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PubMed2005

Membranous osteogenesis system modeled with KUSA-A1 mature osteoblasts.

Several stromal cells were established from murine bone marrow cultures. One of the KUSA subclones, KUSA-A1 cells, displays osteogenic characteristics in vitro and in vivo. The calcium deposition, osteocalcin release, and parathyroid hormone (PTH) responsiveness of KUSA-A1 cells indicate that they are mature osteoblasts or osteocytes. Bone had formed in subcutaneous tissue 1 week after subcutaneous injection of cells into immunodeficient mice. The osteogenesis by KUSA-A1 was not mediated by chondrogenesis and thus was considered to be membranous ossification. These unique characteristics of KUSA-A1 cells provide an opportunity to analyze the process of membranous ossification in detail.

Animals↗
PubMed2002

Animal models of pathologic calcification.

Recent progress in genetics and mouse genomics enables researchers to unveil the molecular basis for mouse phenotypes that express pathologic calcification in soft tissue and/or articular tissues. A newly identified multipass transmembrane protein, ANK, appears to function as an inorganic pyrophosphate (PPi) transporter or regulator of PPi transport. Abnormal extracellular PPi (ePPi) metabolism has been implicated in abnormal calcification, decreased concentrations predisposing to basic calcium phosphate (BCP) deposition, and increased concentrations promoting calcium pyrophosphate dihydrate (CPPD) crystal deposition in articular tissues. The chromosomal location of human ANK overlaps the locus identified in several kindreds affected with familial chondrocalcinosis. Deficient generation of ePPi by the ectoenzyme nucleoside triphosphate pyrophosphohydrolase also results in excessive ossification and ectopic deposition of BCP crystals in tiptoe-walking mice and PC-1 null mice. Recent studies reinforce the important regulatory role of ePPi in pathologic and physiologic calcification.

Animals↗
PubMed2002

A new rapid radiological procedure for routine teratological use in bone ossification assessment: a supplement for staining methods.

BACKGROUND: Presently, bone ossification is assessed by the study of single-stained fetal bones (alizarin red-S) or double-stained bones and cartilaginous structures (alcian blue followed by alizarin red-S). Both methods, especially double-staining, are labor-intensive, time-consuming, and provide qualitative information regarding skeleton ossification. Quantitative evaluation of ossification is more difficult and is usually based on determination of calcium and other minerals in the bone by means of atomic absorption spectrometry. Here we introduce a simple new method that allows quantitative determination of skeleton ossification before routine staining examination. METHODS: Fetuses delivered by laparotomy on the 16th and 21st day of gestation as well as 1-day-old rat pups were examined. The fetuses and pups were prenatally subcutaneously exposed to sodium valproate or to physiological saline. Lateral, prone, and supine digital radiograms of each fetus were taken using the Digora-Soredex digital radiography system and the Planmeca Intra intraoral X-ray machine. According to the best visualization, the data concerning vertebra were analyzed. All the fetuses were then routinely double-stained using alcian blue and alizarin red-S. RESULTS: Malformations of axial skeleton (rib, sternum, and thoracic and sacral vertebra) were found in valproate-treated groups. Unlike cartilage malformations, the bone changes were detected in similar frequency in radiological and staining methods. Differences in densities according to the degree of ossification in the vertebral arches and bodies at different levels of the vertebral column, between drug-treated and negative control groups were noted. CONCLUSIONS: The preliminary results suggest that digital radiography examination is a useful method in determining delaying of skeleton ossification not detectable by other methods. It balances qualitative and quantitative aspects of the presently used methods and is also simple, objective, fast, and relatively inexpensive.

Animals↗
PubMed2006

Relationship between formation of the femoral bicondylar angle and trochlear shape: independence of diaphyseal and epiphyseal growth.

During hominin evolution, an increase in the femoral bicondylar angle was the initial change that led to selection for protuberance of the lateral trochlear lip and the elliptical profile of the lateral condyle. No correlation is found during ontogeny between the degree of femoral obliquity and of the prominence of the lateral trochlear lip. Might there be a relationship with the elliptical profile of the lateral condyle? On intact femoral diaphyses of juvenile humans and great apes, we compared the anteroposterior length of the lateral and medial sides of the distal metaphysis. The two diaphyseal pillars remain equal during postnatal growth in great apes, while the growth of the lateral pillar far exceeds that of the medial pillar in humans. Increase in bicondylar angle is correlated with disproportionate anteroposterior lengthening of the lateral pillar. The increased anteroposterior length of the lateral side of the metaphysis would contribute to increasing the radius of the curvature of the lateral condyle, but not to the projection of the lateral trochlear lip. The similar neonatal and adult femoro-patellar joint shape in humans prompted an assessment of the similarity during growth of the entire neonatal and adult epiphyses. We showed that the entire epiphysis undergoes drastic changes in proportions during postnatal growth. Finally, we emphasize the need to distinguish the cartilaginous phenotype and the ossified phenotype of the distal femoral epiphysis (and of any epiphysis) during postnatal growth. This crucial distinction applies to most postcranial bones, for they almost all develop following the process of endochondral ossification.

