Chondroitinase-resistant sulfated glycosaminoglycans synthesized by cartilages of chick embryos and of newborn chickens and rats.

Biosynthesis of chondroitinase-resistant glycosaminoglycans as minor components was studied in the cartilages of chick embryos and of newborn chickens and rats. Sternal and knee cartilages were labeled in vitro with 35SO42-, and then 35S-labeled glycosaminoglycans were analyzed. In rats up to 2 weeks old, only one glycosaminoglycan could be detected as heparan sulfate. In the chick embryos and the newborn chickens, however, keratan sulfate as well as heparan sulfate could be detected. As chondroitinase-sensitive glycosaminoglycans, large amounts of both chrondroitin 4- and 6-sulfates were synthesized in the chick cartilage, but the synthesis of chondroitin 6-sulfate could scarcely be seen in the rat cartilage. The results seem to indicate that the biosynthesis of keratan sulfate has some relation to that of chondroitin 6-sulfate.     

Glycosaminoglycans in patellar cartilage

We obtained samples of patellar cartilage at the time of surgery from 20 patients: 11 with normal and 9 with macroscopic cartilaginous degeneration. The glycosaminoglycans were separated by cellulose acetateelectrophoresis, and the relative content of major glycosaminoglycans was measured by optic scanning. The total content of glycosaminoglycans was estimated by uronic acid analysis. We found that degenerated cartilage contained an increase in dermatan sulfate and hyaluronic acid and a decrease in chondroitin 6-sulfate and total uronic acid concentration, with only slight changes in chondroitin 4-sulfate and keratan sulfate compared with normal cartilage.     

Glycosaminoglycans in patellar cartilage

We obtained samples of patellar cartilage at the time of surgery from 20 patients: 11 with normal and 9 with macroscopic cartilaginous degeneration. The glycosaminoglycans were separated by cellulose acetate electrophoresis, and the relative content of major glycosaminoglycans was measured by optic scanning. The total content of glycosaminoglycans was estimated by uronic acid analysis. We found that degenerated cartilage contained an increase in dermatan sulfate and hyaluronic acid and a decrease in chondroitin 6-sulfate and total uronic acid concentration, with only slight changes in chondroitin 4-sulfate and keratan sulfate compared with normal cartilage.     

Keratan sulfate content and articular cartilage maturation during postnatal rabbit growth

This article describes the macromolecular changes in keratan sulfate and proteoglycan that occur in rabbit articular cartilage during postnatal development. Articular cartilage glycosaminoglycans from femoral condyles and the tibial plateaus of rabbits at 8, 12, 18, and 26 weeks and 2 years of age were extracted, fractionated, and quantified. The predominant glycosaminoglycan present in articular cartilage at 8 weeks was chondroitin sulfate. During subsequent maturation the relative proportions of keratan sulfate and chondroitin sulfate varied inversely. The greatest increase in the amount of keratan sulfate present in cartilage was observed between 12 and 26 weeks of age. Hyaluronic acid content was measurable at 12 weeks; afterward the amount remained relatively constant with age. Proteoglycans, extracted from 6-, 12-, and 22-week-old rabbit femoral and tibial cartilage in the presence of protease inhibitors, were analyzed on columns of Sepharose CL-2B. Cartilage proteoglycans decreased in hydrodynamic size between 12 and 22 weeks, corresponding to the period of maximal change in content of keratan and chondroitin sulfate.     

Studies on normal and pathological human cartilage proteoglycans with metrizamide density gradient ultracentrifugation

In order to study human articular cartilage proteoglycans (PG) in aging and pathological states, PG extracts of normal cartilage, osteoarthritic cartilage, chondrosarcoma and osteochondroma were subjected to the density gradient ultracentrifugation in 23% metrizamide-6M urea system. Normal cartilages have chondroitin sulfate rich PG and keratan sulfate rich PG. The latter has a higher buoyant density and increases markedly with aging, but is missing in all of the pathological cartilages tested which have juvenile profiles corresponding to the normal cartilage of a child under 10 years of age. No differences in the chain lengths of glycosaminoglycans were observed in any of the cartilages tested, but marked differences were observed in the aggregation PG and hyaluronate in the pathological cartilages. The molecular sizes of hyaluronate are probably small in normal adults but large in the pathological state. Determinations of hexose and uronate distribution in the density gradient characterize easily the differences in aging and pathological cartilages. This system provided more information than the CsCl-guanidine HCl system.     

