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PubMed1995

Shared phenotypic expression of osteoblasts and chondrocytes in fracture callus.

Endochondral ossification in fracture healing of rats at 4, 8, 11, 14, and 21 days was analyzed using immunological and molecular probes for markers of the chondrocyte and osteoblast phenotype. These markers were osteocalcin, type I and type II collagen, including the probes homologous to the alternatively spliced forms of alpha 1 type II collagen, type IIA and type IIB. Histologic examination was performed on serial sections of the same tissue blocks to correlate cellular morphology with the immunohistochemical and in situ hybridization findings. At the junction of the cartilaginous and osseous tissue, an overlap of phenotype and morphology was noted. At the 8-day time point, the cells with chondrocyte morphology expressed intracellular message for osteocalcin and type I collagen. Immunohistochemical analysis of these cells also demonstrated intracellular osteocalcin. However, high levels of the type IIA collagen mRNA, which has previously been associated with less differentiated mesenchymal precursor cells, were expressed in both chondrocytes and osteoblasts. At the later time point (21 days) there was a substantial decrease in the number of cells displaying shared phenotypic characteristics. In situ hybridization and immunohistochemistry have permitted identification of an overlapping or shared phenotype in osteoblasts and chondroblasts in fracture callus. The findings raise important questions regarding the possible plasticity of mesenchymal cell phenotypes within the dynamic environment of fracture healing. Additional examination of these issues will further define factors involved in origin, differentiation, and maturation of bone and cartilage cells.

Animals↗

PubMed1996

Fourier transform infrared microspectroscopic analysis identifies alterations in mineral properties in bones from mice transgenic for type X collagen.

Type X collagen has been implicated in the morphogenetic events of endochondral ossification (EO), including the calcification of hypertrophic cartilage and trabeculae prior to their replacement by bone and marrow. Recently, transgenic mice, which expressed a truncated collagen X protein, were reported to exhibit morphologic alterations in all tissues arising through EO. Specifically, the growth plates were compressed within the zone of cartilage hypertrophy, and the number and size of calcified trabeculae were reduced. The condition in the mouse is comparable to Schmid metaphyseal chondrodysplasia in humans for which, to date, 20 defined type X collagen mutations have been reported. The transgenic mouse showed no alterations in mineralization by conventional histology, however, it did show a decrease in newly formed bony trabeculae, and a thinning of periosteal bones. Fourier transform infrared (FTIR) spectroscopy has previously been shown to provide quantitative and qualitative information about the relative amount of mineral and carbonate present, mineral composition, and crystal perfection. To determine whether the expression of abnormal collagen X molecules had an effect on mineral properties, the "quality" of mineral crystals was analyzed in thin sections of tibia from day 17 and day 25 genotypically negative (normal) and positive (mutant) mice from several independent transgenic mouse lines showing varying degrees of the mutant phenotype, by means of Fourier transform infrared microscopic analysis (FTIRM). The results indicate definite differences between normal and transgenic mice calcified cartilage mineral, both in the amount present and the "quality" of the crystals. Calcified cartilage mineral from transgenic mice exhibited less crystallinity and higher acidic phosphate content than the corresponding mineral from normal specimens.

Animals↗

PubMed2006

Microvascular adaptation to growth in rat humeral head.

The aim of this work is to investigate the growth of the vasculature in the rat humeral head cartilage after the initial development of the secondary ossification centre until the adult organization. Rats aging from 5 weeks to 12 months were used. Histological observations on humeral heads were implemented with morphometrical analysis. Subsequently, vascular corrosion cast, that permits a three-dimensional observation of the vasculature, were prepared and observed by scanning electron microscopy. In young animals the epiphysis contains thin bone trabeculae and most of the epiphysis is occupied by bone marrow spaces. With age, the bone trabeculae progressively enlarge up to double their thickness. The percentage of bone tissue increases from 33.6 to 58.6% of the entire epiphysis, while the bone marrow spaces tend to increase very little in their mean dimension. Vascular corrosion casts show that the epiphyseal microcirculation is well distinguished from that of the diaphysis, and arises from the vessels present in the capsule and the periosteal networks. In young animals the only capillaries are bone marrow sinusoids and few subchondral capillaries. In adult animals small vessels run between the clusters of sinusoids forming the trabecular circulation. Capillary sprouts from sinusoids are always observed both in the young and adult animals. Thus, in adult rats different proper microcirculatory districts can be distinguished in the epiphysis: (a) the sinusoidal network, that supplies the hematopoiesis of the bone marrow and the adjacent osteogenic tissue; (b) the bone tissue microcirculation, limited to small vessels that supply the metabolism and the remodelling of the bone tissue. The reported microvascular organization and its adaptation to the epiphyseal growth represent the morphological basis for understanding the reciprocal interaction among the different tissues in developing and adult rat epiphysis.

