Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.     

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.     

The influence of high ambient glucose level on the production of pericellular glycosaminoglycans by cultured endothelial cells

The 14C-acetate metabolic labeling of glycosaminoglycans (GAGs) was used to investigate the effect of high glucose level on the production of hyaluronic acid (HA), heparan sulphate (HS), chondroitin sulphate (CS) and dermatan sulphate (DS) by human immortalized umbilical vein endothelial cells. It is demonstrated that 30 mM glucose decreased the accumulation of HS and increased the accumulation of CS and DS in the cell layer, pericellular matrix and conditioned medium in 48 h of incubation. The modulation of the overall metabolism of sulphated GAGs by high glucose is in contrast to the observed redistribution of HA from the conditioned medium to the pericellular matrix of endothelial cells. The preincubation at 30 mM glucose increased also the attachment of hyaluronidase-treated endothelial cells to HA-coated surface and had no effect on the cell attachment to poly-D-lysine, indicating the alterations of CD44 binding to immobilized HA. The treatment of endothelial cells with p-nitrophenyl-beta-D-xylopyranoside, which inhibits the coupling of CS to the core protein, attenuated high glucose-induced pericellular HA accumulation and decreased cell attachment to HA-coated surface. It is supposed the implication of CD44-related CS in the accumulation of pericellular HA by endothelial cells exposed to high glucose level.     

Erythrocytes express chondroitin sulphate/dermatan sulphate, which undergoes quantitative changes during diabetes and mediate erythrocyte adhesion to extracellular matrix components

Glycosaminoglycans (GAGs) such as chondroitin sulphate/dermatan sulphate (CS/DS) are complex molecules that are widely expressed on the cell membrane and extracellular matrix (ECM). They play an important role in wide range of biological activities especially during pathological conditions. Diabetes, a metabolic disorder characterized by sustained hyperglycemia, is known to affect GAGs in different tissues and affect erythrocyte adhesion. The present investigation was aimed at exploring the nature of GAGs present in erythrocytes and its role on adhesion of erythrocytes from control and diabetic rats to major extracellular matrix components. GAGs isolated from erythrocytes were demonstrated to be CS/DS and a 2-fold increase was observed in erythrocytes from diabetic rats. Disaccharide composition analysis by HPLC after depolymerization by the enzyme, chondroitinase ABC showed the presence of 4-O sulphated disaccharide units with small amounts of non-sulphated disaccharides, in both control and diabetic erythrocytes. Erythrocytes from diabetic rats, however, showed significantly increased binding to poly-l-ornithine (P-orn), type IV collagen, laminin and fibronectin, which was abrogated on treatment with chondroitinase ABC to various degrees. This study sheds new light on CS/DS in erythrocytes and its likely biological implications in vivo.     

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

Glycosaminoglycans (GAGs) and their changes in early corneal development of Bufo raddei Strauch (from stage 16, neural tube, to stage 25, operculum completely closed) were studied with electron microscopic cytochemical method. Results show that synthesis of GAGs changes from non-sulfated to sulfated, and its content increased gradually with the development of cornea. Hyaluronic acid (HA) in each part of cornea begins to increase gradually from stage 16 to 21 (mouth open stage), with its peak at stage 20 (gill circulation stage) to 21, then decreases. In the mean time, contents of dermatam sulfate (DS), chondroitin sulfate (CS), heparan sulfate (HS) and heparin (Hep) increase gradually. It is considered that HA, HS and collagen may be related to the migration of mesenchymal cells, and HA promotes the expansion and hydration of corneal stroma; sulfated GAGs are correlated with dehydration of cornea, cell density and corneal transparency; DS, CS, HS and Hep deposited among collagen fibrils could adjust their arrangement. All these changes would enhance transparency of cornea.     

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.     

Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

Summary The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S, still exist in mineralized cartilage matrix and lamina limitants.     

