Chicken Growth Plate Research Articles

Identification of a Family of Noncanonical Ubiquitin-Conjugating Enzymes Structurally Related to Yeast UBC6

Ubiquitin-conjugating enzymes (UBCs) selectively target proteins for proteasomal degradation by the covalent attachment of ubiquitin moieties. Yeast UBC6 is unusual in having an active site distinct from all other UBCs and in possessing a transmembrane domain that anchors it to the cytoplasmic surface of the endoplasmic reticulum. During a differential display analysis on chick growth plate chondrocytes we isolated a cDNA encoding a noncanonical ubiquitin-conjugating enzyme (NCUBE1) structurally similar to yeast UBC6. Chick NCUBE1 transcripts were detected in all tissues examined and decreased threefold during chondrocyte terminal differentiation. Database searches identified other related proteins; the human and mouse orthologues of NCUBE1, a second human homologue of yeast UBC6 (NCUBE2), and related proteins from S. pombe, C. elegans, and P. mariana. Together with yeast UBC6 these proteins constitute a distinct family of UBCs sharing a conserved noncanonical active site sequence and a C-terminal transmembrane domain. By analogy with yeast UBC6 they are likely to be localised to the endoplasmic reticulum where they may be involved in targeting retrotranslocated, ER-associated proteins for proteasomal degradation.

A novel integral membrane protein is differentially expressed in the chick growth plate and maps to chromosome 1.

The growth plate is a specialised region of cartilage located at the growing ends of long bones in higher vertebrates. It is responsible for longitudinal bone growth and is under the control of many local and systemic factors. The growth plate consists of an orderly arrangement of small proliferative and larger mature hypertrophic chondrocytes. This paper describes the isolation by differential display of a 988-bp cDNA fragment derived from a transcript that is more highly expressed in proliferating rather than hypertrophic chondrocytes of the chick growth plate. Using 3' RACE, a further 939 bp of cDNA sequence was obtained. The 1.9 kb sequence contains a 924-bp open reading frame encoding an unknown 308 amino acid protein. This protein has a putative transmembrane domain near its N-terminus and three dileucine motifs at its carboxy tail. This gene was expressed in all other tissues examined. A polymorphism was identified by SSCP analysis and the gene was mapped to the centromeric region of the short arm of chicken chromosome 1, close to the locus for autosomal dwarfism.

Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.

Expression patterns of candidate genes for tibial dyschondroplasiain chicken growth plate chondrocytes separated on percoll density gradients

Expression patterns of candidate genes for tibial dyschondroplasiain chicken growth plate chondrocytes separated on percoll density gradients

Chondrocytes in the endochondral growth cartilage are not hypoxic.

From an anatomic viewpoint, the blood supply to chondrocytes in the growth cartilage is limited. As a result, it has been suggested that these cells are hypoxic and that this condition regulates chondrocyte maturation and cartilage mineralization. We examined the state of chondrocyte oxygenation in the chick growth plate using a hypoxia-sensing drug, EF5. EF5 is a pentafluorinated derivative of the 2-nitroimidazole, etanidazole, that is metabolically reduced by oxygen-inhibitable nitroreductase(s). Reduced EF5 covalently forms adducts with cellular macromolecules that can be visualized with a highly specific fluorochrome-conjugated monoclonal antibody. When EF5 was injected into chicks and tissues were subsequently examined by immunohistochemical techniques, chondrocytes in articular, proliferating, and hypertrophic cartilage exhibited a low level of fluorescence-detectable binding, suggesting the absence of significant hypoxia. We confirmed that the results were not confounded by tissue-specific factors related to low-chondrocyte nitroreductase activity or problems from drug diffusion into cartilage. Using in vitro systems, we showed that, under conditions of imposed hypoxia, EF5 diffused into the tissue and was bound to chondrocytes. With the use of an in vivo model in which hypoxia was artificially induced by death, chick chondrocytes were found to bind the drug. Although the EF5-binding method is not optimally suited for determining the precise oxygen partial pressure in heterogeneous tissues, such as the growth plate, we concluded that chick chondrocytes are not oxygen deficient in vivo.

