fourier Transform InfraRed (FTIR) spectroscopy has previously been shown to provide quantitative and qualitative information about the relative amount of mineral and carbonate present, mineral composition, and crystal perfection. To determine the role of collagen X in mineralization, the "quality" of mineral crystals was analyzed in thin sections of calcified cartilage from tibia obtained from several independent transgenic mouse lines showing varying degrees of the mutant phenotype and mice without type X collagen expression, by means of Fourier Transform InfraRed microscopy (FTIRM). In the present paper, the term "mineral quality" is employed to describe crystallinity/crystal maturation, and acid phosphate content. The results indicate significant differences between normal and transgenic mice bone mineral, both in the amount present and the "quality" of the crystals. In contrast, the analysis of the mineral in mice without type X collagen expression was not different from their age/sex-matched controls.

Animals↗
PubMed1978

Fibrodysplasia ossificans progressiva. Biochemical changes in blood serum, urine, skin, bone, and ectopic ossification.

Increased urinary output of total hydroxyproline, hydroxylysine and uronic acid was found in two patients suffering from fibrodysplasia ossificans progressiva. Before treatment of one of the patients, the high molecular weight peptide fraction deriving from newly synthesised collagen was particularly increased. Treatment with disodium etidronate (diphosphonate) reduced the values. The urinary values of sodium, potassium and calcium were also depressed during treatment, and, in both patients, serum phosphate was high, while serum calcium was normal. The hydroxyproline and hydroxylysine contents in skin and bone of FOP patients did not differ from controls, while ectopic ossifications showed a considerable increase in hydroxylysine in relation to normal bone.

Adolescent↗
PubMed1983

Coronal cleft of vertebrae, a variant of normal enchondral ossification.

In 157 spontaneously aborted human embryos and fetuses the incidence and evolution of coronal cleft formation was investigated radiologically and histologically. Complete clefts were formed in nine cases and incomplete clefts in 16 cases. There was a predominance of males. It is concluded that coronal clefts are variations of normal enchondral ossification and that the notochord plays no role in its pathogenesis. Coronal clefts seen on radiographs of newborn should not be interpreted as a malformation. Thus no preventive or treatment regimen is indicated.

Abortion, Spontaneous↗
PubMed1995

Ossification of the anterior longitudinal ligament and Forestier's disease: an analysis of seven cases.

A retrospective review was conducted on the records and radiographs of six symptomatic patients and one asymptomatic patient with Forestier's disease. No other series of patients with this disease is found in the neurosurgical literature. Forestier's disease, also known as diffuse idiopathic skeletal hyperostosis (DISH), is an idiopathic rheumatological abnormality in which exuberant ossification occurs along ligaments throughout the body, but most notably the anterior longitudinal ligament of the spine. It affects older men predominantly; all of our patients were men older than 60 years of age. The disease is usually asymptomatic; however, dyspnea, dysphagia, spinal cord compression, and peripheral nerve entrapment have all been documented in association with the disorder. Five of the six symptomatic patients presented with dysphagia due to esophageal compression by calcified anterior longitudinal ligaments, and one patient developed recurrent spinal stenosis when scar tissue from a previous decompressive laminectomy became calcified. All patients responded well to surgery. Two of the four patients who underwent removal of cervical osteophytes required several months following surgery for the dysphagia to resolve. This would support the hypothesis that not all cases of dysphagia in Forestier's disease are due to mechanical compression. Dysphagia may result from inflammatory changes that accompany fibrosis in the wall of the esophagus or from esophageal denervation. Evaluation of dysphagia even in the presence of Forestier's disease must rule out occult malignancy. The authors' experience suggests that dysphagia in the setting of Forestier's disease is an underrecognized entity amenable to surgical intervention.

Aged↗
PubMed2001

Ossification of autologous pericranium used in duraplasty. Case report.

Pericranium is frequently used in duraplasty and is considered superior to the many other alternatives because of its easy availability and because it offers a watertight dural closure while minimizing the problems of adhesion, infection, and rejection. Although the osteogenic potential of all periosteal tissues is recognized, a review of the literature did not reveal a reported case of osseous formation following use of pericranium for duraplasty. The authors report the case of a 17-year-old man who presented with a self-inflicted gunshot wound to the head. He was obtunded, but moving all extremities purposefully. Computerized tomography scanning demonstrated bifrontal injury. A bicoronal craniotomy with debridement was performed on an emergency basis, with vascularized pericranium used for a duraplasty. Follow-up cranioplasty demonstrated significant ossification of the pericranium 5 months after the original surgery. Pericranium is an attractive material for duraplasty; however, its osteogenic potential may interfere with future cranioplasty and cosmesis. This may be especially relevant in young persons.

