The effects of proteoglycans from different cartilage types onin vitro hydroxyapatite proliferation

Summary Proteoglycans extracted from nasal and articular cartilages have previously been shown to inhibit hydroxyapatite proliferationin vitro. This study now demonstrates that proteoglycans isolated (dissociatively extracted and reaggregatedin vitro) from bovine fetal growth plate and bovine occipital condyle, as well as those from bovine nasal cartilage, all retard hydroxyapatite seeded-growthin vitro. On a weight basis (1 mg/ml), the growth plate proteoglycan preparation had a significantly greater inhibitory effect. The greater inhibitory effect of the growth plate proteoglycans as compared to the other cartilage proteoglycan preparations may be related to the unique properties of the proteoglycans in the growth plate, a tissue that undergoes physiological calcification.     

Changes in proteoglycan aggregates during cartilage mineralization

Summary The dimensions of proteoglycan aggregates, aggregated monomers, and nonaggregated monomers, and the proportion of aggregated monomers found in the different zones of bovine rib growth plate have been defined by the electron microscopic monolayer technique. Growth plates were divided into the following 1 mm thick transverse slices; the hypertrophic zone, the lower proliferative zone, the upper proliferative zone, a transitional zone, and epiphyseal cartilage. Proteoglycans prepared by associative extraction followed by equilibrium density gradient centrifugation under associative conditions were examined by electron microscopy. Proteoglycan aggregate size decreased sharply in the lower proliferative and hypertrophic zones, as indicated by decreases in hyaluronate filament length and in the number of monomers per aggregate. Aggregated proteoglycan monomers did not show evidence of proteolytic degradation. Nonaggregated monomers were shorter than aggregated monomers, but their mean length did not decrease in the lower proliferative and hypertrophic zones. However, the proportion of nonaggregated monomers increased in these zones. Thus, before the cartilage matrix mineralized in the lower proliferative zone and as the cartilage matrix began to mineralize in the hypertrophic zone, proteoglycan aggregate size decreased and the proportion of aggregated monomers decreased. These changes in matrix proteoglycans may be one of the events that allow cartilage mineralization.     

Localization of type X collagen in canine growth plate and adult canine articular cartilage

Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl2. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x 10(4) was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis.     

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 × 10⁶ cells per 35-mm plate and grown for 17 days in BGJ_b medium supplemented with 10% fetal bovine serum, 50 μg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive ⁴⁰Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.     

Structural changes in reassembled growth plate aggregates

To investigate possible structural changes in reassembled proteoglycan aggregates during cartilage mineralization, we examined the molecular architecture and dimensions of growth plate proteoglycan aggregates by electron microscopy. The ends of fetal bovine femurs and tibias were separated into three regions: the epiphysis; the cartilage growth plate, consisting of the proliferative zone and the unmineralized portion of the hypertrophic zone; and the calcified portion of the hypertrophic zone along with part of the metaphysis. Aggregates from all three regions had the same molecular architecture. They consisted of central hyaluronic filaments with multiple attached monomers. Monomers consisted of two segments: a peripheral thick segment, which represents primarily the chondroitin sulfate-rich region, and a thin segment attached directly to the hyaluronic acid filament. The length of aggregated monomers did not differ between the growth plate cartilage and the metaphysis, nor did the lengths of the thin and thick segments, indicating that the chondroitin sulfate-rich region of aggregated monomers is not degraded during cartilage mineralization. Between the growth plate cartilage and the metaphysis, aggregates became shorter and had fewer monomers and wider spacing between monomers. These structural alterations in proteoglycan aggregates may be one of the events that prepares the matrix for mineralization.     