Expression of growth factors in chicken growth plate with special reference to tibial dyschondroplasia

Immunoreactive growth factors were identified in chick embryonic cartilage and bone, and in the growth plate of normal tibiotarsi and tibiotarsi affected with tibial dyschondroplasia (TD). A specific pattern of temporal and spatial expression was observed for each growth factor. Transforming growth factor beta and alpha (TGF beta and TGF alpha) and epidermal growth factor (EGF) were briefly expressed in chondrocytes of early chick embryos. Immunolabelling for TGF beta then gradually shifted into cartilaginous matrix and was not observed in cytoplasm of hypertrophic chondrocytes until the late embryonic and post-hatch stages. The distribution and intensity of TGF beta labelling was the same in chondrocytes of the TD and normal growth plate. Insulin-like growth factor I (IGF-I) labelling persisted from the early embryonic stage to the end of the mid-stage and then disappeared from chondrocytes. IGF-I appeared again in chondrocytes 1-2 days before hatching. After hatching, the labelling intensified in prehypertrophic and hypertrophic chondrocytes. TD lesions displayed IGF-I in the distal region, mainly in chondrocytes around small blood vessels. EGF reappeared in proliferative and hypertrophic chondrocytes of the mid-embryonic stage. By day 18 after hatching, EGF was present mainly in prehypertrophic and hypertrophic chondrocytes. EGF was demonstrated only in distal proliferative and early prehypertrophic chondrocytes of the dyschondroplastic growth plate. TGF alpha was identified in hypertrophic chondrocytes adjacent to the periosteum and in the distal tip of the mid-embryonic growth plate. With progressing ossification, TGF alpha labeling intensified in the embryonic hypertrophic chondrocytes. In the TD growth plate at day 18 after hatching, TGF alpha expression was limited to 1-3 concentric layers of chondrocytes surrounding blood vessels.

Effects of calcitonin and parathyroid hormone on calcification of primary cultures of chicken growth plate chondrocytes.

Few studies have been directed toward elucidating the action of calcitonin (CT) and parathyroid hormone (PTH) on growth plate chondrocytes, cells directly involved in longitudinal bone growth and provisional calcification. In this study, primary cultures of avian growth plate chondrocytes that calcify without the supplement of beta-glycerophosphate were used to investigate the effects of synthetic human CT and 1-34 bovine PTH on (1) cell division and growth; (2) the deposition of Ca2+ and inorganic phosphate (Pi); (3) the activity of alkaline phosphatase (AP), an enzyme long associated with the mineralization process; (4) the levels of proteoglycans; and (5) the synthesis of collagens. Added continually to preconfluent cultures from day 6 until harvest, CT (1-30 nM) and PTH (0.1-1.0 nM) increased mineral deposition; the maximal increase was seen between days 18-21 at 10 nM CT (175-260%) and 0.5 nM PTH (approximately 170-280%), both p < 0.001. CT had no significant effect on cellular protein, or AP-specific activity, whereas PTH increased cellular protein, DNA, proteoglycan, and collagen content of the cultures in a dosage-dependent manner. AP activity and levels of Type II and X collagens and fibronectin in the culture medium showed a biphasic response to PTH; maximal increases were seen at 0.5 nM between days 15-18. Longer exposure (days 21-27) to PTH at higher levels (5-10 nM) caused a marked decreased in AP activity but a lesser decrease in the collagens. These results indicate that CT and PTH can act directly on chondrocytes to stimulate mineralization, but that PTH specifically stimulated cell division and synthesis of cellular and extracellular proteins by growth plate chondrocytes. The implications of these findings with regard to Ca2+ homeostasis and bone formation are discussed.

Lack of chondroitin sulphate epitope in the proliferating zone of the growth plate of chicken tibia.

