Hypertrophic Region Research Articles

Expression of NRL/NGAL ( neu-related lipocalin/neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation

The neu-related lipocalin (NRL) is a protein overexpressed in rat mammary cancer induced by activated neu (HER-2/c-erbB2). This protein belongs to the family of the lipocalins or low molecular weight proteins able to bind and transport small hydrophobic molecules. The NRL homologue in mouse is SIP24, an acute phase protein induced in the animal by turpentine injection; the human homologous protein is NGAL expressed in granulocytes and epithelial cells in pathological conditions, such as inflammation and malignancy. We have investigated NRL expression in developing rat embryos. By immunolocalization we have shown localization of the protein in the hypertrophic region of growth plate cartilage. NRL was particularly enriched in prehypertrophic chondrocytes. In addition, we observed localization of the protein in forming skeletal muscle fibres and in the myocardium of developing heart. In agreement with the immunolocalization data, by in situ hybridization we have demonstrated the presence of the specific mRNA in the same tissues. At an early stage of differentiation, cultured rat embryo-derived chondrocytes did not express NRL; nevertheless expression of the protein was induced in these cells by treatment with an inflammatory agent, such as LPS. By Western blot analysis with specific antibodies we showed protein synthesis by cultured myoblasts also in the absence of LPS treatment, but only when forming myotubes were observed in culture. Stimulation of myoblast cultures with LPS resulted in an enhancement of the NRL expression in well formed myotubes. Our data suggest a role of NRL in cartilage and muscle differentiation. NRL expression was induced by inflammatory agents. We wish to propose that the expression of NRL in hypertrophic chondrocytes and forming myotubes is part of a "physiological" acute phase response occurring during cartilage and muscle development. In this manuscript we also report that NRL is not detectable by immunolocalization in adult cartilage (both articular and tracheal) from normal subjects. On the contrary articular cartilage from osteoarthritic patients was highly positive for the presence of NRL/NGAL. Interestingly the expression of this protein is also activated during neoplastic transformation of chondrogenic lineage cells.

Histomorphometric analysis of the tibial growth plate in a feline model of mucopolysaccharidosis type VI.

Mucopolysaccharidosis type VI (MPS VI) is a genetically inherited lysosomal storage disorder. Severely affected children exhibit a range of skeletal abnormalities including short stature, facial dysmorphia, and dysostosis multiplex. Naturally occurring and transgenic animal models of MPS VI are also found which exhibit pathology similar to the human disorder. In this paper we have characterized the formation of trabecular bone from growth plate cartilage in a feline model of MPS VI. Tibial trabecular bone was shown to be osteopenic in MPS VI animals with a bone mineral volume (BV/TV) of 4.51% compared with a BV/TV of 15.64% in normal animals. In addition to osteopenia, a rearrangement of trabecular bone architecture was also observed in MPS VI tibiae, with fewer, thinner trabeculae noted; bone formation rate was also decreased. These observations support those previously made in the L5 vertebrae of MPS VI animals. When the sequential formation of growth plate cartilage structural elements, their transition into primary bone spongiosa, and remodeling into secondary bone spongiosa was characterized, no difference between normal and MPS VI could be detected in the number of cartilage septae and their arrangement in the proliferative and hypertrophic regions of the growth plate or trabecular elements in the primary spongiosa. However, a deviation from normal was observed in the resting zone of the growth plate and in the secondary spongiosa of bone. Thus, the osteopenia observed in MPS VI bone appears to arise primarily from a defect in bone production within the metaphysis and diaphysis rather than the creation of an abnormal template in the preceding growth plate cartilage.

Folding and Assembly of Type X Collagen Mutants That Cause Metaphyseal Chondrodysplasia-type Schmid: EVIDENCE FOR CO-ASSEMBLY OF THE MUTANT AND WILD-TYPE CHAINS AND BINDING TO MOLECULAR CHAPERONES

Schmid metaphyseal chondrodysplasia results from mutations within the COOH-terminal globular domain (NC1) of type X collagen, a short chain collagen expressed in the hypertrophic region of the growth plate cartilage. Previous in vitro studies have proposed that mutations prevent the association of the NC1 domain of constituent chains of the trimer based upon a lack of formation of a trimeric structure that is resistant to dissociation with sodium dodecyl sulfate. To examine the effect of mutations on folding and assembly within a cellular context, bovine type X cDNAs containing analogous disease causing mutations Y598D, N617K, W651R, and wild-type were expressed in semi-permeabilized cells. We assessed trimerization of the mutant chains by their ability to form a collagen triple helix. Using this approach, we demonstrate that although there is an apparent lower efficiency of association of the mutant NC1 domains, they can drive the formation of correctly aligned triple helices with the same thermal stability as the wild-type collagen. When epitope-tagged mutant and wild-type collagen were co-expressed, heterotrimers could be detected by sequential immunoprecipitation. Both wild-type and mutant type X chains were found in association with the molecular chaperones protein disulfide isomerase and Hsp 47. The implications of these findings on the likely mechanism of Schmid metaphyseal chondrodysplasia will be discussed.

