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PubMed1998

Age changes in the articular tissue of human mandibular condyles from adolescence to old age: a semiquantitative light microscopic study.

In a previous study (Luder, Anat. Rec., 1997;248:18-28), the articular tissue of the adult mandibular condyle was characterized semiquantitatively. However, questions about age changes of mature tissue were not answered, and the time course of tissue maturation from the end of condylar growth to the attainment of the adult appearance remained unknown. These issues are addressed in the present investigation. By using a light microscope, features of the superficial, intermediate, and deep articular tissue zones as well as of the subchondral bone were assessed at nine predetermined condylar sites. The frequencies of these features were recorded as scores from 0 (absent) to 10 (continuous) and were plotted against age. Analysis of covariance served for testing the significance of age and sex effects as well as intracondylar variability. Whereas almost all age-related changes in frequencies of tissue features were similar along the whole lateromedial dimension, changes at the putatively nonload-bearing, posterior slope differed significantly from those at the putatively load-bearing, anterior slope and zenith of the condyle. Two patterns of changes were noted. Frequencies of a first group of tissue features altered mainly during the age period from 15 years to 30 years and remained more or less stable thereafter. This course was characteristic for 1) a progressive cartilaginification of the superficial zone as well as 2) the disappearance of hypertrophic growth cartilage and 3) the appearance of grid-fibrous fibrocartilage in the deep zone, which were accompanied by 4) a decline in endochondral ossification and 5) the formation of a compact, subchondral bone plate. Frequencies of a second group of tissue features disclosed changes that continued up to middle and old age. This pattern was evident regarding 1) a decrease in the prominence associated with 2) a drop in cellularity and 3) progressive fibrosis or even cartilaginification of the intermediate zone. Among the age changes of condylar articular tissue, those affecting the superficial and deep zones as well as the subchondral bone are largely complete by about 30 years of age and seem to be related primarily to a gradual transition from growth to adulthood. In contrast, a second group of alterations, which progress to old age and involve mainly the intermediate zone, appears to be associated with continued maintenance and adaptive articular remodeling as well as possibly senescence. Both maturational and later age changes seem to depend markedly on articular load bearing.

Adolescent↗

PubMed1995

Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: an in vitro and ex vivo study of remodeling bone.

Osteopontin is a phosphorylated glycoprotein believed to be secreted by osteoblasts and deposited into the bone matrix to facilitate osteoclasts adhesion or to initiate osteoid mineralization. Previously we have presented contradictory evidence that osteoclasts express osteopontin mRNA in human remodeling bone. The aim of this study was to ascertain whether osteoclasts synthesize and deposit osteopontin in resorption lucunae. We characterized expression of osteopontin mRNA and protein expression in both intramembranous and endochondral ossification, as well as remodeling bone, in the human osteophyte. Osteopontin mRNA was expressed in osteoclast with tartrate-resistant acid phosphatase (TRAP) positivity within resorption lacunae. The osteoclasts and immediate resorption surfaces also expressed osteopontin. However, osteopontin mRNA and protein were weak (transient) or undetectable in osteoblasts at adjacent bone formation sites; no osteopontin expression was observed in the osteoid, although occasional reactivity was observed in osteocytes and the mineral-osteoid interface. In contrast, osteopontin was highly expressed in the osteoblasts and matrix of woven bone during intramembranous and endochondral ossification. The matrix expression correlated with mineralization; however, in some instances osteopontin deposition was observed prior to mineralization. Similarly, osteopontin expression was evident in cartilage matrix, solely at foci of mineralization. Chondroclasts expressed osteopontin mRNA and protein: the surfaces of resorbed calcified cartilage also expressed osteopontin. Abnormal, unmineralized matrices apparently lacked deposited osteopontin, but were nevertheless resorbed by osteoclasts; the osteoclasts and resorbed surfaces expressed no osteopontin protein. That osteoclasts are responsible for the deposition of osteopontin was confirmed in vitro, whereby resorption pits in whale dentine and bovine bone slices, produced by isolated human osteoclasts, contained deposited osteopontin. Osteopontin may facilitate the adhesion (or detachment) of the osteoclast to the bone surface. Alternatively, the possibility that osteopontin may act as a postresorptive signal to recruit osteoblasts, or to polarize and direct the mineralization of the formed osteoid, is discussed.