Adaptation, Physiological↗

PubMed1982

Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate.

The hypothesis is widely held that, in growth plate during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization, and that this is the mechanism by which a noncalcifiable matrix is transformed into a calcifiable matrix. We have evaluated this hypothesis by examining the immunofluorescent localization and concentrations of proteoglycan monomer core protein and link protein, and the concentrations of glycosaminoglycans demonstrated by safranin 0 staining, in the different zones of the bovine fetal cartilage growth plate. Monospecific antibodies were prepared to proteoglycan monomer core protein and to link protein. The immunofluorescent localization of these species was examined in decalcified and undecalcified sections containing the zones of proliferating and hypertrophic chondrocytes and in sections containing the zones of proliferating and hypertrophic chondrocytes and the metaphysis, decalcified in 0.5 M EDTA, pH 7.5, in the presence of protease inhibitors. Proteoglycan monomer core protein and link protein are demonstrable without detectable loss throughout the extracellular matrix of the longitudinal septa of the hypertrophic zone and in the calcified cartilage of the metaphysis. In fact, increased staining is observed in the calcifying cartilage. Contrary to the prevailing hypothesis, our results indicate that there is no net loss of proteoglycans during mineralization and that the proteoglycans become entombed in the calcified cartilage which provides a scaffolding on which osteoid and bone are formed. Proteoglycans appear to persist unaltered in the calcified cartilage core of the trabeculae, until at last the entire trabeculae are eroded from their surfaces and removed by osteoclasts, when the primary spongiosa is replaced by the secondary spongiosa.

Animals↗

PubMed1998

Vis-à-vis cells and the priming of bone formation.

Bone formation throughout skeletal growth and remodeling always entails deposition of new bone onto a pre-existing mineralized surface. In contrast, the initial deposition of bone in development requires the formation, ex novo, of the first mineralized structure in a nonmineralized tissue. We investigated the cellular events associated with this initial bone formation, with specific reference to the respective role of cartilage and bone cells in bones which form via a cartilage model. The cellular architecture of initial osteogenic sites was investigated by light, confocal, and electron microscopy (EM) in the membranous ossification of fetal calvarial bones (not forming via a cartilage model) and in the membranous ossification of the bony collars of endochondral bones. Bone sialoprotein (BSP), which is expressed during early phases of bone deposition and has been proposed to be involved in the control of both mineral formation and bone cell-matrix interactions, was used as a marker of initial bone formation. We found that at all sites, BSP-producing cells (as identified by intracellular immunoreactivity) are arranged in a characteristic vis-à-vis (face to face) pattern prior to the appearance of the first mineralizing BSP-immunoreactive extracellular matrix. In perichondral osteogenesis, the vis-à-vis pattern comprises osteoblasts differentiating from the perichondrium/periosteum and early hypertrophic chondrocytes located at the lateral aspects of the rudiment. By EM, the first mineral and the first BSP-immunoreactive sites coincide temporally and spatially in the extracellular matrix at the boundary between cartilage and periosteum. We further showed that in an in vitro avian model of chondrocyte differentiation in vitro to osteoblast-like cells, early hypertrophic chondrocytes replated as adherent cells turned on the expression of high levels of BSP in conjunction with the switch to collagen type I synthesis and matrix mineralization. We propose a model for the priming of bone deposition, i.e., the formation of the first bone structure, in which the architectural layout of cells competent to deposit a mineralizing matrix (the vis-à-vis pattern) determines the polarized deposition of bone. For bones forming via a cartilage model, the priming of bone deposition involves and requires cells that differentiate from early hypertrophic chondrocytes.

