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Sonographic appearance of the fetal heel ossification centers and foot length measurements provide independent markers for gestational age estimation.

The sonographic measurement of fetal foot length and the assessment of fetal heel ossification centers were conducted as an additional method for the estimation of gestational age. Such an approach is particularly desirable in cases where measurements of other biometric parameters are precluded, for example, in diseases such as anencephaly, hydrocephalus, and short limb dysplasia. A significant correlation was found between fetal foot length and gestational age (r = 0.9, p less than 0.0001) and between fetal foot length and femur length (r = 0.9, p less than 0.0001). Ossification centers of the calcaneus and talus were all present by the twenty-second week of gestation. However, their measurements discriminated between gestational ages of 18 to 22 weeks. The utilization of fetal foot length and heel ossification centers will serve as a useful adjunct in the estimation of gestational age and possibly in the diagnosis of diseases affecting the fetal foot.

Calcaneus↗

Fontanel size and epiphyseal ossification in neonatal twins discordant by weight.

Size of the anterior fontanel and ossification of the epiphyseal centers at the knee were measured in 13 "discordant" twin pairs. There was a highly significant (p smaller than 0.01) negative correlation between fontanel size and epiphyseal ossification. Severe intrauterine growth retardation (demonstrated by the lighter twin) may result in retardation of membranous as well as enchondral ossification.

Birth Weight↗

Low-intensity ultrasound stimulates endochondral ossification in vitro.

Animal and clinical studies have shown an acceleration of bone healing by the application of low-intensity ultrasound. The objective of this study was to examine in vitro the influence of low-intensity ultrasound on endochondral ossification of 17-day-old fetal mouse metatarsal rudiments. Forty-six triplets of paired metatarsal rudiments were resected 'en block' and cultured for 7 days with and without low-intensity ultrasound stimulation (30 mw/cm2). At days 1, 3, 5, and 7, the total length of the metatarsal rudiments, as well as the length of the calcified diaphysis were measured. Histology of the tissue was performed to examine its vitality. The increase in length of the calcified diaphysis during 7 days of culture was significantly higher in the ultrasound-treated rudiments compared to the untreated controls (P = 0.006). The growth of the control diaphysis was 180 +/- 30 microm (mean +/- SEM), while the growth of the ultrasound-treated diaphysis was 530 +/- 120 microm. The total length of the metatarsal rudiments was not affected by ultrasound treatment. Histology revealed a healthy condition of both ultrasound-treated and control rudiments. In conclusion, low-intensity ultrasound treatment stimulated endochondral ossification of fetal mouse metatarsal rudiments. This might be due to stimulation of activity and/or differentiation of osteoblasts and hypertrophic chondrocytes. Our results support the hypothesis that low-intensity ultrasound activates ossification via a direct effect on osteoblasts and ossifying cartilage.

Animals↗

Impaired ossification in mice lacking the transcription factor Sp3.

Sp3 is a ubiquitously expressed member of the Sp family of transcription factors. Recently, the mouse Sp3 gene has been disrupted by homologous recombination. Sp3 null mice die immediately after birth due to respiratory failure. In addition, these mice show a pronounced defect in late tooth formation. Here we show that Sp3 is also required for proper skeletal ossification. Both endochondral and intramembranous ossification are impaired in E18.5 Sp3-/- embryos. The delay in ossification is reflected by reduced expression of the osteoblast-specific marker gene osteocalcin. The transcription factor - core binding factor 1 (Cbfa1)--that is essential for bone formation, however, is expressed at normal levels. In vitro differentiation studies using Sp3-/- ES cells further support the conclusion that Sp3 is needed for correct bone formation. The capacity of Sp3-/- cells to undergo osteogenic differentiation in vitro is reduced and osteocalcin expression is significantly diminished. Our studies establish Sp3 as an essential transcription factor for late bone development.

Animals↗

The ossification centre of the talus.

The ossification of the talus was studied in plastinated and histological preparations of normal feet of eight newborn children. Quantitative data on the newborn talus were obtained with the IBAS image analysis system and by point counting methods. In the newborn talus up to 24 percent of the talar anlage already consists of bony tissue. The ossification centre is situated in the neck, which includes the non-articulating surfaces of the talus. Periosteal bone joins the endochondral centre below and, in well-differentiated specimens also above. The basal periosteal collar forms the surfaces of the sinus and canalis tarsi, whereas the cranial bony collar is included in the tibiotalar joint. The histological architecture of these periosteal collars differs. Four arteries contribute to the blood supply of the talar ossification centre.