Adaptation, Physiological↗
PubMed1985

Collagen of the calcified layer of human articular cartilage.

The distribution patterns of collagen types I, II and III were studied using immunofluorescent staining techniques in human articular cartilage, including the calcified layer. Tissue taken from femoral heads was stained with the appropriate antiserum. Adjacent sections were stained with von Kossa or Alizarin red to determine the distribution of calcium salts. Results indicate that endochondral ossification at this site occurs by calcium being deposited initially within a matrix of type II collagen.

Calcification, Physiologic↗
PubMed2002

[Biphosphonates in children: present and future].

Biphosphonates are synthetic analogs of the natural pyrophosphate molecule, introduced primarily for the treatment of Paget disease of bone. Their main mechanism of action consisting in an inhibition of osteoclastic activity is critical for treatment of disorders including an increased bone resorption. In childhood osteoporosis (especially osteogenesis imperfecta), hypercalcemia and heterotopic calcifications are the three main situations in which they have been successfully used, with however few follow-up and no controlled studies. The evolution of these compounds generating more potent products, given orally, with limited effect on bone mineralization should allow an extension of their use in pediatric patients. Multicentric studies are now necessary to specify their efficacy and guidelines for use in children.

Administration, Oral↗
PubMed2004

[Action of vitamin D on mineralization].

The effects of 1,25 (OH) (2)D on bone mineralization appear to be indirect by stimulating the calcium and phosphate supply, mainly by absorption from intestine. The disturbance in vitamin D action results in rickets in infancy and osteomalacia in adults. An association has been recently reported between mild vitamin D insufficiency and decreased bone mineral density in the elderly, which enhances the importance of appropriate vitamin D intake.

Animals↗
PubMed2003

FGF-2 stimulation affects calvarial osteoblast biology: quantitative analysis of nine genes important for cranial suture biology by real-time reverse transcription polymerase chain reaction.

Appropriately timed closure of the cranial sutures is a critical factor in normal postnatal morphogenesis of the cranial vault. Suture patency is necessary to permit rapid neonatal expansion of the cerebral hemispheres, and later ossification is important for bony protection of the cerebrum. Premature suture ossification (craniosynostosis) leads to myriad adverse functional and developmental consequences. Several murine studies have implicated dura-derived fibroblast growth factor-2 (FGF-2) paracrine signaling as a critical factor promoting physiologic posterior frontal suture fusion. In this study, the authors used real-time reverse transcription polymerase chain reaction (RT-PCR) to study an in vitro system that models the in vivo stimulation of suture calvarial osteoblasts by dura-derived FGF-2. The authors advocate real-time RT-PCR as a powerful and rapid technique that offers advantages in the highly sensitive, specific, and reproducible analyses of nine genes known to be important in cranial suture biology. The genes studied were growth factors [FGF-2, transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3], growth factor receptors (FGF-R1, FGF-R2, TGF-beta RI, and TGF-beta RII), and a marker of osteoblast differentiation (Co1-I alpha I). These analyses provide a "snapshot" of several important genes involved in suture fusion that is more inclusive and quantitative than that which has been previously reported.

Animals↗
PubMed1997

Enhancement by bFGF of osteogenesis induced by rhBMP-2 in rats.

Subcutaneous implantation of bone morphogenetic protein (BMP) combined with a fibrous glass membrane (FGM) induces cartilage formation in the entire inner area of the membrane within 2 wk. It has been hypothesized that a tight FGM network (1 microm exclusion size) provides immature cells with spaces for penetrating into the membrane, but not for vascular formation, at least until 2 wk. To test this hypothesis, basic fibroblast growth factor (bFGF), known to be a potent stimulant of capillary formation, was applied to the implant. BMP was combined with FGM in the presence or absence of bFGF, and then implanted subcutaneously into the backs of rats. The bFGF-supplemented implant caused 1.3 times higher alkaline phosphatase activity and 3 times higher calcium contents at 2 wk, whereas type II collagen contents decreased, thus indicating that bFGF enhances bone formation in BMP/FGM implants. These results suggest that bFGF induces faster and stronger invasion of capillaries into the FGM and destroys its tight network, resulting in acceleration of the ossification process.

Alkaline Phosphatase↗
PubMed1998

The osteogenic potential of free periosteal autografts in tibial fractures with severe soft tissue damage: an experimental study.