Animals↗

PubMed1992

Development of cartilage and bone tissues of the anterior part of the mandible in cichlid fish: a light and TEM study.

The present paper presents ultrastructural details of chondrogenesis of Meckel's cartilage and of ossification of its associated peri- and parachondral bones in a teleost fish, the cichlid Hemichromis bimaculatus. We have distinguished four stages during chondrogenesis, each of which is characterized by specific cellular and matrix features: blastema, primordium, differentiated cartilage and cartilage surrounded by perichondral bone. The blastema is characterized by prechondroblasts and the lack of cartilage matrix; the primordium by chondroblasts and the onset of secretion of matrix of fibrillar and granular nature; differentiated cartilage is characterized by chondrocytes and larger amounts of typical hyaline cartilage matrix. Once perichondral bone is laid down, the chondrocytes show degenerative features but not true hypertrophy. Differentiation of the cartilage cells is attended with cytoplasmic changes indicative of an increasing secretory activity. There is a regional calcification of the cartilage matrix by fusion of calcospherites. Chondrogenesis of the symphyseal area is continuous with that of the rami but starts slightly later. Formation of perichondral bone at the cartilage surface is attended with the deposition of a transitional zone apparently containing a mixture of the two matrices. The role of the perichondral cells is discussed and it is proposed that they may contribute to the formation of the two matrices. The transitional zone may then result either from a diffusion process or from the simultaneous deposition of elements of the two matrices. Growth of the cartilage is argued to be largely the result of matrix secretion, except in the symphyseal area where appositional growth probably occurs until the region is completely covered by perichondral bone. This paper provides a basis for further studies on the developmental interactions between cartilage, bone and teeth during mandibular development in cichlids.

Animals↗

PubMed2000

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

Animals↗

PubMed2004

[Fluorine as a factor in premature aging].

The use of fluorine compounds in various areas of medicine, particularly in dentistry, as well as in agriculture and industry became very popular in the second half of the 20th century. Fluorine owed this widespread acceptance to observations that its compounds stimulate ossification processes and reduce the prevalence of caries. Unfortunately, growing expectations overshadowed the truth regarding interactions of fluoride on the molecular level. The fact was often ignored that fluoride is toxic, even though laboratory data stood for a careful approach to the benefits of usage. Excessive exposure to fluoride may lead to acute poisoning, hyperemia, cerebral edema, and degeneration of the liver and kidneys. Acute intoxication through the airways produces coughing, choking, and chills, followed by fever and pulmonary edema. Concentrated solutions of fluorine compounds produce difficult to heal necrotic lesions. In spite of these dramatic symptoms, acute intoxications are relatively rare; the more common finding is chronic intoxication attributable to the universal presence of fluorine compounds in the environment. The first noticeable signs of excessive exposure to fluoride in contaminated water, air, and food products include discolorations of the enamel. Dental fluorosis during tooth growth and loss of dentition in adulthood are two consequences of chronic intoxication with fluorine compounds. Abnormalities in mineralization processes affect by and large the osteoarticular system and are associated with changes in the density and structure of the bone presenting as irregular mineralization of the osteoid. Fluorine compounds also act on the organic part of supporting tissues, including collagen and other proteins, and on cells of the connective tissue. These interactions reduce the content of collagen proteins, modify the structure and regularity of collagen fibers, and induce mineralization of collagen. Interactions with cells produce transient activation of osteoblasts, stimulate fibroblasts to produce collagenase, and trigger toxic reactions in osteocytes and chondrocytes of trabecular bone. Growing deformations of the skeleton reduce mobility and result in permanent crippling of the patient. Fluoride increases the mass of non-collagen proteins such as proteoglycans and glucosaminoglycans, accelerating skin aging even though protein biosynthesis is generally suppressed. The final outcome includes progressive vascular lesions and disorders of energy metabolism in muscles. In conclusions, the use of fluoride, particularly by dentists and pediatricians, must be controlled and adapted to individual needs. It is worth remembering that fluoride: is the cause of disability due to bone deformations and abnormalities in the musculoskeletal system; reduces the incidence of caries but do not protect against tooth loss; exerts an adverse effect of metabolic processes in the skin; accelerates calcification of vessels and thus reduces their elasticity; inhibits bioenergetic reactions, in particular oxidative phosphorylation, reducing physical activity of muscles. These findings suggest that fluorine may be yet another factor in accelerated aging and revive the dispute started more than two and half thousand years ago whether aging is a physiologic or pathologic process. The understanding of factors modifying the process of aging is the basis for preventive measures aimed at extending life and maintaining full psychosocial activity.

