Primary Ossification Research Articles

Impaired endochondral bone development and osteopenia in Gli2-deficient mice

Mice homozygous for targeted disruption of the zinc finger domain of Gli2 (Gli2 zfd/zfd) die at birth with developmental defects in several organ systems including the skeleton. The current studies were undertaken to define the role of Gli2 in endochondral bone development by characterizing the molecular defects in the limbs and vertebrae of Gli2 zfd/zfd mice. The bones of mutant mice removed by cesarian section at E16.5 and E18.5 demonstrated delayed endochondral ossification. This was accompanied by an increase in the length of cartilaginous growth plates, reduced bone tissue in the femur and tibia and by failure to develop the primary ossification centre in vertebral bodies. The growth plates of tibiae and vertebrae exhibited increased numbers of proliferating and hypertrophic chondrocytes with no apparent alteration in matrix mineralisation. The changes in growth plate morphology were accompanied by an increase in expression of FGF2 in proliferating chondrocytes and decreased expression of Indian hedgehog (Ihh), patched (Ptc) and parathyroid-hormone-related protein (PTHrP) in prehypertrophic cells. Furthermore, there was a reduction in expression of angiogenic molecules in hypertrophic chondrocytes, which was accompanied by a decrease in chondroclasts at the cartilage bone interface, fewer osteoblasts lining trabecular surfaces and a reduced volume of metaphyseal bone. These results indicate that functional Gli2 is necessary for normal endochondral bone development and that its absence results in increased proliferation of immature chondrocytes and decreased resorption of mineralised cartilage and bone formation.

Double child burial from Sunghir (Russia): pathology and inferences for upper paleolithic funerary practices.

The double child burial from Sunghir (Russia) is a spectacular Mid Upper Palaeolithic funerary example dated to about 24,000 BP. A boy (Sunghir 2) and a girl (Sunghir 3), about 12-13 and 9-10 years old, respectively, were buried at the same time, head to head, covered by red ocher and ornamented with extraordinarily rich grave goods. Examination of the two skeletons reveals that the Sunghir 3 femora are short and exhibit marked antero-posterior bowing. The two femora do not show any asymmetry in the degree of shortening and bowing. Bowing affects the whole diaphysis and shows a regularly incurved profile, with the highest point at midshaft. Pathology is confined to the femora, and no other part of this well-preserved specimen shows abnormality. The isolated nature of the Sunghir 3 anomalies points to cases reported in the medical literature under the label of "congenital bowing of long bones" (CBLB). These are a group of rare conditions exhibiting localized, sometimes bilateral, bowing and shortening which are nonspecific and may result from different causes, including abnormalities of the primary cartilaginous anlage (i.e., the aggregation of cells representing the first trace of an organ). Localized ossification disturbances, possibly linked to a diabetic maternal condition, might explain the shortening and the coincidence of maximum midshaft curvature with the position of the primary ossification center, as well as the lack of involvement of other skeletal parts. This scenario, rather than other possibilities (early bilateral midshaft fracture, acute plastic bowing deformities, or faulty fetal posture), provides the most likely explanation for the Sunghir 3 femoral deformities. The intriguing combination of a pathological condition apparent since birth with a spectacular burial of unusually positioned young individuals of different sexes recalls significant aspects of the triple burial from the contemporary site of Dolní Vestonice (Moravia), evoking a patterned relationship between physical abnormality and extraordinary Upper Paleolithic funerary behavior.

Expression of NG2 proteoglycan during endochondral and intramembranous ossification.

We have used immunohistochemistry to study the distribution of the NG2 proteoglycan during bone development in the mouse. At embryonic day 15.5, NG2 was strongly detected in the immature cartilage of developing limbs. After transient down-regulation in mature chondrocytes, NG2 was up-regulated during primary ossification, colocalizing with alkaline phosphatase and tenascin C. In the epiphyseal growth plates of newborn mouse tibia, NG2 and alkaline phosphatase exhibited overlapping patterns of expression by hypertrophic chondrocytes and by osteoblasts surrounding newly formed bone trabeculae. NG2 was down-regulated after puberty, being only faintly detectable in the tibial growth plates of 3-month-old mice. In cranial sutures, NG2 was strongly labeled in osteogenic bone fronts and in the suture matrix. Our results indicate that NG2 expression is up-regulated during both endochondral and intramembranous ossification, but is down-regulated as ossification is completed.