Acid Phosphatase↗

PubMed2004

Osteoclast deficiency results in disorganized matrix, reduced mineralization, and abnormal osteoblast behavior in developing bone.

UNLABELLED: Studies of the influence of the osteoclast on bone development, in particular on mineralization and the formation of the highly organized lamellar architecture of cortical bone by osteoblasts, have not been reported. We therefore examined the micro- and ultrastructure of the developing bones of osteoclast-deficient CSF-1R-nullizygous mice (Csf1r(-/-) mice). INTRODUCTION: Colony-stimulating factor-1 receptor (CSF-1R)-mediated signaling is critical for osteoclastogenesis. Consequently, the primary defect in osteopetrotic Csf1r(-/-) mice is severe osteoclast deficiency. Csf1r(-/-) mice therefore represent an ideal model system in which to investigate regulation by the osteoclast of osteoblast-mediated bone formation during development. MATERIALS AND METHODS: Bones of developing Csf1r(-/-) mice and their littermate controls were subjected to X-ray analysis, histological examination by light microscopy and transmission electron microscopy, and a three-point bending assay to test their biomechanical strength. Bone mineralization in embryonic and postnatal bones was visualized by double staining with alcian blue and alizarin red. Bone formation by osteoblasts in these mice was also examined by double-calcein labeling and in femoral anlagen transplantation experiments. RESULTS AND CONCLUSIONS: Frequent spontaneous fractures and decreased strength parameters (ultimate load, yield load, and stiffness) in a three-point bending assay showed the biomechanical weakness of long bones in Csf1r(-/-) mice. Histologically, these bones have an expanded epiphyseal chondrocyte region, a poorly formed cortex with disorganized collagen fibrils, and a severely disturbed matrix structure. The mineralization of their bone matrix at secondary sites of ossification is significantly reduced. While individual osteoblasts in Csf1r(-/-) mice have preserved their typical ultrastructure and matrix depositing activity, the layered organization of osteoblasts on the bone-forming surface and the direction of their matrix deposition toward the bone surface have been lost, resulting in their abnormal entrapment by matrix. Moreover, we also found that (1) osteoblasts do not express CSF-1R, (2) the bone defects in Csf1r(-/-) embryos develop later than the development of osteoclasts in normal embryos, and (3) the transplanted Csf1r(-/-) femoral anlagen develop normally in the presence of wildtype osteoclasts. These results suggest that the dramatic bone defects in Csf1r(-/-) mice are caused by a deficiency of the osteoclast-mediated regulation of osteoblasts and that the osteoclast plays an important role in regulating osteoblastic bone formation during development, in particular, in the formation of lamellar bone.

Animals↗

PubMed1997

Calcification of the lumbar vertebrae during human fetal development.

The calcification rate of the human fetal vertebrae has not been previously documented. In bridging this gap in knowledge, this report will specifically provide reference values for mineral content in predominantly trabecular bone during its formation in the prenatal period. Such information would facilitate early detection of ossification defects which become apparent in the fetal spine as early as 20 weeks of gestation [1]. Lumbar vertebrae were obtained at autopsy from 33 human fetuses aged 17-41 weeks of gestation. Vertebral demineralization was effected using an acid-alcohol solution. The extracted concentrations of calcium and phosphorus were determined spectrometrically from the solutions. Atomic absorption spectroscopy was used to determine calcium concentration and phosphate concentration was assessed by ultraviolet colorimetry. Mineralization could be described by a linear relationship over the age range studied. Calcium was taken up by the fetal vertebrae at twice the rate of phosphorus (0.2% per week). When the fetuses were classed according to similar gestational ages, the Ca/P weight ratios increased from 1.7 in the youngest group to 2.2 in the oldest group. The time of onset of calcification, calculated by extrapolation of the calcium data, appeared to be earlier than ultrastructural studies by other investigators suggest, but was subject to many uncertainties.

Calcification, Physiologic↗

PubMed2003

Inactivation of menin, the product of the multiple endocrine neoplasia type 1 gene, inhibits the commitment of multipotential mesenchymal stem cells into the osteoblast lineage.

