Localization of a dermatan sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in other tissues

A monoclonal antibody to a core-protein-related epitope of a small dermatan sulfate-rich proteoglycan (DS-PGII) isolated from adult bovine articular cartilage (22) was used to localize this molecule, or molecules containing this epitope, in bovine articular cartilages, in cartilage growth plate, and in other connective tissues. Using an indirect method employing peroxidase-labeled pig anti-mouse immunoglobulin G, DS-PGII was shown to be present mainly in the superficial zone of adult articular condylar cartilage of the metacarpal-phalangeal joint. In fetal articular and epiphyseal cartilages, the molecule was uniformly distributed throughout the matrix. By approximately 10 months of age it was confined mainly to the superficial and middle zones of articular cartilage and the inter-territorial and pericellular matrix of the deep zone. DS-PGII was not detected in the primary growth plate of the fetus except in the proliferative zone, where it was sometimes present in trace amounts. In contrast, it was present throughout the adjacent matrix of developing epiphyseal cartilage. In the trabeculae of the metaphysis, strong staining for DS-PGII was seen in decalcified osteoid and bone immediately adjacent to osteoblasts. Staining was also observed on collagen fibrils in skin, tendon, and ligament and in the adventitia of the aorta and of smaller arterial vessels in the skin. These observations indicate that DS-PGII and/or molecules containing this epitope are widely distributed in collagenous tissues, where the molecule is intimately associated with collagen fibrils; in adult cartilage this association is limited mainly to the narrow parallel arrays of fibrils which are found in the superficial zone at the articular surface. From its intimate association and other studies, this molecule may play an important role in determining the sizes and tensile properties of collagen fibrils; it may also be involved in the calcification of osteoid but not of cartilage.     

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.     

Lysozyme in preosseous cartilage VII. Evidence for physiological role of losozyme in normal endochondral calcification

Previous work demonstrated that micropuncture aspirates from rat epiphysical plate cartilage contain a nucleating agent for Ca₃(PO₄)₂ mineral growth, and that the nucleation is inhibited by proteoglycan aggregates. In this report data are described which show that mammalian lysozyme inactivates the inhibition. When micropuncture aspirates are incubated in vitro with mammalian lysozyme, a rapid, spontaneous initiation of mineral growth occurs. Incubation of proteoglycan aggregate preparations in the presence of cartilagea lysozyme, but not hen egg white lysozyme, causes a marked decrease of the sedimentation coefficients of the proteoglycans, usually to values close to those obtained with proteoglycan monomer preparations. The inhibition of this effect of mammalian lysozyme by a specific inhibitor of the enzyme tri(N-acetyl-D-glucosamine) suggests that it may be enzymatic in nature.     

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage matrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.     

Distribution of lipids associated with mineralization in the bovine epiphyseal growth plate

Calcium-acidic phospholipid-phosphate complexes, known to induce in vitro hydroxyapatite formation from metastable calcium phosphate sotutions, have been isolated from the morphologically defined zones of the bovine epiphyseal growth plate. The changes in zonal distribution of these complexes in epiphyseal cartilage correlate directly with other biochemical changes which occur prior to cartilage calcification. The concentration of calcium-acidic phospholipid-phosphate complexes increases going from the morphologically defined reserve zone to the proliferative zone, peaking in the hypertrophic zone, where mineralization is initiated, and decreasing in primary spongiosa and diaphyseal bone. Expressed as milligrams of calcium-phospholipid-phosphate complex per milligram hydroxyproline the concentration ranged from 19 (articular cartilage) to 535 (hypertrophic cell zone) decreasing to 43 (diaphyseal bone) with parallel changes being seen when the concentration was expressed per gram of demineralized dry tissue, per total lipid, per DNA, or, per 5′-AMPase activity.     

Biosynthesis and metabolism of prostaglandins in chick epiphyseal cartilage.