Glycosaminoglycans in urothelial carcinomas

Summary The glycosaminoglycan (GAG) content in human urothelial carcinomas was biochemically determined and compared to that of normal urothelium and bladder wall of the calf. The total GAG content was elevated in urothelial carcinomas, and the distribution pattern differed from that of normal urothelium and bladder wall. Whereas urothelial carcinomas contained heparan sulphate, dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate, only heparan sulphate could be detected in the normal urothelium. The GAG determination was based on hexosamine analysis and thin layer chromatography after elution on Dowex 1 X 2 columns.Abbreviations ABC chondrointin ABC lyase - AC chondroitin AC lyase - CH4-S chondroitin 4-sulfate - CH6-2 chondroitin 6-sulfate - DS dermatan sulfate - GAG glycosaminoglycan (s) - Gal-N galactosamine - Glc-N glucosamine - HS heparan sulphate - KS keratan sulphate - TLC thin layer chromatography     

Alterations in glycosaminoglycan concentration and sulfation during chondrocyte maturation

We have used antibodies to chondroitin 4- and 6-sulfate and keratan sulfate along with Alcian blue staining of sulfated proteoglycans to investigate changes in content and sulfation within the avian growth plate. In normal chicks, chondroitin 4- and 6-sulfate content were similar in the proliferating and transitional zones but in the hypertrophic zone, chondroitin 4- and 6-sulfate were slightly lower (13% and 18%, respectively) and keratan sulfate was markedly lower (58%). Compared with the proliferative zone, Alcian blue staining of sulfated glycosaminoglycans was markedly lower in both the transitional (46%) and hypertrophic (22%) zones. In tibial dyschondroplasia, where chondrocyte maturation is arrested at the transitional zone, there was no difference in the chondroitin 4- and 6-sulfate or keratan sulfate staining between the proliferative and transitional zones, which were similar to normal birds. With Alcian blue staining there was no difference in the intensity of the staining within the proliferating zone compared with normal birds but stainign in the transitional chondrocytes was markedly higher (39%). These results suggest that in the early steps of chondrocyte maturation there may be a decrease in the degree of glycosaminoglycan sulfation without any alteration in glycosaminoglycan concentration, and that further maturation may be accompanied by a change in the nature of the proteoglycans which may also affect the level of sulfation.     

Analysis of dentine glycosaminoglycans using high-performance liquid chromatography

Puppy dentine was prepared using ultracentrifugation of tooth powder in organic density gradients. The glycosaminoglycans of the obtained tissue fraction were prepared after papain digestion and beta-elimination, using preparative chromatography on DEAE-cellulose and CPC-cellulose. These polysaccharide fractions were analyzed using highly sensitive HPLC procedures. One such HPLC procedure allowed hyaluronic acid to be determined in less than microgram amounts. The glycosaminoglycans thus prepared consisted only of chondroitin-4-sulfate, chondroitin-6-sulfate, and small amounts of highly hybridized dermatan sulfate, while the experiments failed to demonstrate even trace amounts of keratan sulfate, hyaluronic acid or heparan sulfate.     