Aging↗

PubMed2005

Identification of temporal pattern of mandibular condylar growth: a molecular and biochemical experiment.

OBJECTIVES: Based on the phenomenon that expression of type X collagen and capillary endothelium correlates with endochondral ossification, the prime aim of this study was to establish the temporal pattern of condylar growth in Sprague-Dawley rats by biochemically identifying the expression of these two factors. DESIGN: Sprague-Dawley rats were divided into five groups representing five different stages during somatic pubertal growth. In situ hybridization and immunoperoxidase were performed to examine expression of type X collagen in hypertrophic zone and capillary endothelium in erosive zone of condylar cartilage. Computer-assisted imaging analyses were conducted to allow for a quantitative assessment of the expression of these two factors, from which the temporal pattern of condylar growth was inferred. RESULTS: (1) Synthesis of type X collagen and emergence of capillary endothelium were critical factors during the transition of condylar cartilage from chondrogenesis into osteogenesis, a biological pathway that leads to endochondral bone formation, the mode through which the condyle grows. (2) Quantitative analyses revealed the temporal pattern of the expression of these two factors, indicating that the thrust of natural growth of the condyle in the rats occurred in concomitance with somatic pubertal growth, featured by an acceleration starting from day 38, a maximum growth rate on day 56, followed by a decrease afterwards. CONCLUSION: It is suggested that the biochemical examination of growth markers, such as type X collagen, might be a new approach to accurately depict temporal pattern of condylar growth which is too delicate to be reflected by gross measurement not only in Sprague-Dawley rats but potentially also in other species.

Animals↗

PubMed1994

Perspectives: a vital biomechanical model of the endochondral ossification mechanism.

BACKGROUND: Mechanical usage effects could explain many features of endochondral ossification and related processes. Mineralization of growth plate cartilage could reduce its mechanical strains enough to make its resorption begin and to guide it in space. By removing most of its mineralized vertical septae, resorption could overload the remainder enough to increase woven bone formation on them and construct the primary spongiosa. After it finishes mineralizing, the primary spongiosa could become stiff enough to begin partial disuse in strain terms, so BMU-based remodeling would begin replacing it with lamellar bone. This would construct the secondary spongiosa. In transferring loads from the growth plate to the cortex, the central metaphyseal spongiosa becomes deloaded. This disuse would make remodeling remove it in the diaphyseal marrow space. METHODS: The slow growth of epiphyses and apophyses gives their spongiosas more time to adapt to their loads than the metaphyseal spongiosa beneath faster growing growth plates. Compared to metaphyseal trabeculae, this leads to fewer and thicker epiphyseal trabeculae that turn over more slowly and should persist for life because they carry loads for life. RESULTS: Rapid turnover of metaphyseal cortex in very young subjects could let it strain enough to form woven bone. Increased thickness and slower turnover of this cortex in older subjects could reduce its strains enough to make lamellar bone form there instead. This would compose this cortex mostly of woven bone in the very young and of lamellar bone in adults. CONCLUSIONS: This model assigns particular importance to the stiffness and strains of tissues (as distinguished from their strength and stresses), to the relative rates of some processes, and to responses of the skeleton's biologic mechanisms to a tissue's typical largest mechanical strains (as distinguished from their stresses).

Adult↗

PubMed2002

Cartilage calcification studied by proton nuclear magnetic resonance microscopy.