A macromolecular inhibitor of in vitro calcification of tendon matrix

Summary Bovine and human tendon tissue do not induce calcification in vitro. However, extraction of those tissues with 3% Na₂HPO₄ converts them to calcifiable matrices. The supernatant fraction derived from the extraction contains a nondialyzable, perchloric acid soluble component that inhibits calcification of the extracted matrix. This inhibitory substance is characterized by a molecular weight in the range of 85,000–100,000. Exposure to pronase or hyaluronidase did not alter the inhibitory potency but did render the inhibitor dialyzable. Commercial sources of hyaluronic acid, chondroitin-6-sulfate, chrondroitin-4-sulfate, dermatan sulfate, heparin and lysozyme did not inhibit calcification of the extracted matrix. Phosvitin, a phosphoglycoprotein is a potent inhibitor. Although phosvitin and the tendon extract also inhibit calcification of previously calcified matrix, they have no detectable effect on the rate of decalcification. We conclude that the mechanism of inhibition is characterized by a degree of specificity and that phosvitin and a macromolecular component of tendon tissue blocks conversion of an intermediate matrix-bound CaP complex to crystalline apatite. It seems reasonable that the tendon inhibitor could function in situ and possibly in vivo to control calcification of tendon tissue.     

Electron microscopic analysis of articular cartilage proteoglycan degradation by growth plate enzymes

To assess the effect of intracellular growth plate chondrocyte enzymes on proteoglycan structure, we examined enzyme-treated articular cartilage proteoglycans and untreated articular cartilage proteoglycans with the electron microscopic monolayer technique. The untreated proteoglycan monomers ranged in length from less than 20 nm to more than 700 nm, with a mean length of 224.5 +/- 101.6 nm in one experiment and 224.6 +/- 95.7 nm in a second experiment. Incubation with growth plate enzymes reduced proteoglycan monomers to fragments with lengths that varied from less than 5 nm to 143 nm, increased the variability in monomer length, and destroyed proteoglycan aggregates. The enzyme treated monomers had an average length of 29.5 +/- 17.9 nm in one experiment and 35.2 +/- 17.0 nm in a second experiment. The smallest common fragments were 15 nm long and would be expected to contain about 15 glycosaminoglycan chains. This experiment demonstrates that enzymes extracted from growth plate chondrocytes can degrade the chondroitin sulfate-rich region of proteoglycan monomer core proteins, produce a range of monomer fragment sizes with less than 2% of the fragments shorter than 5 nm or longer than 100 nm, increase the variability in monomer length, and degrade proteoglycan aggregates.     

Soluble and resistant proteoglycans in epiphyseal plate cartilage

The proteoglycans of cartilage occur in a form which is readily extracted (soluble) and in form which is relatively difficult to extract (resistant). Following the extraction of the soluble proteoglycans from slices of epiphyses from young rats, the distribution of the resistant proteoglycans are visualized by staining with toluidine blue. Daily quantitative recoveries of uronic acid over 7 days are used as an index of the rate and completeness of extraction. In contrast to other cartilages (nasal, costal, ear, articular) in which the resistant proteoglycans are restricted to perilacunar localizations, the resistant proteoglycans in epiphyseal plate extend across the plate as a continuous stratum and occupy extraterritorial regions. This stratum of resistant proteoglycans is difficult to identify with a specific zone in the plates of young individuals, because of primitive columniation. In more highly organized, older human and porcine epiphyseal plates, however, the stratum is clearly seen at the junction of the zones of resting and proliferating chondrocytes. It dips down a short distance between the columns, disappears and then reappears again at the level of the zone of provisional calcification. These observations are discussed in the context of endochondral growth.