A computer model of myocardial disarray in simulating ECG features of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) was simulated with a computer heart model having a realistic shape and rotating fiber orientation in order to elucidate possible mechanisms for abnormal ECG findings. The disarray of myocardial muscle in HCM was simulated by assigning random fiber direction and isotropic electrophysiologic properties to abnormal hypertrophic regions, in contrast to the anisotropic modeling for normal myocardium. With these models, main ECG features including abnormal Q wave and QS pattern were reproduced and were comparable with clinical findings. This study suggests that the change in anisotropy in the hypertrophic myocardium is likely to be the main factor responsible to the ECG features of HCM.

Cell Death in Avian Tibial Dyschondroplasia

Tibial dyschondroplasia (TD) is a local defect of growth plates in fast-growing poultry where the transitional zone cartilage fails to resorb and persists as an avascular plug that prevents endochondral bone formation. We compared the differences in the cartilages from normal and TD-affected growth plates using the reduction of MTS to assess cartilage viability. Chondrocyte apoptosis was determined using biochemical measurement of DNA fragmentation, and in situ labeling of nuclei with fluorescein-dUTP using terminal deoxynucleotide transferase (TdT)-mediated nick end labeling (TUNEL) of isolated chondrocytes and growth plate sections. The TD-affected cartilage showed a significantly lower level of MTS reduction and a decrease in trichloroacetic acid (TCA)-precipitable DNA content. The TD cartilages had a higher percentage of fragmented DNA, which was also evident with agarose gel electrophoresis. A significantly higher number of chondrocytes isolated from TD-affected cartilages had condensed morphology, shrunken nuclei with little cytoplasm, and were TUNEL positive as identified by the incorporation of fluorescein-dUTP into the nuclei. In vivo results similarly showed a significant population of chondrocytes in transition zones undergoing condensation and apoptosis as determined by in situ TUNEL staining of growth plate sections. Normal growth plates, under similar conditions, showed no significant apoptosis of chondrocytes from hypertrophic and chondrolyzing zones. The condensation and apoptotic cell death may be responsible for the reduction of growth plate viability as well as the reduction in DNA content and increased DNA fragmentation. While the cause of the pathogenesis of TD is unknown, it appears that the aberrant death of chondrocytes in hypertrophic regions of growth plates may be responsible for the accumulation of cartilage and the arrest of endochondral bone formation.

Intrinsic control of vascularization in developing cartilage rudiments.

The vascularization of developing cartilage rudiments is temporally and spatially defined. By using an in vivo angiogenesis model, the chorioallantoic membrane (CAM) of the chick embryo and chick embryo cartilage rudiments, we conclude that the factors controlling the vascular invasion of cartilage rudiments are intrinsic. Intact rudiments, separate hypertrophic zones and separate rounded cell zones, when grafted onto the CAM, become vascularized in the same temporal and spatial manner as occurs in ovo. When grown as organ cultures prior to CAM grafting, rudiments still become vascularized in the same temporal and spatial manner. The integrity of the extracellular matrix and the presence of the periosteum are two physical factors regulating the control of vascularization. Removal of the periosteum from hypertrophic regions caused a cessation of the invasion. Insults to the matrix via brief enzymatic degradation of extracellular matrix components resulted in invasion and erosion of rounded cell zones at an earlier time than is ordinarily seen both in ovo and on the CAM.

Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic bone.