Monoclonal antibodies specific to chondroitin sulphate (CS-56) and keratan sulphate (AH12) were used to localize proteoglycans in the proximal tibial articular cartilage and growth plate of broiler chickens. There was no CS-56 labelling in the proliferative zone of the growth plate. In contrast, intense labelling with this antibody was observed in the transitional and hypertrophic zones of the growth plate and the articular cartilage. This was confirmed by extracting chondroitin sulphate fractions from different zones of the growth plate and articular cartilage, and examining their antigenicities to CS-56 by ELISA inhibition assay. It was suggested that the maturation of chondrocytes in the growth plate is related to the production of chondroitin sulphate with CS-56 epitope, which may be a prerequisite for normal endochondral bone formation in the chicken tibia. The role of chondroitin sulphate recognized by CS-56 in the articular cartilage is unknown.

Lipopolysaccharide alters aggrecan metabolism in the growth plate.

In this study, we examined the influence of lipopolysaccharide (LPS) on aggrecan metabolism and structure in the growth plate. Two experimental approaches were used: (i) in vivo administration of LPS to 10-day-old chicks; and (ii) in vitro addition of LPS to explant culture of normal chick growth plate. Twelve-day-old male broiler chicks were killed 48 hr after intravenous injection of LPS (3 mg/kg) or saline (control), and growth plate from the femur or tibia was cultured or frozen. Tissue for explant culture was (i) cultured for 5 days with daily medium change (glycosaminoglycan release into the medium estimates proteoglycan breakdown rates), or (b) incubated with 35SO4 to determine the rate of proteoglycan synthesis. Proteoglycan structure was determined by associative (0.5 M sodium acetate) and dissociative (4 M guanidine HCl) Sepharose CL2B chromatography. Explant culture of growth plate from LPS-injected chicks (in vivo) showed a decrease (P < 0.05) in the rate of proteoglycan synthesis. There were a greater proportion of small monomers and a reduced ability to aggregate in growth plate from LPS-injected chicks. In vitro addition of LPS (100 micrograms/ml) to explant culture medium reduced proteoglycan synthesis (P < 0.02), and the rate of release was increased (P < 0.001). In addition, the total and newly synthesized proteoglycans released into the medium from LPS-treated explant culture had a reduced aggregation and a majority of monomers that were smaller than control. These results demonstrate that LPS disrupts the normal metabolism and structure of growth plate aggrecan, and we hypothesize that this may adversely influence longitudinal growth.

Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate

In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxpyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibia] dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur.

Expression and distribution of cartilage matrix macromolecules in Avian tibial dyschondroplasia

Tibial dyschondroplasia (TD) is a disorder of endochondral ossification characterized by the presence of an avascular, non-mineralized cartilage lesion extending from the growth plate into the metaphysis. Cells within the TD growth plate fail to differentiate to full hypertrophy, and instead appear to maintain a 'pre-hypertrophic' or 'transitional' status. The synthesis and distribution of aggrecan, biglycan, decorin, and collagen types II and X in the growth plates of normal and tibial dyschondroplastic chickens have been investigated using in situ hybridization and immunolocalization. Marked reductions in the amount of aggrecan and biglycan core protein mRNAs were observed in the tibial dyschondroplastic lesion by in situ hybridization. Reduction in mRNA production seemed to be specific to the extracellular matrix components since total mRNA expression showed no significant difference between normal and dyschondroplastic cartilage. In addition, expression of collagen type II and decorin did not differ significantly between normal and TD cartilage. Distribution of aggrecan biglycan, decorin, type II and X collagens were examined using immunohistochemistry. Normal hypertrophic cartilage showed a strong matrix labelling for aggrecan and biglycan. Type X collagen in the normal hypertrophic cartilage showed strong pericellular and matrix distribution, whereas in TD cartilage labelling for aggrecan, biglycan and collagen X was located intracellularly with a very low level of signal in the matrix. In contrast, collagen type II was found to be present throughout the extracellular matrix of both the normal growth plate and the TD lesion, suggesting that the differences observed in aggrecan, biglycan and type X collagen distribution are specific to these proteins and not a general disturbance of matrix macromolecular metabolism. The reduced deposition of these macromolecules may have implications in normal and pathological bone development.