Newly synthesized proteoglycans in pseudoachondroplasia

In an attempt to elucidate the biochemical defect in pseudoachondroplasia, proteoglycan metabolism was investigated in cartilage from a patient with the dominant form of this condition. Iliac-crest cartilage was radioactively labeled with ³⁵S-sulfate and the newly synthesized proteoglycans examined for their hydrodynamic size and glycosaminoglycan composition. The banding pattern of the purified proteoglycans was analyzed by gel-electrophoresis using large pore polyacrylamide-agarose. We found a normal chain-length of glycosaminoglycans and a normal ratio of chondroitin-6-sulfate to chondroitin-4-sulfate. The proteoglycans were not enriched in keratan sulfate. Gel electrophoretic analysis of the proteoglycans disclosed a banding pattern comparable to that of two normal controls. In contrast to the findings of other authors no differences between the proteoglycans of pseudoachondroplastic and normal cartilage were detected.     

Renal and urinary glycosaminoglycans in an experimental model of chronic renal failure in rats

The present paper reports the glomerular and renal individual glycosaminoglycan levels in an experimental model of chronic renal failure (CRF) that was induced in Wistar rats by five-sixths mass ablation. Glycemia, body weight, blood systolic pressure and urinary excretions of creatinine, albumin and glycosaminoglycans were measured for 12 weeks. At the end of the experiment, the weight and the glycosaminoglycan composition of the kidneys were determined. In control rats, heparan sulfate was the main glycosaminoglycan found both in whole kidney and isolated glomeruli, with trace amounts of dermatan sulfate. Isolated glomeruli presented higher heparan sulfate concentrations than whole kidney (expressed as mg/g dry weight). In CRF rats, albuminuria appeared from the 2 week on, and dermatan sulfate and chondroitin sulfate contents of the kidney increased, whereas heparan sulfate levels remained unaltered. Changes in urine glycosaminoglycans (heparan sulfate, chondroitin sulfate and dermatan sulfate) were not statistically significant. The increase in glomerular dermatan sulfate and chondroitin sulfate observed in this experimental model could be related to the mechanisms involved in the glomerulosclerosis and proteinuria that occur in CRF.     

Mucopolysaccharides and collagen in prenatal and postnatal human development

Mucopolysaccharides (MPS) and collagen have been analyzed in different types of cartilage and some other tissues at various stages of prenatal and postnatal human development. The concentration of the chondroitin sulfate-type MPS increased from the 38th embryonal day, the 6-sulfate exceeding in amount the 4-sulfate. The increase in collagen concentration was accompanied by a decrease in the proportion of neutral salt-soluble collagen. There was no accumulation of chondroitin sulfates in the calvaria. A maximal concentration of MPS of a low molecular weight occurred in the skin and in the ear pinna after the 10th prenatal week. Postnatally, the MPS-concentration in the iliac crest and in the tibial articular cartilages decreased gradually, the highest values being reached in the newborn; the collagen-concentration showed an approximately reciprocal course.     

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.     

Biosynthesis of rat growth plate proteoglycans in diabetes and malnutrition

Insulin is an important growth factor in man and mammals. In the present investigation, we have studied the incorporation of (35S)-sulfate into growth plate proteoglycans in normal, diabetic, insulin-treated diabetic, and marasmic rats. We found that diabetes leads to an all-but-total stop in the synthesis of sulfated glycosaminoglycans. The glycosaminoglycan chains actually synthesized were shorter than in normal rats. The proteoglycan monomers were smaller and did not form large aggregates in vitro. Marasmic rats and insulin-treated diabetic rats were intermediate between normal and diabetic rats with respect to sulfate uptake by cartilage, incorporation of cartilage sulfate into glycosaminoglycans, and the chain length of glycosaminoglycans. We conclude that insulin and nutrition play important but different roles in the biosynthesis of growth plate proteoglycans and thus for the longitudinal growth of skeletal bones.     