Adolescent↗
PubMed1991

The effects of particle size and origin of calcium carbonate on performance and ossification characteristics in broiler chicks.

The following physico-chemical characteristics of various calcium sources, differing in origin and particle size were determined: mineral composition, sieve and image analysis, apparent solubility (AS), surface area (SA), porous volume, specific gravity, and compressibility (C). The AS, SA, and C values were related more to the calcium particle size than to its origin and were higher in ground calcium sources. Calcium retention of seashells treated with phosphoric acid, oyster shells, and limestone using two particle sizes, ground or particulate, was assayed in 98 broiler chicks. Particulate marble was also tested in this experiment. Calcium retention expressed as a percentage of calcium ingestion was decreased when coarse particles of calcium were supplied in lieu of pulverized sources (40 versus 49%). An experiment with a 3 x 3 x 3 factorial arrangement of treatments were tested using 576 broiler chicks. Treatments included three calcium sources (phosphorus-treated shell, oyster shell, and marble), three particle sizes [ground (less than .15 mm), medium (.6 to 1.18 mm) and coarse (greater than 1.18 mm)] and three levels of calcium (.5,.7, and .9%) with ground limestone as a reference. Performance, tibial morphometry, breaking strength variables, and ash content were measured at 4 wk of age. Weight gain and feed conversion were ameliorated with ground particles of calcium. Cortical thickness, length of the tibia, stiffness, stress, and tibia ash were diminished when coarse particles of calcium were incorporated in the diets. Conversely, the origin of the calcium source hardly affected these criteria. Additionally, the incorporation of phosphorus-treated shells was assayed in 112 broiler chicks. Coarse particles decreased calcium retention. Consequently, ultimate stress, the modulus of elasticity, and stress were impaired. It is concluded that differences in utilization of calcium carbonate sources by the broiler chick is primarily a result of particle size rather than origin. Ground particles of calcium (pan to .15 mm) significantly improves performance and tibial ossification characteristics in broiler chicks.

Animals↗
PubMed1992

Maternal body weight and feed allowance of breeders affect performance of dwarf broiler breeders and tibial ossification of their progeny.

Four hundred and eight dwarf broiler breeder hens were raised collectively in a floor pen to 21 wk of age. At this age they were classified into four groups with reference to their individual BW as heavy (1.95 +/- .1 kg), medium (1.80 +/- .1 kg), light (1.69 +/- .1 kg), and ultralight (1.57 +/- .1 kg). All groups were individually caged at 23 wk of age. During the reproductive period, each group was divided into three subgroups fed on liberal, intermediate, or severe feed restriction (reaching up to 135, 125, and 115 g of daily feed allowance at 29 wk of age, respectively). Intergroup differences in BW were maintained throughout the experiment (21 to 61 wk) but tended to decrease with age. Hen-day egg production was depressed by the lower feed allowance. Fertility and hatchability were impaired when hens received the largest quantities of food. Hen size influenced female breeder performance only slightly. Shell quality and albumen quality were affected by the level of feed consumption. Egg weights as well as BW of the progeny at hatching were enhanced by increased maternal BW and feed allowance. This positive maternal effect was still present at 40 days of age. Despite better overall BW performances of the male versus female broilers, the abdominal fat pad of female broilers was heavier than that of males and tended to increase with breeder size and breeder feed allowance. Accordingly, tibial breaking strength and percentage ash of the progeny at hatching were markedly improved in proportion to the breeders' BW and to their feed allowance. The effect of breeder size on broiler tibial quality was maintained up to 40 days of age but the effect of breeder feed intake tended to disappear with increasing age of the broilers. Tibial strength and mineralization were higher in male than in female broilers at 40 days of age. Dyschondroplasia was higher in broilers hatched from heavier breeder hens, but was not influenced by breeder feed intake. The incidence of varus and valgus in progeny was similar whatever the breeders' treatment. It is concluded that performance of dwarf breeders in a given flock depends mainly upon breeder feed allowance but that broiler performance and, especially, tibial ossification of broilers is greatly influenced by maternal size and, to a lesser extent, by maternal feed intake.

Animals↗
PubMed1994

Serum total calcium, phosphorus, 1,25-dihydroxycholecalciferol, and endochondral ossification defects in commercial broiler chickens.