The physiological roles of menin, the product of the multiple endocrine neoplasia type 1 gene, are not known. Homozygous menin knockout mice exhibit cranial and facial hypoplasia. We, therefore, investigated the role of menin in the regulation of osteoblastic differentiation. Menin antisense oligonucleotides (AS-oligo) reduced endogenous menin expression in the C3H10T1/2 (10T1/2) mouse mesenchymal stem cells and antagonized alkaline phosphatase (ALP) activity and the expression of type I collagen, Runx2/cbfa1 (Runx2), and osteocalcin (OCN) induced by bone morphogenetic protein 2 (BMP-2). AS-oligo did not affect adipogenic markers (Oil red staining and PPARgamma expression) and chondrogenic markers (Alcian blue staining and type IX collagen) induced by BMP-2 in 10T1/2 cells. Menin co-immunoprecipitated with Smad1 and Smad5, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5. In osteoblastic MC3T3-E1 cells, AS-oligo affected neither BMP-2-stimulated ALP activity nor the expression of Runx2 and OCN. Stable inactivation of menin in MC3T3-E1 cells increased ALP activity, mineralization, and the expression of type I collagen and OCN. In 21-day cultures of MC3T3-E1 cells and BMP-2-treated 10T1/2 cells, endogenous menin expression increased up to day 14 and declined thereafter. These data indicate that menin inactivation specifically inhibits the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage, mediated by menin and Smad1/5 interactions. Menin is important for both early differentiation of osteoblasts and inhibition of their later differentiation, and it might be crucial for intramembranous ossification.

3T3 Cells↗

PubMed2007

What triggers cell-mediated mineralization?

Mineralization is an essential requirement for normal skeletal development, but under certain pathological conditions organs like articular cartilage and cardiovascular tissue are prone to unwanted mineralization. Recent findings suggest that the mechanisms regulating skeletal mineralization may be similar to those regulating pathological mineralization. In general, three forms of cell-mediated mineralization are recognized in an organism: intramembranous ossification, endochondral ossification and pathological mineralization. This review summarizes recent work that tried to elucidate how cell-mediated mineralization is initiated and regulated. To explain mineralization, several theories have been proposed. One theory proposes that mineralization is initiated within matrix vesicles (MVs). A second, not mutually exclusive, theory proposes that phosphate induces apoptosis, and that apoptotic bodies nucleate crystals composed of calcium and phosphate. A third theory suggests that mineralization is mediated by certain non-collagenous proteins, which associate with the extracellular matrix. Regardless of the way mineralization is initiated, the organism also actively inhibits mineralization by specific proteins and removal of an inhibitor may also induce mineralization. Although many studies greatly contributed to a better understanding of the mechanisms regulating cell-mediated mineralization, many questions remain about the mechanisms that trigger cell-mediated mineralization and how this process is regulated. Further investigation is necessary to develop in the future novel therapeutic strategies to prevent pathological mineralization.

Animals↗

PubMed1999

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.

Animals↗

PubMed1999

Association of matrix acid and alkaline phosphatases with mineralization of cartilage and endochondral bone.

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50-100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralizing zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.

Acid Phosphatase↗

PubMed2004

Relationships between dental and skeletal maturity in Turkish subjects.

The aim of this study was to investigate the relationships between the stages of calcification of various teeth and skeletal maturity stages among Turkish subjects. The samples were derived from dental panoramic and hand-wrist radiographs of 500 subjects (215 males and 285 females). Calcification of the mandibular canines, first and second premolars, and second and third molars was rated according to the system of Demirjian. To evaluate the stage of skeletal maturation of each hand-wrist radiograph, nine ossification events were determined according to the systems of Björk, and Grave and Brown. Statistically significant relationships were determined between dental calcification and skeletal maturity stages according to Spearman rank-order correlation coefficients. Correlations between dental development and skeletal maturity ranged from .490 to 0.826 for females and .414 to .706 for males (P < .01). The second molar showed the highest correlation and the third molar showed the lowest correlation for female and male subjects. For both sexes, root formation of the canine as well as the first premolar was completed in the majority of the subjects at the MP3cap, PP1cap, Rcap stages. Because of the high correlation coefficients, this study suggests that tooth calcification stages from panoramic radiographs might be clinically useful as a maturity indicator of the pubertal growth period. It is appropriate to put these skeletal and dental maturation relationships into daily orthodontic diagnostic practice, when treating a Turkish patient.