Bone Development↗

A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification.

In vivo expression of the type III sodium-dependent phosphate transporter (NaPiT) Glvr-1 during endochondral ossification, suggests a functional role for inorganic phosphate (Pi) transport in cartilage calcification. For further analysis of this relationship, an in vitro model of endochondral ossification is required. In this context, we investigated the characteristics of Pi transport in the new chondrogenic cell line ATDC5 in relation to extracellular matrix (ECM) formation and mineralization. Pi uptake in ATDC-5 cells and in isolated matrix vesicles (MVs) is mediated by an Na-dependent Pi transporter with a pH dependency characteristic of a type III Pi carrier (lower activity at alkaline pH). Northern blot analysis indicated that ATDC-5 cells express Glvr-1 transcripts during the various stages of their maturation with a maximal level during the proliferating stage. In isolated MVs, Pi transport activity was maximal at day 21, concomitant with the beginning of type X collagen messenger RNA expression. These events preceded the initiation of matrix mineralization, which was apparent at day 25, and then gradually increased until day 47. This temporal relationship between maximal Pi transport activity in MVs and the expression of a marker of mineralizing chondrocytes is compatible with the possible involvement of Pi transport in the ECM calcification observed in ATDC-5 cell cultures. In conclusion, these observations suggest that ATCD-5 cells in culture represent a promising model for the analysis of a functional role of Pi transport in the initial events of endochondral ossification.

Biological Transport↗

Epidermal growth factor receptor-deficient mice have delayed primary endochondral ossification because of defective osteoclast recruitment.

The epidermal growth factor receptor (EGFR) and its ligands function in diverse cellular functions including cell proliferation, differentiation, motility, and survival. EGFR signaling is important for the development of many tissues, including skin, lungs, intestines, and the craniofacial skeleton. We have now determined the role of EGFR signaling in endochondral ossification. We analyzed long bone development in EGFR-deficient mice. EGFR deficiency caused delayed primary ossification of the cartilage anlage and delayed osteoclast and osteoblast recruitment. Ossification of the growth plates was also abnormal resulting in an expanded area of growth plate hypertrophic cartilage and few bony trabeculae. The delayed osteoclast recruitment was not because of inadequate expression of matrix metalloproteinases, including matrix metalloproteinase-9, which have previously been shown to be important for osteoclast recruitment. EGFR was expressed by osteoclasts, suggesting that EGFR ligands may act directly to affect the formation and/or function of these cells. EGFR signaling regulated osteoclast formation. Inhibition of EGFR tyrosine kinase activity decreased the generation of osteoclasts from cultured bone marrow cells.

Animals↗

The effect of prenatal indium chloride exposure on chondrogenic ossification.

Daily indium chloride doses of control (0) or 400 mg/kg were administered orally to pregnant Sprague-Dawley (SD) rats by gavage, on d 20 of gestation. Indium concentration was determined in the maternal and fetal blood, livers, kidneys, skulls, and femurs by atomic absorption spectrometry. Further groups of pregnant rats were treated with control (0) or 400 mg/kg indium chloride orally, during the whole gestation period. The fetuses were examined on d 21 of gestation, using histological and histochemical methods. Four hours after the administration indium concentration was found to be significant in the blood, liver, and kidneys of the dams. Twenty-four hours later it increased in the blood but not in the liver and kidney. Fetal indium concentrations were 40-50% of the maternal levels due to a barrier of the placenta. In the skull and the femur, indium was already detectable at 4 h after the administration, and by the end of 24 h, metal concentration was several times higher than that at 4 h, indicating accumulation. Furthermore, it was found that the birefringency of collagen detectable by picrosirius red staining in polarized light around the chondrocytes disappeared and became irregular. In the matrix of the epiphyseal cartilage, the regular, birefringent network demonstrable by Rivanol reaction became irregular and hardly recognizable. In the cytoplasm of the chondrocytes, the diffuse, evenly distributed positive Ricinus communis agglutinin reaction became irregular or disappeared. Similar but much weaker changes were observed with concanavalin A and wheat germ agglutinin stainings. It was concluded that the missing femur and micromelia diagnosed by alizarin staining is the consequence of a specific toxic effect of indium that inhibits chondrogenic ossification. No similar histochemical changes were observed in the bones of the skull developing by desmogenic ossification, despite the presence of indium. Data indicate that the mechanisms of the effects of indium causing retardation and/or malformation differ in the bones developing through desmogenic or chondrogenic ossification.