The research described in this paper relates the changes in serum concentration of calcium, phosphorus, and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] to changes in tibial ash percentage and the incidence of endochondral ossification defects (EOD) in flocks of commercially reared broiler chickens at 14 d of age. Sequential studies of six Australian broiler flocks representing three major genetic lines were undertaken at weekly intervals from 1 to 28 d of age. Serum collected from birds was analyzed for total calcium, inorganic phosphorus, and 1,25(OH)2D3. Tibial ash percentage was also determined at weekly intervals, and the incidence of EOD was determined at 14 d of age by examining sagittal sections of the proximal tibiotarsus. The EOD observed in the 14-d-old broiler chickens were characterized by enlarged zones of proliferating chondrocytes, similar to that which occurs during calcium- or vitamin D-dependent rickets. Three flocks had a 50% incidence of EOD at 14 d of age and were classified as severely affected. The other three flocks had incidences ranging from 12 to 16% and were classified as mildly affected. Broiler flocks severely affected with EOD (50% incidence at Day 14) had lower (P < or = .05) concentrations of 1,25(OH)2D3 than flocks mildly affected (12 to 16% incidence). Tibial ash percentages were lower (P < or = .05) in the severely affected flocks between Days 14 to 28, and it is likely that a lower rate of ash accretion between Days 7 to 14 precedes the development of the EOD.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Prenatal diagnosis of neural tube defects: origin of midtrimester vertebral ossification centers as determined by sonographic water-bath studies.

A normal vertebral column was removed from a 17-week stillborn fetus and suspended in a water bath. The specimen was sequentially dissected, with spinous processes, transverse processes, and remaining neural arch removed. After dissection, each specimen was imaged by ultrasound in axial, sagittal, and coronal planes. The resultant images clearly demonstrated the origin of the three ossification centers as one at each lamina-pedicle junction and one in the vertebral body. Corollary X-ray studies confirmed the ultrasound findings and, in a case of open spina bifida, demonstrated the superiority of sonography for making this diagnosis.

Female↗
PubMed1999

[Development of hoof cartilage with special considerations of its ossification].

The pre- and perinatal development of the hoof cartilage is described concerning the histological structure and surrounding vessels. Beginning in the third month of fetal development, the anlage of the hoof cartilage is still present in typical shape and location. It is built out of mesenchymal connective tissue. During further fetal development, the connective tissue cells will differentiate into two cell populations, fibroblasts and chondroblasts. Vessels, traversing the hoof cartilage, are surrounded by loose connective tissue, which will partially develop fibrocartilage. At birth, hoof cartilage consists of hyaline cartilage, which is disrupted by vessels, embedded in fibrous connective tissue and/or fibrocartilage. The development of the hoof cartilage is the base to understand its structure and the predisposing locations for its ossification.

Aging↗
PubMed1999

Unusual fan shaped ossification in a female fetus with radiological features of boomerang dysplasia.

We report on a female fetus of 24 weeks whose clinical and radiological findings were compatible with boomerang dysplasia (BD). However, histopathology was unusual with a lateral fan shaped diaphyseal ossification. This has never been described either in typical atelosteogenesis I (AT-I) or in BD. The purpose of this report is to find out if this condition is a separate lethal bone dysplasia or another histological feature of the nosological group of AT-I and BD.

Bone and Bones↗
PubMed1999

Ethanol effect on the chick embryo ossification: a macroscopic and microscopic study.

This study attempts to analyze anomalies in avian embryos induced macroscopically and microscopically when exposed to ethanol (EtOH) during the first stages of development. Fertilized chicken eggs were employed in this study. The eggs were incubated at 37.8 degrees C. Some of the eggs were treated on day 0 with EtOH (20%, 40% and 60%) by instillation in the air sac. The control group was instilled with 0.1 ml of NaCl at 0.9%. Other eggs were treated on the 4th post-incubation day, employing the same methodology. The embryos in both groups were removed from the eggs on the 11th incubation day and examined using a dissecting binocular microscope. After macroscopic analysis, the samples obtained were fixed in 10% formol, photographed and processed according to common histological techniques and the Picrosirius method. Embryos treated with EtOH demonstrated a significant weight decrease. Microscopic analysis by means of the Picrosirius method revealed that the intra-membranous ossification process presents less development, and therefore there was less type I collagen in trabecular bone in the embryos post-exposure to EtOH with respect to the control.

Abnormalities, Drug-Induced↗
PubMedDate not supplied

Sex variation at the time of sonographic appearance of the proximal tibial epiphyseal ossification center.

The time of sonographic appearance of the proximal tibial epiphyseal ossification center and its sexual variation was assessed in 659 normal pregnancies. Proximal tibial center appears between 34-37 weeks gestation. In female fetuses, it appears between 34-35 weeks and in males, between 35-36 weeks (P < 0.001). There was a tendency of female proximal tibial center appearance to precede male appearance, by approximately one week. This difference, however, didn't show statistical significance.

Embryonic and Fetal Development↗