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs) which are capable of degrading most components of the extracellular matrix. Animal studies suggest that the MMPs play a role in bone turnover. Using specific polyclonal antisera, immunohistochemistry was used to determine the patterns of synthesis and distribution of collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9) and of the tissue inhibitor of metalloproteinases-1 (TIMP-1) within developing human osteophytic bone. The different MMPs and TIMP showed distinct patterns of localisation. Collagenase expression was seen at sites of vascular invasion, in osteoblasts synthesising new matrix and in some osteoclasts at sites of resorption. Chondrocytes demonstrated variable levels of collagenase and stromelysin expression throughout the proliferative and hypertrophic regions, stromelysin showing both cell-associated and strong matrix staining. Intense gelatinase B expression was observed at sites of bone resorption in osteoclasts and mononuclear cells. Gelatinase A was only weakly expressed in the fibrocartilage adjacent to areas of endochondral ossification. There was widespread but variable expression of TIMP-1 throughout the fibrous tissue, cartilage and bone. These results indicate that MMPs play a role in the development of human bone from cartilage and fibrous tissue and are likely to have multiple functions.

Structural changes in the large proteoglycan, aggrecan, in different zones of the ovine growth plate.

The large cartilage proteoglycan, aggrecan, was found to vary throughout the ovine physis corresponding to the maturational state of the resident chondrocytes. Two populations of proteoglycan monomer were observed in articular, epiphyseal, and in the resting zone of growth plate cartilage. These proteoglycans contained chondroitin sulfate glycosaminoglycan chains sulfated predominantly in the 4 position along with lesser amounts of chondroitin-6-sulfate and keratan sulfate. In the proliferative zone of the growth plate, chondrocytes synthesize one population of proteoglycan monomer which was significantly larger than monomer populations in articular, epiphyseal, or resting zone and this size increase could be attributed to an increase in its constituent chondroitin sulfate side chains. As these chondrocytes progress through their life cycle they continue to modify the structural characteristics of the aggrecan molecule they synthesize. Thus, in the hypertrophic region of the growth plate, the proteoglycan monomer is larger again than in the proliferative region. Variation in sulfation pattern on aggrecan chondroitin sulfate side chains is also observed in the hypertrophic region with an increasing proportion of unsulfated residues present, which may play a role in the initiation of mineralization. In addition, increasing amounts of the carbohydrate sequence recognized by monoclonal antibody 7-D-4 are observed in the hypertrophic zone.

Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion.

In previous work we showed that the chondrodysplastic phenotype of mice homozygous for a null mutation of the PTH-related peptide (PTHrP) gene was due in part to reduced proliferation and aberrant differentiation of growth plate chondrocytes. In the present study we have extended those observations by examining chondrocytes for evidence of PTH/PTHrP receptor expression, proliferation, and programmed cell death. Receptor messenger RNA and protein were expressed in chondrocytes in the resting and proliferative zones of both wild-type and mutant mice. In normal animals, expression was abundant in the area of transition between proliferative and hypertrophic chondrocytes and absent from cells in the lower hypertrophic region. On the other hand, the hypertrophic zone in mutant mice contained nonhypertrophic chondrocytes, which exhibited characteristics of proliferating cells, including PTH/PTHrP receptor expression, [3H]thymidine incorporation, and expression of proliferating cell nuclear antigen. In contrast to the situation in normal animals, some cells adjacent to the zone of vascular invasion in mutant growth plates showed biochemical and morphological evidence of programmed cell death. In addition to these alterations in the maturation of growth plate chondrocytes, homozygous mutants demonstrated signs of aberrant differentiation of periosteal precursor cells. In some specimens, clusters of chondrocytes embedded in a cartilaginous matrix were observed between the layers of periosteal osteoblasts and the bony collar in the sterna and tibiae of mice homozygous for a null mutation of the PTHrP gene. Taken together, these observations indicate that PTHrP plays a pivotal role in the orderly progression of chondrocytes through stages of proliferation, differentiation, and programmed cell death in the epiphyseal growth plate and may also facilitate the commitment of precursors to cells of the chondrocytic or osteoblastic lineages.

Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion

In previous work we showed that the chondrodysplastic phenotype of mice homozygous for a null mutation of the PTH-related peptide (PTHrP) gene was due in part to reduced proliferation and aberrant differentiation of growth plate chondrocytes. In the present study we have extended those observations by examining chondrocytes for evidence of PTH/PTHrP receptor expression, proliferation, and programmed cell death. Receptor messenger RNA and protein were expressed in chondrocytes in the resting and proliferative zones of both wild-type and mutant mice. In normal animals, expression was abundant in the area of transition between proliferative and hypertrophic chondrocytes and absent from cells in the lower hypertrophic region. On the other hand, the hypertrophic zone in mutant mice contained nonhypertrophic chondrocytes, which exhibited characteristics of proliferating cells, including PTH/PTHrP receptor expression, [3H]thymidine incorporation, and expression of proliferating cell nuclear antigen. In contrast to the situation in normal animals, some cells adjacent to the zone of vascular invasion in mutant growth plates showed biochemical and morphological evidence of programmed cell death. In addition to these alterations in the maturation of growth plate chondrocytes, homozygous mutants demonstrated signs of aberrant differentiation of periosteal precursor cells. In some specimens, clusters of chondrocytes embedded in a cartilaginous matrix were observed between the layers of periosteal osteoblasts and the bony collar in the sterna and tibiae of mice homozygous for a null mutation of the PTHrP gene. Taken together, these observations indicate that PTHrP plays a pivotal role in the orderly progression of chondrocytes through stages of proliferation, differentiation, and programmed cell death in the epiphyseal growth plate and may also facilitate the commitment of precursors to cells of the chondrocytic or osteoblastic lineages.