Future orthopedic training,a global watch

A study was made of glycosaminoglycan metabolism of articular cartilage in developing experimental osteoarthrosis caused by immobilization of the rabbit knee. Cartilage samples from various sites in the knee and hip joints were analysed, samples from the mobile limb serving as controls. The concentration of glycosaminoglycans in the tissue was measured by determinations of hexosamine and uronic acid after prior papain proteolysis and subsequent purification. The uptake of ³⁵S-sulphate was used as an indicator of the synthesis rate of sulphated glycosaminoglycans. Metabolic changes characteristic of osteoarthrosis, i.e., glycosaminoglycan depletion and increased uptake of ³⁵S-sulphate, were found in tibial weight-bearing and femoral condylar cartilages. Net synthesis of glycosaminoglycans occurred in the tibial marginal cartilage.     

Characterization of glycosaminoglycans in tubular epithelial cells: calcium oxalate and oxalate ions effects

BACKGROUND: The interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines. METHODS: Glycosaminoglycan synthesis was analyzed by metabolic labeling with (35)S-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry. RESULTS: The main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of (35)S-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased (35)S-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium. CONCLUSION: Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.     

The glycosaminoglycans of the different layers of bovine articular cartilage in relation to age

The incorporationin vitro of³⁵S-sulphate into chondroitin sulphate fractions, quantitatively assessed and separated, was studied in different layers of bovine articular cartilage in relation to age. After incubationin vitro in Tyrode solution with³⁵S-sulphate the specimens were washed for 12 hours in saturated sodium sulphate solution. No loss of chondroitin sulphate occurred in the young and adolescent articular cartilage after the incubation and washing procedures. Thus, the proteoglycans appear to be firmly bound within this cartilage In contrast, large losses of chondroitin sulphate were found from the epiphyseal cartilage. In the cow, no chondroitin sulphate was lost from the superficial layers but increasing losses of both chondroitinand keratansulphate occurred from the middle to the deep layers. In all ages, the articular surface layer 40 μ in thickness showed high “specific activities” in the different chondroitin sulphate fractions and incorporation of³⁵S-sulphate into chondroitin sulphate of predominantly high molecular weight and/or charge density suggesting an increased turnover rate of these fractions. In the matrix of this layer, the chondroitin sulphate consisted of predominantly low molecular weight/low charged fractions. In the other layers, a pattern of³⁵S incorporation was found largely reflecting the actual chondroitin sulphate distribution pattern. This suggests that sulphation is completeted intracellularly. Further, the data suggested similar turnover rates for the different chondroitin sulphates. Increased incorporation of³⁵S-sulphate into chondroitin sulphate at higher ages indicated increased rate of synthesis, probably secondary to increased degradation.     

Glycosaminoglycans of iliac crest cartilage in spondyloepiphyseal dysplasia congenita

Iliac crest cartilage biopsies from five children with spondyloepiphyseal dysplasia congenita contained increased amounts of chondroitin-6-sulfate and keratan sulfate. The increase was similar to that observed in the cartilage of patients with Morquio's disease. However, unlike patients with Morquio's disease, those with spondyloepiphyseal dysplasia congenita did not excrete an abnormal amount of keratan sulfate in urine. Spondyloepiphyseal dysplasia congenita can be considered a mucopolysaccharidosis without mucopolysacchariduria.     

The glycosaminoglycans of dog compact bone and epiphyseal cartilage in the normal state and in experimental hyperparathyroidism