Adolescent↗

PubMed1997

Developmental expression of perlecan during murine embryogenesis.

Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constituent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the potential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that recognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expression and deposition during murine ontogeny. Perlecan appeared early (E10.5) in tissues of vasculogenesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11-13), the greatest deposition of perlecan occurred within the developing cartilage, especially the cartilage undergoing endochondral ossification, where it remained elevated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothelial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosynthesis and turnover rates in the blood vessels vis-à-vis those of cartilaginous and other mesenchymal tissues. During later stages of development (E13-17.5) perlecan mRNA levels progressively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the results indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage.

Animals↗

PubMedDate not supplied

Phenotypic features of the domestic pigs bred in the Roman settlements of Pompeii and Caralis.

A reconstruction of the morphological features of domestic pigs from two Roman settlements is here suggested by means of the study of skeletal and dental remains, with the aim at evaluating their degree of selection in comparison with wild boars. Material was formed by 111 bone and tooth fragments and was uncovered during the excavations of Polybius' House in Pompeii and of Roman buildings in the neighbourhood of Caralis harbour (Sardinia). The remains underwent morphological examination. The eruption of permanent teeth and ossification of epiphyseal cartilages let us establish that most animals were over 18-20 months. When possible, the determination of sex was made by detecting tusk features. The presence of anthropic signs on the bone surface provides some information about slaughtering and cooking procedure in the Roman period and supports the hypothesis that the animal remnants were food remains. Osteometric analysis was carried out on long and short bones and teeth through suitable multiplicative parameters, leading to the assessment of the withers height and other main phenotypic features. Logarithmic deviation pointed out the significant osteometric differences between the domestic pigs from the two Roman settlements. These data were also compared with those from wild boars and modern crossbred wild boars X non-selected pigs. In conclusion, our data show that pigs from Caralis bear much resemblance to wild boars, whereas those from Pompeii appear to be improved, so sharing some phenotypic features of modem improved breeds.

Animal Husbandry↗

PubMed2002

3-Methylcholanthrene, which binds to the arylhydrocarbon receptor, inhibits proliferation and differentiation of osteoblasts in vitro and ossification in vivo.

3-Methylcholanthrene (3MC) is a ligand for arylhydrocarbon receptor (AhR), which binds dioxin. We examined the effects of 3MC on the proliferation and differentiation of osteoblasts using cultures of rat calvarial osteoblast-like cells (ROB cells) and mouse calvarial clonal preosteoblastic cells (MC3T3-E1 cells). Analysis by RT-PCR revealed that the mRNAs for AhR and AhR nuclear translocators were expressed in both ROB and MC3T3-E1 cells. Cell proliferation and the synthesis of DNA by ROB cells and MC3T3-E1 cells were markedly inhibited on exposure of cells to 3MC. Furthermore, 3MC reduced the activity of alkaline phosphatase and the rate of deposition of calcium by cells. The level of expression of mRNA for osteocalcin, which is a marker of osteoblastic differentiation, was also depressed by 3MC. Moreover, when 3MC (1 mg/kg body weight) was administered sc to pregnant mice at 10.5, 12.5, and 14.5 d post coitus, fetuses examined subsequently at 15.5 or 17.5 d post coitus revealed evidence of inhibition of appropriate calcification of bones. The treated metacarpals showed no subperiosteal bone matrix histologically. Our findings indicate that 3MC might have critical effects on the formation of bone both in vivo and in vitro.

Animals↗

PubMedDate not supplied

Metalloproteinases (MMPs -2, -3) are involved in TGF-beta and IGF-1-induced bone defect healing in 20-month-old female rats.