A three-dimensional (3D) mineralizing culture system using hollow fiber bioreactors has been developed to study the early stages of endochondral ossification by proton nuclear magnetic resonance (NMR) microscopy. Chondrocytes harvested from the cephalic half of the sterna from 17-day-old chick embryos were terminally differentiated with 33 nM of retinoic acid for 1 week and mineralization was initiated by the addition of 1% beta-glycerophosphate to the culture medium. Histological sections taken after 6 weeks of development in culture confirmed calcification of the cartilage matrix formed in bioreactors. Calcium to phosphorus ratios (1.62-1.68) from X-ray microanalysis supported electron diffraction of thin tissue sections showing the presence of a poorly crystalline hydroxyapatite mineral phase in the cultures. After 4 weeks of culture, quantitative proton NMR images showed water proton magnetization transfer rate constants (km) were higher in premineralized cartilage compared with uncalcified cartilage, a result suggesting collagen enrichment of the matrix. Notably after 5 weeks mineral deposits formed in bioreactors principally in the collagen-enriched zones of the cartilage with increased km values. This caused marked reductions in water proton longitudinal (T1) and transverse (T2) relaxation times and water diffusion coefficients (D). These results support the hypothesis that mineralization proceeds in association with a collagen template. After 6 weeks of culture development, the water proton T2 values decreased by 13% and D increased by 7% in uncalcified areas, compared with the same regions of tissue examined 1 week earlier. These changes could be attributed to the formation of small mineral inclusions in the cartilage, possibly mediated by matrix vesicles, which may play an important role in cartilage calcification. In summary, NMR images acquired before and after the onset of mineralization of the same tissue provide unique insights into the matrix events leading to endochondral mineral formation.

Animals↗

PubMedDate not supplied

24,25-(OH)(2)D(3) regulates cartilage and bone via autocrine and endocrine mechanisms.

The purpose of this paper is to summarize recent advances in our understanding of the physiological role of 24(R),25(OH)(2)D(3) in bone and cartilage and its mechanism of action. With the identification of a target cell, the growth plate resting zone (RC) chondrocyte, we have been able to use cell biology methodology to investigate specific functions of 24(R),25(OH)(2)D(3) and to determine how 24(R),25(OH)(2)D(3) elicits its effects. These studies indicate that there are specific membrane-associated signal transduction pathways that mediate both rapid, nongenomic and genomic responses of RC cells to 24(R),25(OH)(2)D(3). 24(R),25(OH)(2)D(3) binds RC chondrocyte membranes with high specificity, resulting in an increase in protein kinase C (PKC) activity. The effect is stereospecific; 24R,25(OH)(2)D(3), but not 24S,25-(OH)(2)D(3), causes the increase, indicating a receptor-mediated response. Phospholipase D-2 (PLD2) activity is increased, resulting in increased production of diacylglycerol (DAG), which in turn activates PKC. 24(R),25(OH)(2)D(3) does not cause translocation of PKC to the plasma membrane, but activates existing PKCalpha. There is a rapid decrease in Ca(2+) efflux, and influx is stimulated. 24(R),25(OH)(2)D(3) also reduces arachidonic acid release by decreasing phospholipase A(2) (PLA(2)) activity, thereby decreasing available substrate for prostaglandin production via the action of cyclooxygenase-1. PGE(2) that is produced acts on the EP1 and EP2 receptors expressed by RC cells to downregulate PKC via protein kinase A, but the reduction in PGE(2) decreases this negative feedback mechanism. Both pathways converge on MAP kinase, leading to new gene expression. One consequence of this is production of new matrix vesicles containing PKCalpha and PKCzeta and an increase in PKC activity. The chondrocytes also produce 24(R),25(OH)(2)D(3), and the secreted metabolite acts directly on the matrix vesicle membrane. Only PKCzeta is directly affected by 24(R),25(OH)(2)D(3) in the matrix vesicles, and activity of this isoform is inhibited. This effect may be involved in the control of matrix maturation and turnover. 24(R),25(OH)(2)D(3) causes RC cells to mature along the endochondral developmental pathway, where they become responsive to 1alpha,25(OH)(2)D(3) and lose responsiveness to 24(R),25(OH)(2)D(3), a characteristic of more mature growth zone (GC) chondrocytes. 1alpha,25(OH)(2)D(3) elicits its effects on GC through different signal transduction pathways than those used by 24(R),25(OH)(2)D(3). These studies indicate that 24(R),25(OH)(2)D(3) plays an important role in endochondral ossification by regulating less mature chondrocytes and promoting their maturation in the endochondral lineage.