In normal dogs and in dogs treated with 20 or 30 U.S.P. parathyroid extract for 5 and 3–4 days, respectively, the glycosaminoglycans of compact bone tissue were identified using the cetylpyridinium chloride precipitation method, and the concentrations of total hexosamines and the hexosamines corresponding to cetylpyridinium chloride precipitable acid glycosaminoglycans were determined. Further, the glycosaminoglycan pattern of the epiphyseal plate and the incorporation of³⁵S-sulphate into the glycosaminoglycans of bone tissue and epiphyseal cartilage after administration of³⁵S-sulphatein vivo was studied.In compact bone tissue, the hexosamines corresponding to acid glycosaminoglycans constuted approximately one third of the total hexosamine concentration and approximately 0.05–0.06% of the total dry weight. The main component of the acid glycosaminoglycans in bone was chondroitin-4-sulphate. This was sulphated to a higher degree and also of a higher molecular weight than thechondroitin sulphate of the epiphyseal cartilage, which in accordance with earlier investigations was found to have infrared characteristics of both chondroitin-4-sulphate and chondroitin-6-sulphate, with the former dominating. The molecular weights of the main part of bone chondroitin sulphate ranged from approximately 45,000 to 56,000. A small component of the bone glycosaminoglycans was hyaluronic acid.Large regularly recurring differences in the specific activity of fractions with differences in molecular weight in the condroitin sulphate of bone tissue and epiphyseal cartilage were noted.Treatment of the dogs with parathyroid extract gave no effect on the molecular weights of the chondroitin sulphate of the bone matrix or of the epiphyseal cartilage. Nor was there any unequivocal effect on the concentrations of total hexosamines or on the acid glycosaminoglycans. No evident stimulatory or depressant effect on the incorporation of³⁵S-sulphate into the chondroitin sulphate or in the molecular distribution of newly sulphated and/or synthesized molecules of the condroitin sulphate within these tissues occurd.

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Zusammenfassung An normale Hunden und an Hunden die unter 5 respektive 3–4 Tagen 20 oder 30 U.S.P./kg Parathyreoidea-Extract erhalten hatten, wurden im kompakten Knochen die Glykosaminoglykane unter Anwendung der Cetylpyridiniumchlorid-Fällungsmethode identifiziert und die Konzentration des Total-Hexosamins und des Hexosamins, entsprechend den Cetylpyridiniumchlorid fällbaren sauren Glykosaminoglykanen, wurde bestimmt. Außerdem wurden das Glykosaminoglykanmuster der Epiphysenplatte und der Einbau von³⁵S-Sulfat in die Glykosaminoglykane des Knochengewebes und des Epiphysenknorpels nach Zufuhr von³⁵S-Sulfatin vivo studiert.Im kompakten Knochengewebe macht des Hexosamin, entsprechend den sauren Glykosaminoglykanen, ungefähr ein Drittel der totalen Hexosaminkonzentration und ungefähr 0,05–0,06% des totalen Trockengewichtes aus. Der Hauptanteil der sauren Glykosaminoglykane im Knochen war Chondroitin-4-Sulfat. Dieses war in höherem Grad sulfatiert und hatte ein höheres Molekülgewicht als das Chondroitinsulfat der Epiphysenplatte, welches, in Übereinstimmung mit früheren Untersuchungen, Infrarot spektra kennzeichnend für sowohl Chondroitin-4-sulfat als auch für Chondroitin-6-sulfat, das Erstere überwiegend, hatte. Das Molekülgewicht des Hauptanteiles des Knochen-Chondroitinsulfates lag zwischen ungefähr 45000–56000. Ein kleiner Teil der Knochen-Glykosaminoglykane war Hyaluronsäure.Sowohl im Knochengewebe als auch im Epiphysenknorpel wurde in verschiedenen Fraktionen des Chondroitinsulfates, mit unterschiedlichem Molekülgewicht, grße und regelmäßig reproduzierbare Unterschiede in der spezifischen Aktivität gefunden.Behandlung der Hunde mit Parathyreoideaextrakt gab keinen Ausschlag in den Molekülgewichten des Chondroitinsulfates, weder des Knochens noch des Epiphysenknorpels. Ebenso wurde kein eindeutiger Effekt auf die Konzentration des totalen Hexosamins oder der sauren Glykosaminoglykane gefunden. Kein offenbarer, weder anregender noch senkender, Effekt auf den Einbau von³⁵S-Sulfat in das Chondroitinsulfat oder in der molekularen Verteilung der neulich sulfatierten und/oder synthetisierten Moleküle des Chondroitinsulfates dieser Gewebe wurde gefunden.