Matrix metalloproteinases are important in the physiological and pathological degradation of extracellular matrix including that of bone and cartilage. The process of bone defect healing is associated with formation of cartilage callus and cancelous bone. With maturation and aging, the response of skeletal tissues to injury is limited. The ability of growth factors to enhance bone defect healing in aged rats was studied. Partial bone defects were induced in femurs of aged rats. A single dose of IGF-1, TGF-beta+IGF-1 or saline was inserted in the defect and bones were examined after 2 and 4 weeks. Morphology revealed that after 2 weeks of treatment with TGF-beta the defects were filled with mesenchyme-like tissue and delicate bone trabeculae. Positive staining for metalloproteinase-2 (MMP-2) was shown at the sites of new bone formation. In defects treated with IGF-1 or TGF-beta+IGF-1 nodules of cartilage and fine bone trabeculae along with positive staining for both MMP-2 and MMP-3 were demonstrated in the healing defects. After 4 weeks radiology revealed mineralization in defects treated with TGF-beta and less pronounced mineralization after treatment with IGF-1, or with TGF-beta+IGF-1, whereas only partial healing of the defects was observed in control specimens. MMP-2 and MMP-3 were detected at sites of new bone formation after treatment with TGF-beta, IGF-1, and TGF-beta+IGF-1. It is concluded that TGF-beta and IGF-1 induced bone defect healing in aged rats. TGF-beta induced bone formation while IGF-1 induced cartilage and than bone formation via endochondral ossification. The localization of MMP-2 and MMP-3 in the healing defects reflected the synthesis of bone or cartilage matrices in the defect, reflecting the involvement of MMPs in the process of bone formation and endochondral ossification. The ability to induce bone defect healing in aging is of great clinical importance and understanding the involvement of MMPs in this process can contribute to future treatment with growth factors to enhance bone defect healing in 20-month-old female rats.

Journal Article↗

PubMed1972

Collagen heterogeneity within different growth regions of long bones of rachitic and non-rachitic chicks.

The different anatomical regions involved in osteogenesis in the chick long bone have been examined for heterogeneities in collagen structure that might relate to the mechanism of ossification. Experimentally induced lathyrism was employed to enhance collagen solubility, and vitamin D deficiency to allow accumulation of osteoid, the precursor of bone matrix. The extractable lathyritic collagens of the cartilaginous and osseous regions of growing long bones from rachitic and non-rachitic chicks were examined for alpha-chain type and amino acid composition. In both groups of animals the growth plate and cartilaginous regions of the epiphysis gave collagen molecules of the constitution [alpha1(II)](3), whereas the ossifying regions contained [alpha1(I)](2) alpha2. The degree of hydroxylation of the lysine moieties was increased by approximately 50% in the alpha1(I)-chain and alpha2-chain of rachitic bone collagen. Since uncalcified osteoid is greatly enriched in rachitic bone, it is concluded that the collagen of osteoid has the configuration [alpha1(I)](2) alpha2, similar to that of bone matrix, but has an elevated hydroxylysine content. The possible relationship of this difference to the mechanism of calcification is discussed.

Amino Acids↗

PubMed2003

Orientation of mineral crystallites and mineral density during skeletal development in mice deficient in tissue nonspecific alkaline phosphatase.

Tissue nonspecific alkaline phosphatase (TNALP) is thought to play an important role in mineralization processes, although its exact working mechanism is not known. In the present investigation we have studied mineral crystal characteristics in the developing skeleton of TNALP-deficient mice. Null mutants (n = 7) and their wild-type littermates (n = 7) were bred and killed between 8 and 22 days after birth. Skeletal tissues were processed to assess mineral characteristics (small angle X-ray scattering, quantitative backscattered electron imaging), and to analyze bone by light microscopy and immunolabeling. The results showed a reduced longitudinal growth and a strongly delayed epiphyseal ossification in the null mutants. This was accompanied by disturbances in mineralization pattern, in that crystallites were not orderly aligned with respect to the longitudinal axis of the cortical bone. Among the null mutants, a great variability in the mineralization parameters was noticed. Also, immunolabeling of osteopontin (OPN) revealed an abnormal distribution pattern of the protein within the bone matrix. Whereas in the wild-type animals OPN was predominantly observed in cement and reversal lines, in the null mutants, OPN was also randomly dispersed throughout the nonmineralized matrix, with focal densities. In contrast, the distribution pattern of osteocalcin (OC) was comparable in both types of animals. It is concluded that ablation of TNALP results not only in hypomineralization of the skeleton, but also in a severe disorder of the mineral crystal alignment pattern in the corticalis of growing long bone in association with a disordered matrix architecture, presumably as a result of impaired bone remodeling and maturation.