24,25-Dihydroxyvitamin D 3↗

PubMed2001

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

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

Animals↗

PubMed1993

Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes.

Numerous studies of experimental hypo- and hypervitaminosis A have long suggested that retinoic acid (RA) is involved in chondrocyte maturation during endochondral ossification and skeletogenesis. However, the specific and direct roles of RA in these complex processes remain unclear. Based on recent studies from our laboratories, we tested the hypothesis that RA induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix. Cell populations containing chondrocytes at advanced stages of maturation were isolated from the upper portion of Day 18 chick embryo sterna and grown for 2 weeks in monolayer until confluent. The cells were then treated with low doses (10-100 nM) of RA for up to 6 days in the presence of a phosphate donor (beta-glycerophosphate) but in the absence of ascorbic acid. Within 4 days of treatment, RA dramatically induced expression of the alkaline phosphatase (APase), osteonectin, and osteopontin genes, caused a several-fold increase in APase activity, and provoked massive mineral formation while it left type X collagen gene expression largely unchanged. The mineral had a mean Ca/Pi molar ratio of 1.5; Fourier transform infrared spectra confirmed that it represented hydroxyapatite. Mineralization was completely abolished by treatment with parathyroid hormone; this profound effect confirmed that RA induced cell-mediated mineralization and not nonspecific precipitation. When cultures were treated with both RA and ascorbic acid, there was a slight further increase in APase activity and increased calcium accumulation. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum; however, RA only had minimal effects on mineralization and gene expression in these cells. Thus, RA appears to be a rapid, potent, maturation-dependent, ascorbate-independent promoter of terminal maturation and matrix calcification in chondrocytes.

Alkaline Phosphatase↗

PubMed1994

Changes in parathyroid hormone receptors during chondrocyte cytodifferentiation.

The purpose of this study was to investigate the relationship between changes in parathyroid hormone (PTH) receptor levels and chondrocyte maturation during endochondral ossification. Chondrocytes were isolated from the growth plate of rabbit ribs and maintained in the presence of 10% serum in mass cultures. Treatment with PTH-(1-84) and a PTH-(1-34) fragment suppressed the increases in alkaline phosphatase activity and in type X collagen and 1 alpha,25-dihydroxyvitamin D3 receptor levels and abolished 45Ca incorporation into mineral, all of which occurred in parallel untreated cultures in the hypertrophic (terminal) stage. These effects of PTH were observed at low concentrations (10(-10) to 10(-9) M) and within 24-48 h of treatment. PTH-(1-84) and PTH-(1-34) also increased [35S]sulfate incorporation into newly synthesized proteoglycans. In contrast, the middle and carboxyl-terminal fragments of PTH tested had little effect on proteoglycan synthesis or terminal differentiation. The binding of 125I-PTH-(1-34) to cells in the growth plate was greater than that to cells in liver, skin, muscle, brain, or kidney. When the correlation between binding levels and stage of maturation was examined, we found that 125I-PTH-(1-34) binding to its 72-kDa receptor was low in resting and proliferating chondrocytes, increased 10-fold in matrix-forming chondrocytes, and thereafter decreased in hypertrophic chondrocytes both in vitro and in situ. Scatchard analysis revealed that the changes in PTH binding were due to changes in the number, and not in the affinity, of the receptor. The changes in PTH-(1-34) binding paralleled those in [35S]sulfate incorporation into proteoglycans. These findings suggest that stage-dependent increases in PTH/PTH-related peptide receptor levels localize the hormone stimulation of proteoglycan synthesis and inhibition of precocious hypertrophy in the matrix-forming zone of growth plates.