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Résumé Les glycosaminoglycanes de l'os compact ont été identifiés, par la méthode de précipitation au chlorure de cetylpyridinium, chez le chien normal et des chiens soumis à 20 ou 30 U.S.P. d'extrait parathyroïdien pendant 5 et 3–4 jours. Les concentrations d'héxosamines totales ainsi que les héxosamines, en rapport avec les glycoaminoglycanes acides, précipités par chlorure de cetylpyridinium, sont déterminées. En outre, la répartition du glycosaminoglycane de la zone d'ossification épiphysaire ainsi que l'incorporation du³⁵S-sulfate dans les glycosaminoglycanes du tissu osseux et du cartilage épiphysaire, après administration de³⁵S-sulfatein vivo, ont été étudiées.Dans l'os compact, les héxosamines, en rapport avec les glycosaminoglycances acides, constitutent environ un tiers de la concentration totale en héxosamine et environ 0,05–0,06% du poids sec total. Le constituant principal des glycosaminoglycances acides osseux est formé par le chondroitine-4-sulfate. Ce dernier est plus riche en sulfate et présente un poids moléculaire plus élevé que le chondroitine sulfate du cartilage épiphysaire, qui, selon des travaux antérieurs, présente des caractéristiques infra-rouges du chondroitine-4-sulfate et du chondroitine-6-sulfate, avec prédominance du premier. Les poids moléculaires du chondroitine sulfate osseux varient surtout d'environ 45000 et 56000. L'acide hyaluronique constitute une faible fraction des glycosaminoglycanes osseux.Des différences marquées de l'activité spécifique des fractions de cohondroitine sulfate de l'os et du cartilage épiphysaire, à poids moléculaires variables, on tété notées de façon répétée. L'administration d'extrait parathyroïdien à des chiens n'a pas d'effet sur les poids moléculaires en chondroitine sulfate de l'os ou du cartilage épiphysaire. Elle n'influence pas non plus les concentrations en héxosamines totales ou en glycosaminoglycances acides. Dans ces tissus, il ne se produit pas d'effect de stimulation ou de dépression concernant l'incorporation de³⁵S-sulfate dans le chondroitine sulfate our sur la séparation moléculaire de molécules transformées en sulfates et/ou de molécules synthétiques de sulfate chondroitine.

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The nomenclature ofBalasz andJeanloz (1965) has been followed.This work was reported, in part, at the meeting of Svenska Patologföreningen in Stockholm, Sweden, December 26th–27th, 1965; see Nordisk Medicin 75, 434–435 (1965); and at the Fourth European Symposium on Calcified Tissues, Leiden, Noordwijk aan Zee, The Netherlands, March 28th–April 1st. 1966; see Exerpta Medica Foundation, International Congress Series No 120, 1966, p. 55–56.     

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

The large cartilage proteoglycan, aggrecan, was found to vary throughout the ovine physis corresponding to the maturational state of the resident chondrocytes. Two populations of proteoglycan monomer were observed in articular, epiphyseal, and in the resting zone of growth plate cartilage. These proteoglycans contained chondroitin sulfate glycosaminoglycan chains sulfated predominantly in the 4 position along with lesser amounts of chondroitin-6-sulfate and keratan sulfate. In the proliferative zone of the growth plate, chondrocytes synthesize one population of proteoglycan monomer which was significantly larger than monomer populations in articular, epiphyseal, or resting zone and this size increase could be attributed to an increase in its constituent chondroitin sulfate side chains. As these chondrocytes progress through their life cycle they continue to modify the structural characteristics of the aggrecan molecule they synthesize. Thus, in the hypertrophic region of the growth plate, the proteoglycan monomer is larger again than in the proliferative region. Variation in sulfation pattern on aggrecan chondroitin sulfate side chains is also observed in the hypertrophic region with an increasing proportion of unsulfated residues present, which may play a role in the initiation of mineralization. In addition, increasing amounts of the carbohydrate sequence recognized by monoclonal antibody 7-D-4 are observed in the hypertrophic zone. Received: 7 July, 1995 / Accepted: 17 June 1996