The present study was undertaken on 80 adult male New Zealand rabbits to assess the effect of free nonvascularized, autologous, periosteum transplants on bone healing in a rabbit fracture model comparable to a tibial fracture with severe soft tissue damage. Comparison was made between transplantation of free autologous periosteal grafts on the anteromedial side of the tibia and nontransplantation on the contralateral tibia (control). We produced a standardized transverse osteotomy of both tibial diaphyses. The medullary cavity was reamed and nailed ; a 1-cm segment of periosteum was excised from either side of the osteotomy. Periosteal and extraosseous ingrowth was prevented at the osteotomy site by a silastic sheet wrapped around two-thirds of the circumference of the tibia. On the silastic-free bone window, in one group we spanned the osteotomy with a free nonvascularized longitudinally-oriented autologous periosteum sewn to the adjacent periosteum proximally and distally. In the second group, the periosteum was placed transversely with a gap between it and the adjacent periosteum proximally and distally. Revascularization of the graft was determined with the colored microsphere technique. Our data suggest that orthotopically-placed autologous nonvascularized periosteum retains its osteogenic potential in a poorly vascularized environment comparable to a tibial fracture with severe soft tissue damage ; the effect is enhanced if the graft is in contact with intact periosteum. Histologically, callus formation after periosteal grafting resembles endochondral and intramembranous ossification.

Animals↗
PubMed2001

Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice.

We have developed a transgenic mouse line, NJ.1638, which expresses high levels of IL-5 from T cells, with profound hematological consequences. Eosinophils comprise more than 60% of circulating white blood cells in these animals, with the total peripheral white blood cell counts increasing more than 40-fold relative to wild-type littermates. This extraordinary proliferative capacity is sustained by expanded sites of extramedullary hematopoiesis and is accompanied by multifocal, ectopic bone formation in the spleen. Histology of the splenic nodules revealed the presence of osteoid matrices and osteocytes trapped within mineralized trabecular plates. In addition, polarized light microscopy of calcified tissue sections revealed both woven bone and areas of organized lamellar bone. Morphometric assessments demonstrated that both the growth and mineralization of splenic bone occurred at rates nearly an order of magnitude higher than in skeletal bone. Skeletal bone metabolic parameters were also perturbed. We also observed heterotopic ossification of the spleen and perturbation of skeletal bone homeostasis following adoptive engraftment of transgenic marrow to wild-type recipients. These data suggest that IL-5 overexpression mediates bone formation through the mobilization of marrow-derived osteogenic progenitors and/or the inhibition of recruited osteoclasts.

Animals↗
PubMed2003

Developmental origin of the frontoparietal bone in Bombina variegata (Anura: Discoglossidae).

Development of the frontoparietal bone in the European yellow-bellied toad, Bombina variegata, was followed on the basis of histological analysis of transverse serial sections through the larval skulls to recognize early stages of ossification represented by osteoid (uncalcified bone matrix) and on cleared and stained specimens to investigate more advanced stages. Ossification of the frontal begins as three tiny areas of osteoid (F(1), F(2), F(3)) adjoining the dorsal surface of the orbital cartilage, which are separated by areas without osteoid. F(3) is the largest (most advanced). Prior to calcification, F(3) extends to fuse with F(2) and then with F(1), but it does not expand over the prootic fissure posteriorly. As calcification begins the strip of bone is joined posteromedially by F(4). Only then does a single ossification center corresponding to the parietal arise on the anterodorsal surface of the otic capsule. This ossification sequence corresponds to those observed in the Actinopterygii and in caudate amphibians.

Animals↗
PubMed2004

Variation in the ossification process of the anconeal and medial coronoid processes of the canine ulna.

This morphological and radiographic study investigates the ossification process of the anconeal and medial coronoid processes of the ulna in a sample of 142 dogs ranging in age from neonatal to 44 weeks. The anconeal process was noted to develop by appositional ossification, formation of a separate ossification center, or a combination of both. Several developmental stages of the ossification center of the anconeal process as well as its anatomic position and radiographic appearance are described. Differences have been noted in the shape of this ossification center as well as the ossification process itself. The medial coronoid process develops exclusively by appositional ossification. Unlike ossification of the anconeal process, ossification of the medial coronoid process was completed earlier (p < 0.05) in smaller than in the larger dogs. In smaller dogs, both the medial coronoid and anconeal processes were found to be mature by the age of 16 weeks. In the larger dogs, ossification of the anconeal process was completed not before 14 weeks of age and ossification of the medial coronoid process was completed about 6 weeks later.

Animals↗
PubMed1997

Type X collagen expression and hypertrophic differentiation in chondrogenic neoplasias.

Little is known about matrix biochemistry and cell differentiation patterns in chondrogenic neoplasms. This is the first description of the focal expression of collagen type X by neoplastic chondrocytes in situ and its incorporation into the extracellular matrix of cartilaginous tumors. This shows that neoplastic chondrocytes have the potential to undergo the full program of cell differentiation, including hypertrophy, comparable to their physiological counterparts in the growth plate. However, only in benign osteochondromas was a zonal expression of type X collagen found similar to that observed in the growth plate, where the cells immediately above the ossification frontier are selectively positive for type X collagen. In enchondromas and chondrosarcomas, the expression was randomly distributed within the tumors. Surprisingly, in less differentiated chondrosarcomas with spindle-shaped cells and non-cartilaginous extracellular matrix, exceptional expression of collagen type X was observed, which indicates potential uncoupling of collagen type X expression from the differentiated chondrocytic phenotype in neoplastic chondrocytes in vivo.

Bone Neoplasms↗
PubMed2000

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.

Animals↗