Increased cell diameter precedes chondrocyte terminal differentiation, whereas cell-matrix attachment complex proteins appear constant.

Chondrocytes in specific areas of chick sterna have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix, whereas middle and caudal chondrocytes remain cartilagenous throughout development, continuing to secrete collagen types II, IX, and XI. In this report, we ask if the cell size and cytoarchitecture of chondrocytes differ in cephalic, middle, and caudal portions of whole sterna prior to and during hypertrophy. In addition, what is the distribution of integrin subunits and actin associate proteins in differentiating chondrocytes? Phalloidin was used to stain filamentous actin, and immunohistochemistry was used to localize the distribution of collagen molecules, integrin receptor subunits, and actin-associated proteins. Chondrocytes stained for filamentous actin demonstrated that on day 14 cephalic chondrocytes had a significantly larger diameter than middle and caudal chondrocytes. Day 17 chondrocytes in nonhypertrophic cephalic and middle regions of sterna were significantly smaller than hypertrophic chondrocytes and significantly larger than caudal chondrocytes. In contrast to day 14 chondrocytes, day 17 chondrocytes in the hypertrophic region demonstrated similar diameters at all cartilagenous depths. The beta 1 integrin subunit appeared punctate and associated with cell membranes, allowing nonpolarized interactions with extracellular matrix molecules. The distribution of alpha integrin subunits was similar to the beta 1 integrin subunit, although alpha integrin subunits also appeared cytoplasmic. Actin-associated proteins, vinculin, and alpha-actinin, were associated with F-actin, but vinculin was more specifically localized to the ends of the actin filaments. Focal adhesion kinase was diffusely distributed throughout the cytoplasm but also demonstrated areas of colocalization with vinculin. Zyxin and paxillin demonstrated a punctate distribution, although paxillin was slightly more diffuse. Using immunohistochemical detection, no difference in integrin subunit or actin associated protein distribution could be determined between chondrocytes and hypertrophic chondrocytes. The increased chondrocyte diameter observed in cephalic regions of sterna on day 14 suggests that intracellular changes may precede the specific hypertrophic marker, type X collagen, by several days. In addition, the presence of integrin subunits, which are known to interact with collagen and cytoskeletal proteins, suggests that communication may exist between chondrocytes and their extracellular matrix via these receptor molecules.

End labeling studies of fragmented DNA in the Avian growth plate: Evidence of apoptosis in terminally differentiated chondrocytes

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.

Synthesis and phosphorylation of osteopontin by avian epiphyseal growth-plate chondrocytes as affected by differentiation

The regulation of synthesis and phosphorylation of osteopontin in relation to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture. Osteopontin gene expression was evaluated in the tibia growth-plate of 3-week-old chickens by in situ hybridization. The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated. Within the hypertrophic region, a poorly labeled area separated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation. In culture, proliferative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activity. Chondrocytes not treated with ascorbic acid, exhibited lower levels of osteopontin mRNA than the treated cells. The phorbol ester TPA—an activator of protein kinase C—and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression. Chondrocytes secreted low levels of phosphorylated osteopontin to the medium. EGF treatment resulted in the appearance of phosphorylated osteopontin in the medium, without affecting the synthesis of other proteins. FGF and TGF β, but not IGF-I or IGF-II, also caused phosphorylation of osteopontin. Ascorbic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further. Parathyroid hormone caused a dose-dependent attenuation of osteopontin phosphorylation and inhibited the EGF-dependent osteopontin phosphorylation. The results suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms. The response to the various controlling agents varies with the state of differentiation. Both processes—the synthesis and phosphorylation of osteopontin—are under the control of local growth factors which are involved in bone growth and calcification.

Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: Rapid induction of type X collagen in culture

Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15-20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer-modeling of oxygen supply to cartilage: addition of a compartmental model.

Our previous studies have focussed on the architecture of the avian growth plate and the oxygen consumption of growth plate chondrocytes in order to develop an appropriate computer model for estimating chondrocyte anoxia (Haselgrove et al., 1993). Initially, we used two models: the Krogh cylinder (Silverton et al., 1989), and a second model with similar geometry utilizing a complex oxygen consumption as a function of oxygen concentration (Silverton et al., 1990). For this purpose, we divided the growth plate into two anatomical regions; the region of resting-proliferating chondrocytes and the region of hypertrophic chondrocytes. We modeled the two growth plate regions separately and ignored the transition zone. We also used a two dimensional analysis assuming that the major flow of oxygen was radial rather than axial. To extend our model, we have now used a compartmental model originally developed for modeling the oxygen and carbon dioxide distribution in the microvasculature of the brain (Ye et al., 1993). With this model we have been able to evaluate the contribution of the microvacular structure to oxygen supply of the resting and hypertrophic regions of the growth cartilage and to estimate oxygen and carbon dioxide partial pressure variations in the growth plate.

Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

Computer modeling of the oxygen supply and demand of cells of the avian growth cartilage.

We have used computer modeling studies to investigate the oxygen supply to the prehypertrophic and hypertrophic regions of avian growth plate. We measured experimentally the characteristics of the oxygen consumption of chondrocytes at different oxygen tensions. The oxygen consumption decreases at low oxygen tensions. This relation between oxygen tension and oxygen consumption serves as a protective mechanism that prevents the cells in the prehypertrophic zone from becoming anoxic in the regions farthest from the blood vessels. The results of the calculations, when combined with redox measurements of the cells in the growth plate, indicate that the metabolism of the chondrocytes is not controlled simply by the available oxygen supply.

Measurement of metabolic events in the avian epiphyseal growth cartilage using a bioluminescence technique.

We developed a technique to map the distribution of selected metabolites in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidoreductase. Chick tibial epiphyses were freeze-trapped, sectioned, and freeze-dried. For evaluating lactate, luciferase was suspended in a buffer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydrogenase (LDH). The buffer was frozen into a layer 800 microns thick and placed in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was focused through a photographic lens and collected on film. We found that lactate was synthesized by cells in all regions of the growth plate. The highest concentration of the metabolite was observed in the calcified hypertrophic region. Substantial levels of lactate were also present in articular cartilage. By modifying the composition of the buffer solution, we were able to map the distribution of glucose and glucose-6-phosphate and the activity of LDH. Maximal levels of each of the three components were present in hypertrophic cartilage. Chemical analysis of the tissue section confirmed the luminographic studies and provided further evidence that there was reliance on glycolytic metabolism in terminally differentiated chondrocytes. Use of enzyme couples similar to those described above should permit the technique to be used to study most, if not all, of the major metabolic components of cartilage.

Intron-exon structure, alternative use of promoter and expression of the mouse collagen X gene, Col10a-1.

The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was approximately 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.

Influence of trace metal ions on matrix vesicle calcification

Since the proposal by Roivison that alkaline phosphatase (ALP) has a role in calcification [ 11, and the later demonstration that its activity was dependent on the presence of Zn2” [2], a role for trace metals in biological mineralization has been indicated. Nutritional studies in poultry in the 1950’s and 60’s showed that dietary deficiencies of Mn2+, Cu2+, or Zn*+ resulted in severe skeletal disorders [3]. Subsequently, it was found that excessive amounts of these metal ions could a!so be harmful to the skeletal system. In vitro studies indicated an inhibitory effect of metal ions on endochondral mineralization [4]. In more recent years, toxic metal ions such as Cd2+ and A13+ have been identified as etiological agents in a number of bone diseases [S-6]. Prior to the discovery of matrix vesicles (MV) by Anderson [7] and Bonucci [8], histochemical studies showed a colocalization of Zn2+ and Sudanophilic material in the hypertrophic region of the epiphyseal growth plate [9]. The discovery of MV readily explained the Sudanophilic reaction, however, it has been only recently that these structures were found to contain substantial amounts of Zn2+ [lo]. Since our discovery of the importance of Zn*+ in MV mineralization, we have investigated a number of aspects of MV function which may be affected by the presence of trace metal ions, including: 1) MV enzyme activities; 2) mineral ion transport; and 3) mineral nucleation, maturation, and growth.