The expression of transforming growth factor-β by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P < 0.05) and 10(-10) M (20.7%; P < 0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P < 0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.

Metallothionein induction in calcifying growth plate cartilage chondrocytes.

Metallothionein (MT) induction was studied in mineralizing cultures of chicken growth plate chondrocytes and quantitated using a Cd-saturation assay. In serum free media, MT induction was observed for Cd concentrations of 0.1 microM and greater and at Zn concentrations of 100 microM and greater. Supplementation of culture media with cysteine and/or methionine resulted in higher levels of MT induction and reduced toxicity during Cd exposure. Maximum MT induction appeared to coincide with the earliest culture stages during which important enzymes and matrix components are being synthesized. Of non-metal MT inducers tested, sodium butyrate caused a low level induction of MT while interleukin-1 had no effect on basal MT levels. 1,25-dihydroxyvitamin D increased MT induction. The steroid hormone dexamethasone caused a reduction in basal and induced MT levels. These findings suggest that MT regulation in growth plate chondrocytes differs significantly from what is known in other cell types and that this difference may be related to the mineralization of this tissue.

PTH and extracellular nucleotides synergistically induce c-fos expression in osteoblasts via distinct signal transduction pathways.

Mineralisation of bone and cartilage is essential for skeletal development and function. We have previously reported a novel gene (PHOSPHO1); a member of the large haloacid dehalogenase superfamily of hydrolases which has an active site indicative of a phosphatase. Its high expression in skeletal tissues has led us to speculate that PHOSPHO1 may be involved in the mineralisation process. Therefore, in this study, we have determined that PHOSPHO1 is localized to sites of mineralisation in both cartilage and bone. Recombinant derived PHOSPHO1 protein was produced and affinity purified PHOSPHO1 antiserum was generated and used to immunostain a range of skeletal and soft avian tissues. In addition, PHOSPHO1 gene expression was determined in SaOS-2 and MG-63 osteoblast-like cells by RT-PCR. In diaphyseal cortical bone, immunohistochemistry localized PHOSPHO1 protein to the osteoid layer of the periosteum, forming surfaces of growing osteons, and newly formed osteocytes, whereas the endosteum and closed osteons were negative. In growth plate cartilage, immunoreactivity was limited to the early hypertrophic chondrocytes and the ossification groove of Ranvier. Cartilage remnants and trabecular bone within the primary spongiosa exhibited strong immunoreactivity on their mineralising surfaces. In 17-day-old embryonic calvaria, the osteoid present on the intramembranous and periosteal bone surfaces stained positively for PHOSPHO1. All soft tissues examined were negative. PHOSPHO1 gene expression was detected in mineralising SaOS-2 but not in the non-mineralising MG-63 osteoblast-like cells and gene expression levels were unchanged by dexamethasone, estradiol, 1,25-dihydroxyvitamin D3 or PTHrP treatment. Western analysis of chick growth plate cell lysate yielded bands (30.4 and 28.6 kD) corresponding to transcripts initiated at each of two possible initiation codons indicating the presence of alternative transcripts for PHOSPHO1 in growth cartilage. These results confirm that the PHOSPHO1 protein and gene expression profile is consistent with a role for PHOSPHO1 in bone and cartilage matrix mineralisation.