Animals↗

A solid cystic tumor of the pancreas with ossification and possible malignancy, coexisting nonfusion of the pancreatic ducts.

We report the case of a 34-year-old woman with a solid cystic tumor (SCT) of the pancreas accompanied by ossification and possible malignancy, coexisting nonfusion of the pancreatic ducts. There was a 24 x 29 x 33-mm mass with a prominent calcified lesion in the tail of the pancreas detected by abdominal ultrasonography, computed tomography, and magnetic resonance imaging. There were no distal metastases detected. Endoscopic retrograde pancreatography revealed nonfusion of the pancreatic ducts. The resected tumor consisted of solid and cystic components. The tumor was not encapsulated and included a severely ossified lesion inside. On microscopy, the tumor cells were small, eosinophilic, and proliferated in a solid or pseudo-papillary pattern. The tumor cells infiltrated into the surrounding normal pancreas parenchyma and invaded part of the mesentery. The immunostaining was positive for alpha-1-antitrypsin, neuron-specific enolase, vimentin, and chromogranin A. In the literature, only a few cases of SCT of the pancreas described ossification. As far as we know, only three cases of SCT of the pancreas, which demonstrated nonfusion of the pancreatic ducts, have been reported. Thus, SCT of the pancreas with ossification, possible malignancy, and coexisting nonfusion of the pancreatic ducts is extremely rare.

Adult↗

[Chronology of the postnatal ossification of the shoulder and elbow joints in the Siamese cat (Felis catus L.)].

A study was done by radiological techniques to show the chronology of ossification of the shoulder and cubit joints in the Siam cat, from birth up to the 25th week of postnatal development. For this experiment we used 40 little cats (19 males and 21 females) belonging to 11 litters subjected to different controls: pattern race, healthy, feeding, growing up and radioactivity. The time of the appearance and the evolution of the ossification centers is determined as well as the phenomenon of fusion during this time. The postnatal ossification from both joints of the Siam cat is compared with that of the common cat.

Animals↗

[Chronology of the postnatal ossification of the thoracic limb in the Siamese cat (Felis catus L.)].

A study was done by radiological techniques to show the chronology of ossification of the thoracic autopodo of the Siam cat from birth up to the 25th week of postnatal development. For this experiment we used 40 little cats (19 males and 21 females) belonging to 11 litters subjected to different controls: pattern race, healthy, feeding, grow up and radioactivity. The time of the appearance and evolution of the centres that form the basipodo, metapodo and acropodo of the thoracic limb is determined. Likewise it is analyzed the phenomenon of fusion during that time to define the main ossification sequence in the carpo. The aspects of the postnatal ossification of the thoracic autopodo in the Siam cat are compared with the literature of different authors about the common cat.

Animals↗

Ossification of the appendicular skeleton in the Spanish Ibex Capra pyrenaica Schinz, 1838 (Artiodactyla: Bovidae), with regard to determination of age.

We studied ossification macroscopically in 35 Spanish ibex (Capra pyrenaica) appendicular skeletons in order to establish the skeletochronology in both sexes of the species. The age of animals had been determined by means of horn segment counts, teeth replacement and cementum layer counts from first incisors and ranged from <1 to 12 years. Females showed a faster ossification process than males. Moreover, for each age or year class considered, regarding both sexes, different stages of epiphyseal fusion could be found. Therefore, if we try to use data from bone remains in population studies (e.g. differential mortality rates), determination of the ossification stage for ageing bone samples must be carried out with caution.

Age Determination by Skeleton↗

Detailed examination of cartilage formation and endochondral ossification using human mesenchymal stem cells.