Identification, quantification and fine structural characterization of glycosaminoglycans from uterine leiomyoma and normal myometrium

The type, amount and fine chemical composition of glycosaminoglycans (GAGs) present both in human normal myometrium and uterine leiomyoma have been studied. GAGs were fractionated by ion-exchange chromatography on DEAE-Sephacel, isolated by gel-permeation chromatography on Sepharose CL-6B and characterized using electrophoresis in cellulose acetate membranes, specific enzymic treatments and analysis by high-performance capillary electrophoresis (HPCE). No statistical intrabatch differences in total GAG content in both tissues were identified, whereas significant interbatch differences between normal myometrium and uterine leiomyoma were recorded. Hyaluronan (HA), chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate (HS) and keratan sulphate (KS) were identified in both tissues. Statistically significant (P 相似文献   

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

本文用钌红显示氨基多糖的电镜细胞化学方法,较细致地研究了花背蟾蜍(Bufo raddeiStrauch)胚胎发育过程中(16—25期,即神经管至鳃盖完全封闭期)氨基多糖(GAG)在角膜早期发育中的变化。结果表明:GAG的合成由非硫酸化向硫酸化转变,且随着角膜的发育其含量逐渐增加。角膜各部位的HA在16—21期(胚胎开口期),其含量逐渐增加,且20(鳃血循环期)-21期达最高水平,此后含量下降,同时DS、CS、HS和Hep的含量上升。据分析,HA、HS和胶原与间充质细胞迁移有关,HA还可促使基质的膨胀和水化。硫酸化GAG在角膜的脱水、致密、细胞密度及角膜透明中起作用。胶原纤维间的硫酸化GAG能调节胶原纤维的规则化,故也有助于角膜的透明。     

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.     

Glycosaminoglycans from bovine eye vitreous humour and interaction with collagen type II

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (K _D) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity.     

In vitro control of neuronal polarity by glycosaminoglycans.

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondro?tin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.     

Structure and proteoglycan composition of specialized regions of the elastic tendon of the chicken wing

The elastic tendon of the avian wing has been described by others as a unique structure with elastic properties due to the predominance of elastic fibers in the midsubstance. Further analyses of the tendon have shown it to possess five anatomically distinct regions. Besides the major elastic region, a distally located fibrocartilage and three tendinous regions are present. The tendinous regions connect: (1) the muscle to the elastic region, (2) the elastic region to the fibrocartilage and (3) the latter to the insertion site. The elastic region possesses thick and abundant elastic fibers and very thin, interconnecting collagen fibers. The collagen fibers in the sesamoid fibrocartilage are thick and interwoven, defining spaces occupied by fibrochondrocytes embedded in a non-fibrillar and highly metachromatic matrix. Biochemical analyses have shown that the fibrocartilage has about tenfold the amount of glycosaminoglycans (GAGs) found in the other regions. The main GAG in this region was chondroitin sulfate (CS) (plus keratan sulfate as detected immunocytochemically), while the other regions showed variable amounts of CS, dermatan sulfate (DS) and heparan sulfate. Further analyses have shown that a large CS-bearing proteoglycan is found in the fibrocartilage. The elastic region possesses two main proteoglycans, a large CS-bearing proteoglycan (which reacted with an antibody against keratan sulfate after chondroitinase ABC treatment) and a predominant DS-bearing proteoglycan, which showed immunoreactivity when assayed with an anti-biglycan antibody. The results demonstrate that the elastic tendon is a complex structure with complex regional structural and compositional adaptations, suited to different biomechanical roles.     

Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma

Glycosaminoglycans (GAGs) in proteoglycan (PG) forms or as free GAGs are implicated in the growth and progression of malignant tumors. These macromolecules were investigated in human gastric carcinoma (HGC) and compared with those in human normal gastric mucosa (HNG). We report that HGC contained about 2-fold increased amounts of GAGs in comparison to HNG. Specifically, HGC showed 3- and 2.5-fold net increase in chondroitin sulphate (CS) and hyaluronan (HA) contents, respectively. Dermatan sulphate (DS) was slightly increased, but the amount of heparan sulphate (HS) was decreased. Of particular, interest were the quite different sulphation profiles of CS and DS chains in HGC in which, non-sulphated and 6-sulphated disaccharide units were increased 10 and 4 times, respectively, in comparison to HNG. On PG level, three different populations were identified in both HNG and HGC, being HSPGs, versican (CS/DS chains) and decorin (CS/DS chains). In HGC, the amounts of versican and decorin were significantly increased about 3- and 8-fold, respectively. These PGs were also characterized by marked decrease in hydrodynamic size and GAG content per PG molecule. Analysis of Delta-disaccharide of versican and decorin from HGC showed an increase of 6-sulphated Delta-disaccharides (Delta di-6S) and non-sulphated Delta-disaccharides (Delta di-0S) with a parallel decrease of 4-sulphated Delta-disaccharides (Delta di-4S) as compared to HNG, which closely correlated with the increase of CS content. In addition, the accumulation of core proteins of versican and decorin in HGC was also associated with many post-translational modifications, referring to the number, size, degree and patterns of sulphation and epimerization of CS/DS chains. Studies on the modified metabolism of PGs/GAGs are under progress and will help in deeper understanding of the environment in which tumor cells proliferate and invade.     

The relationship of mechanical properties to morphology in patellar tendon autografts after posterior cruciate ligament replacement in sheep.

In a sheep model the posterior cruciate ligament (PCL) was replaced by a patellar tendon autograft (PTAG) using the central one-third of the ipsilateral patellar tendon (PT). The sheep were sacrificed at 16, 26, 52 and 104 weeks postoperation. The PTAG, and, as controls, the contralateral PCL and PT were harvested. These were examined using biomechanical testing as well as light and transmission electron microscopy, including immunohistological techniques. The material properties (maximum stress, elastic modulus) were compared to the morphological features. The cellular distribution, the distribution of glycosaminoglycans (GAGs), the collagen fibril diameter and the occurrence of Type III collagen were studied. Prior to transplantation, the PTAG was shown to be superior in maximum stress (57.2 +/- 5.5 MPa vs 41.3 +/- 1.9 MPa) and elastic modulus (368.8 +/- 49.3 MPa vs 172.3 +/- 14.6 MPa) to the PCL. The early decline in material properties of the PTAG (maximum stress 22% and elastic modulus 42% of the control) after free grafting paralleled a cell- and capillary-rich PTAG tissue with remnants of necrosis and a poorly organized extracellular matrix. Two years after implantation, with progressive alignment of the tissue matrix, maximum stress and elastic modulus acquired approximately 60 and 70% of the control, respectively. However, there was also an evidence of degenerative changes characterized by acellular areas, loss of the normal bundling pattern of collagen fibers and abnormal accumulation of GAGs. Ultrastructurally, there was a predominant shift to thin collagen fibrils in the PTAG compared to PCL and PT, both consisting of thick and thin collagen fibrils. Thin fibrils were demonstrated to be, in part, split thick fibrils as well as newly formed fibrils. Most of these thin fibrils revealed a positive reaction with antibodies to Type III collagen.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells

Chondroitin/dermatan sulphate (CS/DS) sulphation motifs on cell and extracellular matrix proteoglycans (PGs) within stem/progenitor cell niches are involved in modulating cell phenotype during the development of many musculoskeletal connective tissues. Here, we investigate the importance of CS/DS chains and their motifs in the chondrogenic differentiation of bone marrow mesenchymal stem cells (bMSCs), using p-nitrophenyl xyloside (PNPX) as a competitive acceptor of CS/DS substitution on PGs. Comparison of cultures grown in control chondrogenic medium, with those grown in the presence of PNPX showed that PNPX delayed the onset of chondrogenesis, characterised by cell rounding and aggregation into spheroidal beads. PNPX reduced gene expression of SOX-9, aggrecan and collagen type II, and caused reduced levels of collagen type II protein. PNPX-treated cultures also showed delayed expression of a native CS/DS sulphation motif epitope recognised by antibody 6C3. This epitope appeared associated with a range of PGs, particularly biglycan, and its close association was lost after PNPX treatment. Overall our data show that perturbation of PG glycosylation with CS/DS GAGs using PNPX significantly delays the onset of chondrogenic differentiation of bMSCs, highlighting the importance of CS/DS during the initial stages of chondrogenesis. The delayed expression of the CS/DS sulphation motif recognised by 6C3 suggests that this motif, in particular, may have early involvement in chondrogenesis. The mechanism(s) by which CS/DS chains on PGs contribute to early chondrogenic events is unknown; however, they may be involved in morphogenetic signalling through the capture and cellular presentation of soluble bioactive molecules (e.g. growth factors).     