Growth dependent changes in the chemical composition of proximal tibial articular cartilage and growth plate of broiler chickens

Chemical composition of the proximal tibial articular cartilage and growth plate of broiler chickens from seven age groups (1, 8, 14, 22, 29, 36, and 43 d old) was studied. In the articular cartilage, the contents of dry matter, collagen measured as hydroxyproline, and keratan sulfate increased (P &lt; 0.05), and the content of sialic acid decreased (P &lt; 0.05) as age advanced. The content of chondroitin sulfate measured as uronic acid and the proportion of hyaluronic acid in total glycosaminoglycan (GAG) were relatively constant (P &gt; 0.05) among the age groups. In the growth plate, the contents of chondroitin sulfate and keratan sulfate increased (P &lt; 0.05) with age, while the contents of dry matter, sialic acid, and collagen were similar among the age groups. The proportion of hyaluronic acid in total GAG decreased at the age of 8 d, and remained relatively constant thereafter. The growth dependent changes observed in the concentration of chondroitin sulfate in the broiler chicken cartilage were different from those in other species which have been reported to show a decrease during early postnatal growth. Key words: Tibia, epiphyseal cartilage, growth plate, broiler chicken, age

Regulators of chondrocyte differentiation in tibial dyschondroplasia: an in vivo and in vitro study.

Tibial dyschondroplasia (TD) is a disorder of endochondral bone growth and results in the retention of a mass of unmineralized, avascular cartilage extending into the metaphysis. We have studied various parameters of chondrocyte differentiation, both in isolated chick chondrocytes and growth plate sections, in an attempt to determine whether the inhibition in chondrocyte differentiation seen in TD is a consequence of an inherent incapability of chondrocytes to differentiate terminally and mineralize. Results from in vitro experiments indicated that both normal and lesion chondrocytes synthesized a matrix that stained with antibodies to types II and X collagen and displayed foci of mineralization. Alkaline phosphatase activity in lesion chondrocytes was significantly increased in comparison to that in normal hypertrophic chondrocytes. In addition, normal and lesion chondrocytes in culture synthesized transforming growth factor-beta and 24,25(OH)2D3 but not 1,25(OH)2D3. There was no significant difference in the production rate of these growth regulators between normal and lesion chondrocytes. In contrast, in growth plate sections, alkaline phosphatase activity was markedly reduced in the lesion chondrocytes and sites of mineralization were not evident. Type II collagen was located throughout the growth plate and lesion, but type X collagen was not present within the lesion except at sites of vascularization. These results indicate that, in culture, lesion chondrocytes have the ability to differentiate terminally and mineralize, and suggest that the primary abnormality in TD is related to a developmental fault which is only operative in vivo. This may include a defect in cartilage vascularization and/or impairment of chondrocyte differentiation by mechanisms that have not yet been elucidated but may involve the abnormal production of regulatory factors.

Ultrastructural Changes of Chondrocytes of Growth Plates of Young Broiler Chickens Predisposed to Tibial Dyschondroplasia

Growth plates from the proximal tibiotarsal bones of growing broiler chickens from lines exhibiting high and low incidences of tibial dyschondroplasia (TD) were compared for ultrastructural differences. Samples of growth plates were collected from five chickens of each line at 2, 7, 14, and 21 d of age. Each sample was prepared for ultrastructural evaluation after initial fixation in 2.5% glutaraldehyde and secondary fixation in 1% osmium tetroxide, each containing .7% ruthenium hexamine trichloride. No gross TD lesions were observed. The ultrastructure of growth plates of birds at 2, 7, and 14 d of age was similar between the groups. However, at 21 d of age, chondrocytes in the prehypertrophic zone of the high incidence group contained large lipid inclusions and vesiculated and disarranged stacks of rough endoplasmic reticulum with greatly dilated cisternae. Apoptotic and necrotic chondrocytes were present in the hypertrophic zone. Nuclei of apoptotic chondrocytes contained crescentic caps of condensed chromatin. The cytoplasm contained short segments of dilated granular endoplasmic reticulum, lipid droplets, and vacuoles. Necrotic chondrocytes appeared as amorphous masses with pyknotic and karyorrhectic nuclei. These changes were not observed in chondrocytes of the low TD incidence group and have not been previously reported in nonthickened growth plate cartilage.

Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.

Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia.

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone 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)