Microsomal fractions of cells isolated from chick epiphyseal cartilage catalyzed the synthesis of prostaglandins from radiolabeled delta8,11,14-eicosatrienoic and from arachidonic acids. In addition, the microsomal supernatants contained both 15-hydroxyprostaglandin dehydrogenase and prostaglandin 15-keto delta13,14-reductase activities. Two major classes of prostaglandins (E and F) were synthesized; however, a major product which chromatographically behaves as PGA was also isolated. Synthetase activities were analyzed for pH optima and response to known stimulators and inhibitors of prostaglandin systhesis. The different activators had varying stimulatory effects on prostaglandin synthesis; the anti-inflammatory drugs were all strongly inhibitory. Synthetase activity in the growth plate was highest in the zone of hypertrophy, declining substantially in the more heavily calcified regions. Degradative enzyme activities were highest in the zone of maturation and significantly lower in the adjacent hypertrophic zone. The net effect of these opposing activities would be to elevate prostaglandin levels at the zone of hypertrophy, a finding which suggests that prostaglandins may play a role in the modulation of epiphyseal cartilage metabolism.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18- 19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Zonal analysis of metabolic profiles of articular-epiphyseal cartilage chondrocytes: a histochemical study

Summary Articular—epiphyseal cartilage from the femur of New Zealand rabbits was subjected to histochemistry for determination of the presence of metabolic enzymes along its zonal stratification. Glycolytic enzymes were strongly reactive in all of the zones. Krebs cycle enzymes, enzymes of the hexose monophosphate shunt and the respiratory chain enzymes showed a progressive increase in reactivity from the tangential zone through the top half of the epiphyseal zone. Indicators of lipid metabolism were fairly high in all regions of the cartilage.     

Biosynthesis and metabolism of prostaglandins in chick epiphyseal cartilage

Microsomal fractions of cells isolated from chick epiphyseal cartilage catalyzed the synthesis of prostaglandins from radiolabeled Δ^8,11,14-eicosatrienoic and from archidonic acids. In addition, the microsomal supernatants contained both 15-hydroxyprostaglandin dehydrogenase and prostaglandin 15-keto Δ^13,14-reductase activities. Two major classes of prostaglandins (E and F) were synthesized; however, a major product which chromatographically behaves as PGA was also isolated. Synthetase activities were analyzed for pH optima and response to known stimulators and inhibitors of prostaglandin synthesis. The different activators had varying stimulatory effects on prostaglandin synthesis; the anti-inflammatory drugs were all strongl inhibitory. Synthetase activity in the growth plate was highest in the zone of hypertrophy, declining substantially in the more heavily calcified regions. Degradative enzyme activities were highest in the zone of maturation and significantly lower in the adjacent hypertrophic zone. The net effect of these opposing activities would be to elevate prostaglandin levels at the zone of hypertrophy, a finding which suggests that prostaglandins may play a role in the modulation of epiphyseal cartillage metabolism.     

Identification of a high-molecular-weight (> 400 000) protein in hyaline cartilage

A high-molecular-weight (> 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2–4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the > 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Characterization of collagen types XII and XIV from fetal bovine cartilage.

The structurally related type XII-like collagen molecules TL-A and TL-B were recently identified in fetal bovine epiphyseal cartilage and subsequently shown to be collagen types XII and XIV, respectively. By indirect immunofluorescent staining of cartilage using monoclonal antibodies to the NC3 domains of each molecule, it was shown that type XII collagen was present predominantly around cartilage canals, the articular surface, subperichondrial margins, and the perichondrium, was less so in the remaining cartilage matrix, and was absent from the growth plate region. In the permanent cartilage of trachea, type XII stained somewhat more intensely in the margins beneath the loose connective tissue. Type XIV collagen localized more uniformly throughout the articular cartilage and was also absent from the growth plate region, whereas in tracheal cartilage, its distribution was similar to type XII. We have characterized the structure of these cartilage molecules and compared them with those from fetal bovine skin. Extraction of cartilage with 1 M NaCl and differential NaCl precipitation yields a fraction enriched for these two collagens. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies to the large amino-terminal non-triple-helical domain, NC3, revealed the presence in cartilage of two forms of type XII collagen: type XIIB, the molecule previously identified in chick and bovine tissues, and type XIIA, a much larger form equivalent to the molecule recently identified in WISH-transformed epithelial cell culture medium (Lunstrum, G. P., McDonough, A. M., Marinkovich, M. P., Keene, D. R., Morris, N. P., and Burgeson, R. E. (1992) J. Biol. Chem. 267, 20087-20092). Digestion with bacterial collagenase shows that the increased mass is present in the NC3A domain. Additional purification by velocity sedimentation and observation of rotary-shadowed images demonstrates molecules with extended non-triple-helical arms approximately 80 nm in length analogous to the WISH cell molecules. Electrophoretic mobilities of bands corresponding to type XIIA, but not type XIIB, are sensitive to chondroitinase ABC, indicating that type XIIA is a chondroitin sulfate proteoglycan and that modification occurs predominantly within the NC3A domain distal to NC3B. Neither type XIIB from skin nor type XIIA from WISH cells are chondroitinase-sensitive. By similar analysis, a portion of the type XIV collagen chains in cartilage was also sensitive to chondroitinase digestion. Chondroitin sulfate is apparently not located on its NC3 domain. As in skin, collagen types XII and XIV have subtly different distributions within cartilage and type XII may have a tissue-specific structure.     

Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.     

Phosphomonoesterases in growth cartilages of the rat

Summary Epiphyseal plate cartilage, epiphyseal cartilage, synchondroseal cartilage and mandibular condylar cartilage were studied morphologically and histochemically in 14 days old rats. Ordinary decalcified paraffin sections were stained with hematoxylin & eosin, van Giesons connective tissue stain, or toluidine blue, and used for morphological studies of the different cartilaginous structures. Undecalcified cryostat sections were used for demonstration of acid and alkaline phosphatase. The enzyme activity was tested for at regular intervals during incubation from 15 sec to 120 min.The morphologic study revealed that a marked similarity of construction exists between epiphyseal plate cartilage and synchrondroseal cartilage. The construction of epiphyseal and condylar cartilage differ from that of the other two structures and also differ mutually.With small variations the reaction for both alkaline and acid phosphatase was found to be identical in the zones of erosion, hypertrophy and maturation of the four structures. Intercellularly, acid phosphatase is present in all zones in the synchondroseal and the epiphyseal plate cartilage, while in the epiphyseal and condylar cartilages it is only present in the zones of erosion, hypertrophy and maturation.The identical reaction for acid phosphatase in the epiphyseal and the condylar cartilage is thought, in all likelihood, to be accidental. When kinetic conditions are taken into account, epiphyseal cartilage seems to react like epiphyseal plate and synchondroseal cartilage, while the condylar cartilage takes up an exceptional position among growth cartilages.     

Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats

 The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation. Accepted: 19 February 1997     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Identification of a high-molecular-weight (greater than 400 000) protein in hyaline cartilage

A high-molecular-weight (greater than 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2-4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the greater than 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Immunohistochemical localization of alkaline phosphatase in the chicken epiphyseal growth cartilage

Summary Alkaline phosphatase of chicken epiphyseal cartilage has been localized by two immunohistochemical methods. Double layer immunofluorescence and peroxidase anti-peroxidase (PAP) methods gave similar results. Alkaline phosphatase in epiphyseal cartilage is extracellular as well as intracellular in the localization. Extracellular reaction was strongest in the lower layers of growth plate and the most intense reaction was noted in the pericellular lacunae of hypertrophic chondrocytes. Also intracellular immunoreaction was noticed through the whole growth plate.     

The calcification of cartilage matrix in chondrocyte culture: studies of the C-propeptide of type II collagen (chondrocalcin)

We have shown that when chondrocytes are isolated by collagenase digestion of hyaline cartilage from growth plate, nasal, and epiphyseal cartilages of bovine fetuses they rapidly elaborate an extracellular matrix in culture. Only growth plate chondrocytes can calcify this matrix as ascertained by incorporation of 45Ca2+, detection of mineral with von Kossa's stain and electron microscopy. There is an extremely close direct correlation between 45Ca2+ incorporation in the first 24 h of culture and the content of the C-propeptide of type II collagen, measured by radioimmunoassay, at the time of isolation and during culture. Moreover, growth plate cells have an increased intracellular content of the C-propeptide per deoxyribonucleic acid and, during culture, per hydroxyproline (as a measure of helical collagen) compared with nasal and epiphyseal chondrocytes. In growth plate chondrocytes 24,25-dihydroxycholecalciferol (24,25-[OH]2D3), but not 1,25-dihydroxycholecalciferol alone, stimulates the net synthesis of the C-propeptide and calcification; proteoglycan net synthesis is unaffected. Together, these metabolites of vitamin D further stimulate C-propeptide net synthesis but do not further increase calcification stimulated by 24,25-(OH)2D3. These observations further demonstrate the close correlation between the C-propeptide of type II collagen and the calcification of cartilage matrix.