Peculiaridades en la organización de la cadena osicular timpánica humana a lo largo de su ontogenia

AbstractWe have studied the development of the tympanic ossicles in 40 embryo-foetal human series aged between 32 days (6 mm) and newborn. Once performed the measurements to date chronologically embryos and foetuses, we did a meticulous dissection of temporal bones. After fix in 10% formol, decalcified with 2% nitric acid, embedded in Paraplast, sectioned in a sequence of 7mm, and stained with Martin´s trichrome. The tympanic ossicles are developed in the mesenchyme of the two first pharyngeal archs. The head of the malleus, the body and the short limb of the incus arise from the first arch while the handle of the malleus, the long limb of the incus and the mass of the stapes arise from the second arch. The vestibular side of the stapedial footplate develops in the otic capsule. The tympanic ossicles develop from endochondral ossification, while anterior process of the malleus has the membranous ossification. In their ontogenia 6 stages are observed. First stage, the formation of their sketch by mesenchimal condensation, in the second stage, "pre-cartilaginous", the cells of the primordia are differentiated into condroblasts, in the third stage "cartilaginous" the ossicles show a cartilaginous structure, in the forth stage the primary ossification centers are developed, in the fifth stage the ossicles arise in the periostic annulus and inside the endochondral bone, and in the last stage the osseous tissue grows until it acquires a compact osseous structure.

Osteomas of the skin revisited: a clinicopathologic review of 74 cases.

Cutaneous ossification is an unusual event that may be primary or secondary to either inflammatory or neoplastic processes. It is classified as primary when it occurs in the absence of a demonstrable preexisting lesion. Secondary lesions have been most commonly reported occurring with pilomatricoma, basal cell carcinoma, acne vulgaris, and melanocytic nevi (nevus of Nanta). Histologically, the osteomas are composed of well-formed bony spicules with prominent cement lines and calcification. They may demonstrate osteoblasts, osteoclasts, and osteocytes and occasionally may even demonstrate bone marrow elements. We searched the files of a reference dermatopathology laboratory to identify cases of either primary or secondary cutaneous ossification. We present a series of 74 cases of primary and secondary cutaneous ossification. Most cases were secondary in nature. Lesions were more common on the head and neck and in whites. Lesions were also more commonly identified in female patients. In addition, included in our series are 19 cases of nevus of Nanta. To our knowledge, this represents the largest series of such cases in the English literature. Cutaneous ossification is seen both in primary and, more commonly, in secondary conditions involving the skin. Benign neoplasms, especially melanocytic nevi, represent the most common cause of secondary osteoma formation. Women are more commonly affected than men, but the reason for this is unclear. The exact reason why osteoma formation occurs is unclear and requires further study.

Calcaneal apophysitis: a quantitative radiographic evaluation of the secondary ossification center.

Calcaneal apophysitis in children is a self-limited condition that may interfere with walking and physical performance in sports, thus causing concern to the patient and parents. There is still controversy about the significance of the radiographic changes in children with heel pain, since the report of Sever in 1912. One of the reasons is that normal children may display a considerable variation in the radiographic aspects of the secondary ossification center of the calcaneus at different ages. In this investigation, the developmental aspects of primary and secondary ossification centers of the calcaneus were studied in radiographs obtained from healthy boys and from boys with calcaneal apophysitis. The normal population comprised 392 children and adolescents ranging in age from 6 to 15 years. There were 69 individuals with calcaneal apophysitis ranging in age from 8 to 14 years. Lateral standard radiographs were obtained of both heels, and a copper step wedge was used as a calibration to determine bone density. The following parameters were analyzed on the plain films: time of appearance, fusion and number of fragments of the secondary nucleus, area and bone densitometry of the primary and secondary ossification centers of the calcaneus. In the normal population, the ossification of the secondary nucleus began at 7 years of age, and at 15 years of age, the nucleus was fused in all individuals. In the apophysitis group, the secondary ossification center was present and not fused in all individuals. Both secondary nuclei increased in size with age with no difference between the two groups. Regarding bone density, both the primary and secondary nuclei were less dense in the apophysitis group than their counterparts in the normal population. The most significant difference between the two populations referred to the degree of fragmentation, which was greater in the apophysitis group. Our data showed that the sclerotic aspect of the secondary nucleus of the calcaneus is a normal feature and, therefore, should not be used to establish the diagnosis of Sever's disease. The most consistent difference between the normal and apophysitis group was related to the more fragmented aspect of the secondary nucleus in the latter individuals, which may suggest a mechanical etiology for that condition.

Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis.

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF(120/120) mice. VEGF(120/120) mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.

The Urokinase Plasminogen Activator Receptor–Associated Protein/Endo180 Is Coexpressed with Its Interaction Partners Urokinase Plasminogen Activator Receptor and Matrix Metalloprotease-13 during Osteogenesis

The urokinase plasminogen activator receptor–associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V.

Prenatal development of the human mandible.

In an effort to better understand the interrelationship of the growth and development pattern of the mandible and condyle, a sequential growth pattern of human mandibles in 38 embryos and 111 fetuses were examined by serial histological sections and soft X-ray views. The basic growth pattern of the mandibular body and condyle appeared in week 7 of fertilization. Histologically, the embryonal mandible originated from primary intramembranous ossification in the fibrous mesenchymal tissue around the Meckel cartilage. From this initial ossification, the ramifying trabecular bones developed forward, backward and upward, to form the symphysis, mandibular body, and coronoid process, respectively. We named this initial ossification site of embryonal mandible as the mandibular primary growth center (MdPGC). During week 8 of fertilization, the trabecular bone of the mandibular body grew rapidly to form muscular attachments to the masseter, temporalis, and pterygoid muscles. The mandible was then rapidly separated from the Meckel cartilage and formed a condyle blastema at the posterior end of linear mandibular trabeculae. The condyle blastema, attached to the upper part of pterygoid muscle, grew backward and upward and concurrent endochondral ossification resulted in the formation of the condyle. From week 14 of fertilization, the growth of conical structure of condyle became apparent on histological and radiological examinations. The mandibular body showed a conspicuous radiating trabecular growth pattern centered at the MdPGC, located around the apical area of deciduous first molar. The condyle growth showed characteristic conical structure and abundant hematopoietic tissue in the marrow. The growth of the proximal end of condyle was also approximated to the MdPGC on radiograms. Taken together, we hypothesized that the MdPGC has an important morphogenetic affect for the development of the human mandible, providing a growth center for the trabecular bone of mandibular body and also indicating the initial growth of endochondral ossification of the condyle.

Sox-4 Messenger RNA Is Expressed in the Embryonic Growth Plate and Regulated via the Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor in Osteoblast-like Cells

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.

Influence of muscular activity on local mineralization patterns in metatarsals of the embryonic mouse.

This study addressed the theory that local mechanical loading may influence the development of embryonic long bones. Embryonic mouse metatarsal rudiments were cultured as whole organs, and the geometry of the primary ossification center was compared with that of rudiments that had developed in utero. The mineralization front in vivo was found to be nearly straight, whereas in vitro it acquired a more convex shape due to a slower mineralization rate at the periphery of the mineralized cylinder. A poroelastic finite element analysis was performed to calculate the local distributions of distortional strain and fluid pressure at the mineralization front in the metatarsal during loading in vivo as a result of muscle contractions in the embryonic hindlimbs. The distribution of fluid pressure from the finite element analysis could not explain the difference in mineralization shape. The most likely candidate for the difference was the distortional strain, resulting from muscle contraction, which is absent in vitro, because its value at the periphery was significantly higher than in the center of the tissue. Without external loads, the mineralization process may be considered as pre-programmed, starting at the center of the tissue and resulting in a spherical mineralization front. Strain modulates the rate of the mineralization process in vivo, resulting in the straight mineralization front. These results confirm that disturbances in muscle development are likely to produce disturbed mineralization patterns, resulting in a disordered osteogenic process.