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Zusammenfassung Die Knorpelproteoglycane kommen in einer leicht extrahierbaren (löslichen) und in einer relativ schwer extrahierbaren (resistenten) Form vor. Nach der Extraktion der löslichen Proteoglycane aus Epiphysenschnitten junger Ratten wird die Verteilung der resistenten Proteoglycane durch Toluidinblau-Färbung aufgezeigt. Als Index für die Geschwindigkeit und Vollständigkeit der Extraktion wird die tägliche quantitative Ausbeute von Uronsäure während 7 Tagen verwendet. Im Gegensatz zu anderen Knorpelarten (Nasen-, Rippen-, Ohren- und Gelenkknorpel), bei welchen die resistenten Proteoglycane nur perilacunär vorkommen, gehen die resistenten Proteoglycane der Epiphysenplatte über die Platte als zusammenhängende Schicht hinaus und treten in extraterritorialen Bereichen auf. Diese Schicht resistenter Proteoglycane kann in den Platten junger Individuen wegen der ursprünglichen Säulenbildung nur schwierig als eine bestimmte Zone identifiziert werden. In höher organisierten, älteren Epiphysenplatten des Menschen und des Schweines ist die Schicht jedoch deutlich an der Berührungsstelle der Zonen ruhender und proliferierender Chondrocyten ersichtlich. Sie setzt sich eine kurze Strecke zwischen den Säulen fort, verschwindet dann aber und erscheint wieder auf der Höhe der vorläufigen Verkalkungszone. Diese Beobachtungen werden mit dem endochondralen Wachstum in Zusammenhang gebracht.

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Résumé Les protéoglycanes du cartilage se présentent sous une forme que l'on peut extraire facilement (soluble) et sous une formule difficile à extraire (résistante). Après extraction de protéoglycanes de coupes d'épiphyses de jeunes rats, le répartition des protéoglycanes résistantes est visualisée par coloration au bleu de toluidine. La détermination quantitative quotidienne d' acide uronique pendant 7 jours est utilisée comme indice de la vitesse et de l'efficacité de l'extraction. Contrairement à d'autres cartilages (nasal, costal, oreille, articulaire) où les protéoglycanes résistantes sont limitées à des régions périlacunaires, les protéoglycanes résistantes de la métaphyse s'étendent au-delà sous forme d'une couche continue et occupent des régions extra-territoriales. Cette couche de protéoglycanes résistantes est difficile d'identifier avec une zone spécifique dans la métaphyse de jeunes individus, par suite d'un alignement primitif. Cependant au niveau de métaphyses humaines ou de porcs plus âgés, cette couche est nettement visible à la jonction des zones de chondrocytes au repos et en division. Elle s'étend sur une courte distance entre les cellules sériées, disparait et réapparait à nouveau au niveau de la zone de calcification temporaire. Ces résultats sont discutés en fonction de la croissance enchondrale.

     

Lectin-binding histochemistry of intracellular and extracellular glycoconjugates of the reserve cell zone of growth plate cartilage

The distribution of intracellular and extracellular lectin-binding glycoconjugates of the reserve cell zone of growth plate cartilage was studied in the distal radial growth plate of 4-week-old Yucatan swine using a postembedment method on Epon-embedded sections. Direct comparisons were made to articular, tracheal, and auricular cartilages not involved in endochondral ossification. All patterns of lectin binding that in the growth plate were restricted to the reserve cell zone were also patterns characteristic of tracheal, articular, and auricular cartilages. These included: (a) pericellular binding with peanut agglutinin (PNA) without prior digestion with neuraminidase; (b) pericellular binding with wheat germ agglutinin (WGA) at 24 h; (c) intracellular cytoplasmic binding to concanavalin A (CON-A), Lens culinaris agglutinin (LCA), and Lotus tetragonobolus agglutinin (LTA) after periodic acid oxidation; and (d) a lack of pericellular binding with CON-A and ricin agglutinin 1 (RCA-1) after periodic acid oxidation. We conclude that reserve zone chondrocytes lack specific phenotypic markers as defined by lectin-binding affinity that are found in the cellular zones of the growth plate that undergo calcification and vascularization. The reserve zone has identical lectin-binding affinities to the three structural cartilages used as controls. One interpretation of these results is that the reserve zone may not be involved directly in endochondral ossification, but may have a structural function in growth plate cartilage.     