1. Cartilage formation is one of the most complex processes in biology. The aim of the present study was to produce a simplified in vitro system to resolve its complexities. 2. Human mesenchymal stem cells (hMSC) were maintained in alginate beads with a chondrogenesis-induction medium containing 10 ng/mL transforming growth factor (TGF)-beta3. 3. At days 0, 2, 4, 8, 12, 16 and 19 of culture, we examined the cells using a light microscope and a transmission electron microscope. We also evaluated the cells using immunocryo-ultramicrotomy. 4. The present study demonstrated that hMSC produced numerous extracellular matrices containing abnormal collagen fibres following their exposure to a chondrogenesis-induction medium in alginate beads. At this time, calcification was detected by alizarin red staining and electron-dense particles, composed of hydroxyapatite, appeared in both the cytoplasm and the extracellular spaces. 5. In addition immunocryo-ultramicrotomy revealed that collagen type II, type X and proteoglycan were prominent and that osteocalcin was detectable at day 2. During 8-16 days of culture, collagen type X maintained its strong expression and the expression of osteocalcin increased markedly. In contrast, the expression of collagen type II and proteoglycan decreased with time. 6. These findings demonstrate that hMSC rapidly differentiate into chondrocytes expressing collagen type II and proteoglycan. 7. The expression of collagen type II and proteoglycan then dropped and the activity of collagen type X was the same as before (4-8 days). As a result, the cells developed into the next cell type, so-called hypertrophic chondrocytes. Finally, both osteocalcin activity and the calcification of cell bodies and extracellular matrices became evident, indicating endochondral ossification. Thus, we conclude that hMSC rapidly differentiate into chondrocytes, followed by the development of hypertrophic chondrocytes. Endochondral ossification is the final form in this culture. 8. The findings of the present study indicate that our three-dimensional culture is a convenient in vitro model for the investigation of the regulatory mechanisms of cartilage formation and endochondral ossification.

Adult↗

Calcitonin treatment for intersternocostoclavicular ossification: clinical experience in two cases.

Intersternocostoclavicular ossification is a benign arthro-osteitis of the upper anterior chest of unknown cause. Two patients with acute exacerbation of this disorder were successfully treated with intramuscular injections of an eel calcitonin analogue (40 units three times a week). Besides symptomatic relief of local pain and swelling, serial scintigrams showed quantitative improvement in radiophosphonate uptake. The rapid alleviation of pain implies that the hormone has a central analgesic effect, in addition to its direct influence on bone cells and antiinflammatory action. In one patient the disease was associated with palmoplantar pustulosis, which was cured with oral colchicine, whereas the other patient did not have such skin lesions. Despite a hypothetical link between palmoplantar pustulosis and intersternocostoclavicular ossification, colchicine had no beneficial impact on the bone pain. Salmon calcitonin delivered by nasal spray was tried for the second patient but failed, probably because of insufficient drug delivery. The initial favourable results described here warrant future use of calcitonin injection on a larger number of patients with intersternocostoclavicular ossification.

Aged↗

Ultrasonographic identification of fetal lower extremity epiphyseal ossification centers.

The reliability of the sonographic identification of the fetal distal femoral epiphyseal ossification center (DFE) and the proximal tibial epiphyseal ossification center (PTE) in the estimation of gestational age (GA) was assessed in 84 singleton pregnancies. We used the presence of a DFE to indicate a GA greater than or equal to 33 weeks, and this method was 95% sensitive, 95% specific, and accurate overall in 95% of a selected cohort of 61 cases. In the same cohort, the presence of the PTE was highly predictive (accuracy of a positive prediction 95%) of a GA greater than or equal to 35 weeks. Ultrasonographic evaluation of fetal lower extremity epiphyseal ossification centers is a promising technique for the estimation of GA during the last trimester of pregnancy.

Epiphyses↗

Development of femoral head to the rat from calcification to ossification.

The development of the epiphysis of the femoral head from calcification to ossification was investigated in 50 rats of Wistar strain from an age of 10 to 63 days using radiography and histology. The first deposition of calcium in the cartilage appeared in the central portion of the epiphysis at an age of about 21 days. The region of calcification gradually expanded toward the periphery and after about 3 weeks it occupied the entire epiphysis except for the periphery. After completion of calcification ossification began in the lateral part of the epiphysis at the earliest at an age of 6 weeks. The onset of ossification was characterized by disintegration of hypertrophied cartilage cells and formation of bone trabeculae accompanied by vascular invasion from the lateral side.

Animals↗

Ossification of femoral head in infancy. I. Normal standards.

Observations made in 455 Malmö infants with assumedly normal ossification of the femoral heads were used for establishing reference standards for the age at onset of the ossification and for the size of the ossification centers according to sex and age.

Age Factors↗

Three modes of ossification during distraction osteogenesis in the rat.

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, 'transchondroid bone formation'. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis.

Animals↗