Unravelling the secrets of foetal wound healing: an insight into fracture repair in the mouse foetus and perspectives for clinical application

This study was designed to investigate the nature of mammalian foetal fracture healing in utero. A compound 'pinch' fracture was created in the foetal mouse ulna at the end of the second trimester, prior to mineralisation, and healing observed in whole mount limbs and in histological section. Cartilaginous ends gained initial contact within a perichondrial sleeve by 24 h. Bony union was achieved within 48 h by cartilage remodelling during the phase of primary endochondral ossification in the limb, a process to which adult fracture healing aspires. The molecular response to wounding was investigated using a whole mount in situ hybridisation technique and antisense mRNA probes to three target genes (BMP-2, BMP-4 and GDF-5). These experiments failed to identify altered expression in wounded limbs compared with controls.

INTOXICACION CON ION COBRE EN RATAS PREÑADAS Y SU EFECTO EN LA FORMACION DE CENTROS PRIMARIOS DE OSIFICACION EN FETOS

The social and economic activity in the 2nd Region of Chile is copper mining, generating subproducts which are eliminated in the environment. It has been reported that in industrialized centers, there is a transference of heavy metals from the mother to the fetus by placental transfer. It's proposed to know the bioaccumulation of copper in organs and fluids of pregnant rats and its effects on the primary ossification centers (POC). Three month female albine rats (Sprague Dowley), are cycled and mated in estrous. On the 8th gestation day, they are injected intraperitoneally with 1 ml of CuSO4 in concentrations 1024, 512, 256, 128, 64 ppm and physiological serum. After 18 days, the female are sacrificed and the fetus evaluated and processing with alizarin red stainig skeleton and histologic technique, 5 mm tissue sections are stained with alcian blue and/or toluidine blue. Maternal blood is obtained, as well as, fetal liver, and fetus, placenta, amniochorion membrane, and amniotic fluid for determining copper levels by means spectrophotometry of atomic absorption. In addition, fetus are checked for external visible malformation. The statistical study was carried out through variance analysis ANOVA, multiple comparison test (LSD), bilateral Fisher test, Kruskal Wallis test. The results show a high and statistically significant levels of copper in fetal liver of the intoxicated groups and control (p<0.01). There are no differences of the chemical in maternal blood, amniotic fluid, amniochorion membrane and placenta between intoxicated and control groups. A significant difference in POC formation in cervical and dorsal vertebras was found with the treatment of 256 ppm compared to the control group (p<0.05). It wasn't observed external abnormalities. It is inferred that copper ion is it's placental transferred to the fetus via placenta bioaccumulating in the liver inducing alterations in the POC formation , specially in cervical and dorsal vertebra level, which microscopically shows absence of characteristic hyaline cartilage tissue

Apparent primary ossification of the menisci in a dog

Apparent primary ossification of the menisci in a dog

Osteomyelitis of the scaphoid with sequestration of the primary ossification centre

An 8-year-old boy presented with Staphylococcus aureus osteomyelitis affecting the left (non-dominant) scaphoid. Surgical drainage resulted in the expulsion of the primary ossification centre as a sequestrum. Seven years later the wrist function was minimally impaired and X-rays showed complete ossification of the cartilage remnant with a relatively normal scaphoid.

Postnatal maturation of the sacrum and coccyx: MR imaging, helical CT, and conventional radiography.

The purpose of this paper is to provide a detailed radiologic description of the postnatal developmental anatomy of the sacrum and coccyx as revealed by MR imaging, helical CT, and conventional radiography. One hundred ten imaging examinations of the sacrococcygeal spine were performed in patients who were newborn to 30 years old. Imaging included conventional radiography (n = 63), three-dimensional gradient-recalled echo MR imaging (n = 10), and helical CT with sagittal and angled coronal reformations (n = 37). A detailed analysis was performed of the ossification and fusion of the primary and secondary ossification centers. The sacrum and coccyx were noted to develop from 58 to 60 sacral ossification centers and eight coccygeal centers, respectively. These centers were noted to ossify and fuse in an organized temporal pattern from the fetal period to the age of 30. The sacrum and coccyx are formed by a complex process that fuses primary and secondary ossification centers. Because the maturation process can be asymmetric, an understanding of this process may prove useful for distinguishing physeal plates from fracture lines.