Ultrastructure of type VI collagen in human skin and cartilage suggests an anchoring function for this filamentous network

An mAb was used in conjunction with immunoelectron microscopy to study the ultrastructure and distribution of the type VI collagen network. Type VI collagen in femoral head and costal cartilage was found distributed throughout the matrix but concentrated in areas surrounding chondrocytes. Three-dimensional information gained from high voltage stereo pair electron microscopy showed that the type VI collagen network in skin was organized into a highly branched, open, filamentous network that encircled interstitial collagen fibers, but did not appear to interact directly with them. Type VI collagen was also found concentrated near basement membranes of nerves, blood vessels, and fat cells although in a less organized state. Labeling was conspicuously reduced close to the epithelial basement membrane in the region of the anchoring fibrils. No labeling of basement membranes was seen. Based on these observations it is suggested that the type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into surrounding connective tissues.     

Characterization of calcium accumulation in the brain of rats administered orally calcium: The significance of energy-dependent mechanism

Primary cultures of rat hepatocytes maintained on different matrix proteins such as collagen (Co IV) fibronectin (Fn), Laminin (Ln) or different tissue biomatrices were metabolically labelled with ³⁵[S]-SO₄ and the synthesis of sulphated proteoglycans was studied. The incorporation of the label into total glycosaminoglycan (GAG) was significantly higher in cells maintained on Co IV compared to those maintained on Fn or Ln. Similarly the incorporation of label was maximum in those cells maintained on the aortic biomatrix compared to liver or mammary gland biomatrix. About 80–95% of the GAG synthesised and secreted by cells maintained on individual matrix proteins and liver biomatrix was heparan sulphate (HS). But in the case of cells maintained on collagen IV aortic or mammary biomatrix in addition to HS, significant amount of chondroitin sulphate (CS) was also found. Nearly 50% of the total ³⁵[S]-GAG was associated with the cell layer after 24 h in culture in the case of cells maintained on individual matrix protein while those maintained on tissue biomatrix, retained about 70% of the ³⁵[S]-labelled proteoglycans (PG) with the cell layer. Analysis of the cell surface ³⁵[S]-labelled proteoglycans isolated from cells maintained on different biomatrix showed that it is a hybrid proteoglycan consisting of CS and HS. While the PG isolated from cells maintained on liver biomatrix consists of HS and CS in the ratio of 3:2 that from cells maintained on aorta or mammary gland matrix was about 2:3 indicating an alteration in the nature of the cell surface PGs produced by cells maintained on different tissue biomatrix. These results indicate that depending on the nature of the matrix substratum with which the cells are in contact, the nature and quantity of sulphated proteoglycans produced by hepatocytes vary.     

Differentiation of the secondary elastic cartilage in the external ear of the rat.

The cartilage in the external ear of the rat belongs to the group of secondary cartilages and it has a unique structural organization. The chondrocytes are transformed into typical adipose cells, the proteoglycan cartilage matrix is reduced to thin capsules around the cells and the rest of the extracellular matrix is occupied by a network of coarse elastic fibers. It appears late in development (16-day fetus) and needs more than one month for final development. The differentiation proceeds in several steps which partly overlap: the appearance of collagen fibrils, elastin fibers, the proteoglycan matrix, and the adipose transformation of chondrocytes. The phenotype of this cartilage and the course of its differentiation are very stable, even in very atypical experimental environmental conditions. The only exceptions are explants in organ culture in vitro and perichondrial regenerates. In these conditions the development of elastic fibers is slow and poor while the production of the proteoglycan matrix is abundant. The resulting cartilage then displays structural characteristics of hyaline cartilage rather than those of the initial elastic one.