Alkaline Phosphatase↗

PubMed1981

Transplacental effects of cortisone acetate on calcification and ossification of long bones in mice.

The transplacental effects of cortisone acetate on the skeleton of neonatal mice has been investigated. Pregnant mice were injected intramuscularly with 0.75 mg cortisone acetate daily on days 11-19 of gestation. The mice were allowed to deliver and pups were weighed on days 1, 3, 5, 10, 15, 20 and 30 and compared to controls. Light microscopy of non-decalcified longitudinal sections of bones revealed mineral precipitates throughout the zone of hypertrophic cartilage most prominent on days 1, 3 and 5 and gradually disappearing on days 10-20. Transmission electron microscopy revealed intracellular calcification of maturing and hypertrophic chondrocytes in addition to abnormal calcification of the interstitial substance. Hydroxylapatite crystals were not necessarily associated with matrix vesicles. Scanning electron microscopy of long bones revealed a wide zone of calcified intercartilagenous matrix representing the calcified proliferating and hypertrophic zones. In this area, the calcospherites varied in size (0.5-1.4 mu in diameter) and were found in both longitudinal and transverse septa. No significant differences in the distribution and shape of cartilage matrix vesicles between experimental and control animals was observed. However, the fact that mineral crystals in experimental animals was not necessarily associated with matrix vesicles points to the possibility that this "pathological calcification" may utilize a different mechanism than the normal process through matrix vesicles.

Animals↗

PubMed2003

The immunolocalisation of VEGF in the articular cartilage of sheep mandibular condyles.

INTRODUCTION: Vascular endothelial growth factor (VEGF) has recently been found to be essential for hypertrophic chondrocyte apoptosis and angiogenesis at the growth plate of long bones, indicating a central role in endochondral ossification. VEGF has more recently, also been shown to be expressed in articular cartilage chondrocytes in human osteoarthritic and rheumatoarthritic joints but not healthy adult joints. To investigate the role of VEGF in the fibrocartilage of the temporomandibular joint, this study aimed to document the presence and distribution of VEGF in the condylar articular cartilage of sheep temporomandibular joints. METHODS: Mandibular condyles of the temporomandibular joints of five 18-month old Wether sheep were fixed, decalcified, paraffin embedded and sectioned. The sections were analyzed using immunohistochemistry for VEGF. RESULTS: VEGF was found to be localised predominantly to the proliferative and maturing layers of chondrocytes in the condylar fibrocartilage of the temporomandibular joints. Articular cartilage is an avascular and alymphatic tissue. As such, the localisation of VEGF to the articular cartilage of normal temporomandibular joint condyles suggests a role for VEGF other than angiogenesis. CONCLUSION: VEGF is shown here for the first time to be present in mandibular condylar cartilage, leading us to propose a possible role in non-angiogenic extracellular matrix remodeling.

Animals↗

PubMed1996

Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis.

Mature osteoclasts specifically express the purple, band 5 isozyme (Acp 5) of tartrate-resistant acid phosphatase, a binuclear metalloenzyme that can generate reactive oxygen species. The function of Acp 5 was investigated by targeted disruption of the gene in mice. Animals homozygous for the null Acp 5 allele had progressive foreshortening and deformity of the long bones and axial skeleton but apparently normal tooth eruption and skull plate development, indicating a rôle for Acp 5 in endochondral ossification. Histomorphometry and mineralization density analysis of backscattered electron imaging revealed widened and disorganized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover. Increased mineralization density was observed in the long bones of older animals which showed modelling deformities at their extremities: heterozygotes and homozygous Acp 5 mutant mice had tissue that was more mineralized and occupied a greater proportion of the bone in all regions. Thus the findings reflect a mild osteopetrosis due to an intrinsic defect of osteoclastic modelling activity that was confirmed in the resorption pit assay in vitro. We conclude that this bifunctional metalloprotein of the osteoclast is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.

Acid Phosphatase↗