Ossification and growth rates of the limb long bones during the prehatching period in the quail (Coturnix coturnix japonica).

The timing of ossification and the growth of six long bones of the prehatching period in the quail was studied. Ninety-nine quail eggs were incubated and in total nine fetuses were selected daily from the sixth to the sixteenth day of incubation. The fetuses were stained with alizarine and alcian blue double colouration. The fetuses were studied under the stereoscopic microscope and linear measurements were obtained from the humerus, ulna, radius, femur, tibia and fibula. The first appearance of the primary ossification centres in the diaphysis of the studied bones was found to occur between the sixth and the seventh day of incubation. Different growth patterns between the bones of the leg and of the wing were observed. Humerus and tibia showed the greatest growth rate while the radius and fibula showed the lowest.

Intrachondral microvasculature in the human fetal talus.

The intrachondral microvasculature of the growing talus of human was studied in 16 fetuses aged from 15 to 44 weeks of gestation, using interrupted serial sections and vascular injection of ink. The cartilage model of the talus was shown to be well vascularized throughout by cartilage canals. The cartilage canal contained blood vessels and connective tissue, with vessels originating from the perichondrial vessels. They were covered by a thick connective tissue wall that was continuous with the perichondrium. The functions of the cartilage canals were mainly to nourish the large masses of cartilage and to supply osteogenic tissue, which initiates the primary ossification center. As in the adult, the fetal talus was supplied with four to five main branches originating from the sinus tarsi and the tarsal canal; there were no anastomoses between the vessels of the adjacent cartilage canals and between the branches within the cartilage canal. This type of microvasculature is vulnerable to injury and, if impaired, may cause serious complications.

Bilateral duplication of the primary ulnar ossification center in Ellis-van Creveld Syndrome

A patient with Ellis-van Creveld (EvC) syndrome and bilateral duplication of the primary ulnar ossification center is presented. Abnormal ossification involving secondary ossification centers of the femur and tibia as well as duplication of primary ossification centers in the carpal bones are well described in EvC. No abnormality of ulnar ossification has been noted in patients with EvC or any other skeletal dysplasia. The occurrence of bilateral duplication of ulnar ossification centers extends the clinical findings of the EvC syndrome. The gap between the ossified areas could be misinterpreted as a fracture and thus lead to a suspicion of osteogenesis imperfecta prenatally or traumatic fracture postnatally.

Primary Structure and Tissue Distribution of FRZB, a Novel Protein Related to Drosophila Frizzled, Suggest a Role in Skeletal Morphogenesis

Articular cartilage extracts were prepared to characterize protein fractions with in vivo chondrogenic activity (Chang, S., Hoang, B., Thomas, J. T., Vukicevic, S., Luyten, F. P., Ryba, N. J. P., Kozak, C. A., Reddi, A. H., and Moos, M. (1994) J. Biol. Chem. 269, 28227-28234). Trypsin digestion of highly purified chondrogenic protein fractions allowed the identification of several unique peptides by amino acid sequencing. We discovered a novel cDNA encoding a deduced 36-kDa protein by using degenerate oligonucleotide primers derived from a 30-residue peptide in reverse transcription polymerase chain reactions. Its N-terminal domain showed approximately 50% amino acid identity to the corresponding region of the Drosophila gene frizzled, which has been implicated in the specification of hair polarity during development. Hydropathy and structural analyses of the open reading frame revealed the presence of a signal peptide and a hydrophobic domain followed by multiple potential serine/threonine phosphorylation sites and a serine-rich C terminus. Cell fractionation studies of primary bovine articular chondrocytes and transfected COS cells suggested that the protein is membrane-associated. In situ hybridization and immunostaining of human embryonic sections demonstrated predominant expression surrounding the chondrifying bone primordia and subsequently in the chondrocytes of the epiphyses in a graded distribution that decreased toward the primary ossification center. Transcripts were present in the craniofacial structures but not in the vertebral bodies. Because it is expressed primarily in the cartilaginous cores of developing long bones during embryonic and fetal development (6-13 weeks) and is homologous to the polarity-determining gene frizzled, we believe that this gene, which we named frzb, is involved in morphogenesis of the mammalian skeleton.