Role of proteoglycans in endochondral ossification: Inhibition of calcification

Summary Proteoglycans from bovine nasal septa and from the Swarm rat chondrosarcoma were isolated as aggregates (PGC) and as monomers (PGS). Portions of the PGC preparations were degraded with cathepsin D or chondroitinase AC. Chondroitin sulfates were isolated by differential precipitation from alkaline digests of the PGS from bovine nasal septa. The effects of these preparations at concentrations up to 2 mg/ml on the precipitation of tricalcium phosphatein vitro at pH 7.8 in 16 hours at 25°C were ascertained. To this end, the amounts of calcium and phosphate in the precipitates and in the supernates were determined. The PGC preparations were found to be very effective inhibitors; in the presence of 2 mg/ml, precipitate did not form. The PGS preparations were less effective than the PGC preparations; in the presence of 2 mg/ml, about 20% as much calcium phosphate precipitated as in their absence. The chondroitinase AC-degraded preparations at concentrations equivalent to 2 mg/ml of the PGC preparations were approximately as effective as the PGS preparations. On the other hand, the cathepsin D-degraded PGC preparations and the chondroitin sulfate chains were relatively poor inhibitors. Although the viscosity of the solutions may have influenced the rate at which the precipitates settled to the bottom of the bubes, the amounts of the tricalcium phosphate formed were related to the composition and concentration of the proteoglycan preparations.     

Demonstration of type I and type II somatomedin receptors on bovine growth plate chondrocytes

The chondrocytes of the epiphyseal growth plate are the presumed target cells for hormones regulating skeletal growth. The somatomedins, a family of low molecular weight peptides, are thought to play a stimulatory role in this regulation. The cellular actions of the somatomedins are themselves determined by binding to specific receptors on target cells. Previous studies have characterized a specific receptor for somatomedin-C (Sm-C) or insulin-like growth factor I (IGF-I) on bovine growth plate chondrocytes (GPCs). We now report the characterization of a second type of somatomedin receptor on these cells that is more specific for another class of somatomedin represented by multiplication-stimulating activity (MSA) or rat insulin-like growth factor II (rIGF-II). Binding of [125I]MSA/rIGF-II to isolated GPCs was time dependent and saturable. Unlabeled Mr 7,100 MSA/rIGF-II and Sm-C/IGF-I were approximately equipotent in competing with [125I]MSA/rIGF-II for binding, while Mr 8,600 MSA/rIGF-II was an order of magnitude less potent. Low levels of competition by insulin appeared in some studies at concentrations of 10(-7) M and higher, suggesting displacement of [125I]MSA/rIGF-II binding to the Sm-C/IGF-I receptor. In affinity-labeling studies, [125I]MSA/rIGF-I labeled a complex of Mr greater than 300,000 (unreduced) and of Mr 140,000 (reduced), consistent with a type I somatomedin receptor composed of disulfide-linked subunits. [125I]MSA/rIGF-II labeled a Mr 240,000 moiety (unreduced) and Mr 260,000 (reduced), consistent with a type II somatomedin receptor. Both affinity-labeling and kinetic data revealed cross-binding of MSA/rIGF-II and insulin with the type I receptor and of Sm-C/IGF-I with the type II receptor. In contrast, the type II receptor did not recognize insulin. These data suggest a complex pattern of graded specificity of these receptors for their ligands. These data are consistent with the hypothesis that IGF-II as well as Sm-C/IGF-I participate in the stimulation of skeletal growth.     

Effects of cations on cartilage structure: Swelling of growth plate and degradation of proteoglycans induced by chelators of divalent cations