Alkaline Phosphatase↗

PubMed1996

A novel angiogenic molecule produced at the time of chondrocyte hypertrophy during endochondral bone formation.

Angiogenesis is a pivotal event in endochondral ossification. Vessels grow into the hypertrophic cartilage and erode it to produce a scaffold on which osteoblasts settle to produce woven bone. A new culture system was used to determine whether growth-plate chondrocytes produce an angiogenic molecule. Chondrocytes from primary growth plates of bovine fetuses were separated into maturationally distinct subpopulations. When cultured these cells produce an extensive extracellular matrix and the prehypertrophic cells mature to express the hypertrophic phenotype defined by the synthesis of type X collagen and matrix calcification. The culture medium collected from the hypertrophic cells contains a chemoattractant, nonmitogenic molecule for bovine endothelial cells which can induce angiogenesis in vivo in the rabbit cornea model. This molecule has a Mr of approximately 120 x 10(3). The production of this molecule by hypertrophic cells is enhanced by both 1,25-(OH)2 vitamin D3 and 24,25-(OH)2 vitamin D3 at 10(-8)-10(-12) M, but only in pre- and early hypertrophic cells. In contrast, these metabolites have either no effect or an inhibitory effect on the more mature hypertrophic cells. These results describe for the first time the production of an angiogenic molecule by hypertrophic chondrocytes. They demonstrate an important role for vitamin-D3 metabolites in regulating hypertrophy and angiogenesis during normal skeletal growth and differentiation. Thus, a defective regulation of these processes, due to the lack of vitamin-D metabolites, may explain the observed enlargement of the hypertrophic zone and impairment of skeletal growth in rickets which is induced clinically and experimentally by a deficiency of vitamin D.

24,25-Dihydroxyvitamin D 3↗

PubMed2000

Regulatory roles of zinc in matrix vesicle-mediated mineralization of growth plate cartilage.

Zinc (Zn2+) has long been known to play important roles in mineralization and ossification of skeletal tissues, but the mechanisms of Zn2+ action are not well understood. In this study we investigated the effects of Zn2+ on mineralization in a cell culture system in which terminal differentiation and mineralization of hypertrophic growth plate chondrocytes was induced by retinoic acid (RA) treatment. Addition of Zn2+ to RA-treated cultures decreased mineralization in a dose-dependent manner without affecting alkaline phosphatase (APase) activity. Characterization of matrix vesicles (MVs), particles that initiate the mineralization process, revealed that vesicles isolated from RA-treated and RA/Zn2+-treated cultures showed similar APase activity, but vesicles from RA/Zn2+-treated cultures contained significantly less Ca2+ and Pi. MVs isolated from RA-treated cultures were able to take up Ca2+ and mineralize in vitro, whereas vesicles isolated from RA/Zn2+-treated cultures were not able to do so. Detergent treatment, which ruptures the MV membrane and exposes preformed intravesicular Ca2+-Pi-phospholipid complexes, did not restore the Ca2+ uptake abilities of MVs isolated from RA/Zn2+-treated cultures, suggesting that vesicles from RA/Zn2+-treated cultures did not contain functional Ca2+-Pi-phospholipid complexes. Zn2+ treatment did not affect the content of annexins II, V, and VI in MVs or the Ca2+-dependent, EDTA-reversible binding of these molecules to the membrane surface. However, Zn2+ treatment did affect the EDTA-nonreversible binding of these molecules to the MV membrane, suggesting that Zn2+ interferes with the assembly of annexins in the MV membrane. In addition, Zn2+ inhibited annexin II-, V-, and VI-mediated Ca2+ influx into liposomes. In conclusion, Zn2+ inhibits the mineralizing competence of intravesicular Ca2+-Pi-phospholipid complexes and function of annexin channels, thereby controlling Ca2+ influx into MVs, the formation of the first crystal phase inside the vesicles and initiation of mineralization.

Animals↗

PubMed2003

Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification.

UNLABELLED: Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. INTRODUCTION: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. MATERIALS AND METHODS: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-(EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). RESULTS: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces pi-induced mineralization. CONCLUSIONS: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

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