Summary Slices of fresh ovine and bovine epiphyseal cartilages swell following extraction in 0.05 M disodium ethylenediaminetetraacetate (EDTA) in Tris buffer, pH 5.8 and 7.4, at 4° and 37°. The swelling is strikingly visible to the unaided eye and is most pronounced in the growth plate region of the epiphysis. Other chelators—ethyleneglycolbis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA), and citrate buffer—also induce swelling. Swelling is associated with increased degradation of proteoglycans (PG) especially at pH 5.8, however, collagen seems to be unaffected. These effects are prevented by the addition of certain divalent cations (Ca, Mg, Zn) to the extraction media. At higher concentrations, the monovalent cation sodium also prevents swelling. It is concluded that divalent cations are required to maintain structure and function of cartilage. Freezing and thawing the cartilage did not prevent swelling or degradation, which suggests that these phenomena are not dependent on living chondrocytes. Although PG degradation and loss is markedly increased at 37° as compared with 4°, swelling is unaffected. It is concluded therefore that the degradative effects are enzymatic but the swelling is physicochemical. Other cartilages (nasal, manubrium) also swell and show histochemical evidence of PG degradation. These effects are minimal compared with the effects induced in the growth plate. It is inferred that growth plate contains more proteases than other cartilages and has properties that make it more susceptible to swelling. Swelling of the growth plate occurs even when the metaphysis is attached to it albeit to a lesser extent than when it is freed of underlying bone. A hypothesis is offered which attempts to link these phenomena with chondrocyte and matrical imbibition of water (swelling) in the zone of hypetrophy of the growth plate.     

Regeneration of growth plate cartilage induced in the neonatal rat hindlimb by reamputation

Following primary hindlimb amputations dividing the lower femur or the central tibiofibula, the neonatal rat innately regenerates the distal growth plate(s) with a frequency of about 20-30%. One or two reamputation procedures were performed in an effort to increase the frequency of physeal regeneration, noting that such procedures, and related forms of tissue stimulation, have been repeatedly shown to induce regenerative growth at limb amputation sites of some amphibians that display little innate regenerative capacity. The present reamputation sequences divided the skeletal stump through the cartilaginous mass arising at its distal end. Following first reamputation an approximate three fold increase in the frequency of growth plate cartilage regeneration was observed at transfemoral and transtibiofibular sites. Only after second reamputation, however, did tibiofibular physeal cartilage regeneration equal in frequency that observed after first reamputation through the lower femur. Ectopic growth plate cell architecture was identified in cartilaginous extensions arising from the side of the distal femoral shaft, and also within the regrown secondary cartilage body, which unites the lower tibia and fibula in the shank of the rat. Moreover, among 3 of 11 femoral amputees that had sustained reamputations, regrowth of the distal femoral condylar mass and profile were achieved to varying degrees. It is concluded that a regimen of reamputation, known to induce regenerative growth in the amphibian limb, also induces skeletal regneration in the mammalian limb, and lead to the appearance of ectopic growth plate cell architecture at adjacent sites.     

Extracellular matrix alterations during endochondral ossification in humans

Immunohistochemical methods were employed to examine alterations in the cartilage extracellular matrix constituents associated with endochondral ossification in humans. The distributions of chondroitin 4- and 6-sulfate and keratan sulfate proteoglycan (PG) determinants, cartilage PG link protein, collagen types I and II, and fibronectin were determined in iliac crest growth-plate specimens using the avidin-biotin-horseradish peroxidase system. Collagen type II was distributed throughout the growth plate, providing a framework within which chondrocytes divided and formed clusters of differentiating (hypertrophic) cells. The septa between these clusters and their subchondral extensions into underlying bone trabeculae were rich in PG, PG link protein, and collagen type II and resembled the extracellular matrix of reserve cartilage. The territorial matrix associated with the differentiating cells within the clusters contained reduced amounts of collagen type II, PG link protein, and possibly cartilage PG. Collagen type I and fibronectin were detected within the cytoplasm of the maturing and degenerating cells, and fibronectin localized intensely to the pericellular matrix envelopes of these cells. These alterations presumably facilitate the degradation of the matrix associated with the cell clusters by invading vascular tissue, while the septa, which retain the characteristics of more typical cartilage matrix, are not degraded and firmly anchor the cartilage to the subchondral bone.     

Distribution of acidic glycosaminoglycans in rabbit growth plate cartilage

Chondroitin-4-sulfate (C-4-S), chondroitin-6-sulfate (C-6-S) and keratan sulfate (KS) concentrations were measured in growth plate cartilage derived from rabbits of two different ages, and from three growth plate zones from rabbits of the same age. With increasing age, the concentration of C-4-S increased, C-6-S decreased and KS remained constant. There was no variation when three different growth plate zones were sampled. Radiosulfate uptake into chondroitan sulfate was also measured, and this showed an inverse relationship to concentration with increasing age.Research supported in part by a grant from the Western Chapter (Pennsylvania) Arthritis Foundation; Research Grant AMI 1382-04 from the National Institute of Arthritis and Metabolic Diseases; and the Orthopaedic Research Fund, The Hospital for Joint Diseases.     

Differences in matrix vesicle concentration among growth plate zones

We studied the proximal tibial growth plates of 15-day-old mice to determine if matrix vesicle concentration varies among growth plate zones or between the pericellular and territorial matrix compartment and the interterritorial matrix compartment. Growth plates were examined by electron microscopy and divided into five zones: reserve zone (RZ), upper proliferative zone (UPZ), lower proliferative zone (LPZ), upper hypertrophic zone (UHZ), and lower hypertrophic zone (LHZ) which included the calcifying zone. We measured the diameter and volume fraction of matrix vesicles and calculated their numerical density and volume per cell and number per cell in the pericellular and territorial matrix and in the interterritorial matrix of each zone. In the pericellular and territorial matrix compartment, the matrix vesicle concentration progressively decreased from the RZ to the LHZ. Changes in matrix vesicle concentration in the interterritorial matrix followed a different pattern. Between the RZ and the UPZ, matrix vesicle numerical density declined slightly and then increased to peak values in the LPZ and UHZ, followed by a decline between the UHZ and the LHZ. These changes in matrix vesicle concentration paralleled previously reported changes in intramitochondrial calcium content, suggesting that matrix vesicle production in growth plate may be related to intracellular calcium concentration. The existence of the maximum concentration of matrix vesicles in the LPZ and UHZ longitudinal septa which do not mineralize followed by a decline in matrix vesicle concentration in the LHZ longitudinal septa which mineralize suggests that a high concentration of matrix vesicles may be needed to prepare the matrix for mineralization or to initiate mineralization and that matrix vesicles are depleted during mineralization.     

Steindler lecture. Binding sites in fetal and growth plate cartilage

In addition to genetic and nutritional factors, linear growth during the prenatal and postnatal periods is controlled by peptide, steroid, and thyroid hormones interacting with the receptors present on the membrane or in the cytosol and nuclei of growth plate cartilage. Using standard procedures, insulin and "nonsuppressible insulin-like activity" (a somatomedin) showed significant binding in 600, 15,000, and 105,000 g membrane fractions of epiphyseal cartilage of immature animals. The binding of growth hormone and prolactin was small and probably not significant. Specific uptake of glucocorticoid was demonstrated in viable canine chondrocytes, but not of androgen, estrogen, or vitamin D3 metabolite. A triiodothyronine receptor was present in nuclei from dog epiphyseal cartilage. Hormones that lack binding may affect cartilage only indirectly. Hormone receptors were studied in those portions of fetal growth cartilage that will later evolve into an ossification center, articular cartilage, and epiphyseal cartilage. Cytosol fractions contained a receptor for glucocorticoid but not for androgen or estrogen. Zonal analysis showed a higher level in the peripheral and central sections than in the palisade section. Triiodothyronine binding was also detected in nuclei prepared from whole fetal cartilage. Heterogeneity of cell function was obvious in fetal cartilage. Cell division was high in the central and peripheral zones as well as the upper half of the palisade zone, but low in the lower palisade section. Proline and sulfate incorporation predominated in the palisade section compared with the central and peripheral sections. Disease states with changes of metabolic activities in the cartilage may perhaps be better understood with a clearer knowledge of receptor levels and interactions.     

The influence of vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (chondrocalcin)

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)