Total Amount Of Glycosaminoglycans Research Articles

Long term cyclic compression of articular cartilage alters extracellular matrix turnover

Purpose: Osteoarthritis (OA) is a disease characterized by progressive degradation of articular cartilage extracellular matrix (ECM), Mechanical factors play a large role in the initiation and progression of OA, where mechanical compression at physiologic levels of cartilage is shown to have chondroprotective effects, activating anabolic processes and stimulating matrix synthesis. Previous studies suggest that mechanical loading of cartilage increases aggrecan and type II collagen gene expression, and reduce matrix metalloprotease formation. Biomarkers specifically targeting aggrecan and type II collagen turnover, provides a unique opportunity to study the dynamic effects of mechanical loading on cartilage ECM turnover. A better understanding of these processes might aid the development of novel DMOADs. The aim of this study was to investigate the effects of long-term cyclic compression on the cartilage ECM turnover, through measurement of specific cartilage turnover biomarkers, using a bovine articular cartilage ex vivo model. Methods: Full depth bovine cartilage explants (BEX) were cultured for 2 weeks. The explants were treated 3 times a week with either OSM [10 ng/mL] and TNF-a [2 ng/mL] (O+T), or TGF-B1 [50 ng/mL]. Untreated samples were included as negative controls (w/o). For each condition two groups were established; an unloaded group and a group compressed 3 times a week. Compression was applied using Electroforce 5500® (TA Instruments), in a sine wave with a maximum load per cycle of 1 MPa, at 1 Hz frequency for 1200 cycles. Metabolic activity was measured once a week using AlamarBlue. Biomarkers released to the supernatant were assessed using the following well-described ELISAs: type II collagen was measured by ProC2, MMP-degradation of type II collagen was measured by C2M, and aggrecanase-mediated degradation of aggrecan was measured by AGNx1. Sulfated glycosaminoglycans (GAGs) released to media was measured using a 1.9-dimethylmethylene blue (DMB) assay. Results: Compression of BEX significantly decreased the MMP-degradation of type II collagen measured by C2M at day 14 with 48% and 42% compared to the unloaded samples in the untreated and the O-T treated groups respectively (Figure 1c). The compressed samples release similar levels of AGNx1 and ProC2 to the unloaded explants (Figure 1b+d). The metabolic activity similarly appears to be unaltered by compression (Figure 1a). Compression significantly increased the GAG release in the w/o group by 195% and by 152% in the TGF-B group. Conclusions: Compression of cartilage, and compared to standard cultures, resulted in significant effect on cartilage tissue turnover and metabolic profile. Compression reduced the degradation of type II collagen, which indicates an inhibition of the involved catabolic mechanisms. Furthermore, type II collagen formation was unaltered, which in combination with the reduced degradation may result in an elevated deposition of collagen in the explants. While the AGNx1 release was unchanged by compression, the total amount of GAG was increased in the supernatants, suggesting an increase in proteoglycan turnover in the explants. In conclusion, compression of cartilage resulted in altered cartilage turnover, which was quantified by molecular cartilage turnover biomarkers. This approach may be essential for translation science, selecting and validating the best approaches to regenerate cartilage or block degradation.

Glycosaminoglycanomic profiling of human milk in different stages of lactation by liquid chromatography-tandem mass spectrometry

Glycans in human milk serve several important biological functions that promote infant health. As kind of important glycans, glycosaminoglycans (GAGs) are a complex family of polyanionic carbohydrate, participating in a variety of critical physiological and pathological processes. In this study, the content and the detailed composition of human milk GAGs from Chinese mothers in different stages of lactation, based on a liquid chromatography-tandem mass spectrometry approach was investigated. The results showed that the GAG fraction in the human milk samples was very complex as it was composed of heparan sulfate, chondroitin sulfate, and hyaluronic acid. With lactation extending, the total amount of GAGs in human milk decreased. This study provided an important guide for the demands of GAGs during different stages of lactation. The results were also beneficial for studies on the composition and functional properties of infant formula.

Anti-inflammation response of astaxanthin on inhibiting nuclear factor kappa B activation

minimize the ‘‘variations’’ during manufacturing. Glycosaminoglycans (GAGs) and proteoglycans are one of the major components of extracellular matrix including basement membrane in oral mucosa and havemany biological functions. Thus, they play an important role in homeostasis of the epithelium. Recently, CXylopyranoside derivatives is reported to increase GAGs and PGs biosynthesis and improve tissue morphogenesis in a skin organotypic culture model. Purpose: We hypothesized that one of the C-Xylopyranoside derivatives b-D-xylopyranoside-n-propane-2-one (XPP) stimulates the ability to produce basement membrane related molecules such as basement membrane components and integrins in oral mucosa keratinocytes and fibroblasts, resulting in improvement of epithelial structure. The present study aimed to assess the effects of XPP on the expression level of GAGs and basement membrane related molecules produced by oral keratinocytes and fibroblasts, and the differences of histological appearance in a reconstructed oral mucosa model. Methods: Primary oral keratinocytes and fibroblasts were isolated and serially cultured in EpiLife culture medium containing 0.06mMCa (growthmedium) and Dulbecco’s Modified Eagle Medium containing 10% serum (DMEM), respectively. First, we tested the cell viability of oral keratinocytes cultured in growth medium and EpiLife containing 1.2mMCa(hereafter differentiationmedium) as well as oral fibroblasts cultured in differentiation medium and DMEM supplemented with 0, 2 and 10mM XPP for up to 48 hours using a Cell-Counting Kit. Next, total amount of GAGs produced by oral keratinocytes and fibroblasts into the culture media was quantitated using Blyscan GAG assay. Concurrently, oral keratinocytes and fibroblasts were lysed and immunoblotting was performed to detect the expression levels of basement membrane related molecules. Using an organotypic culture system, three-dimensional oralmucosamodels (3DOMM) inwhich oral keratinocytes and fibroblasts were incorporated were fixed, embedded in paraffin, and then histologically and immunohistochemically examined. Results: The concentration of 2 and 10mM XPP did not affect cell viability of oral keratinocytes and fibroblasts grown in those culture media. Blyscan assay showed the amount of GAGs secreted from both cells into those culture media significantly increased when XPP was added, particularly from keratinocytes cultured in differentiation medium. The expression level of type zW collagen, Nidogen, Integrin a6 and b1 in oral keratinocytes increased when cultured in growth medium. While the histological examinations did not showed significant differences between the XPP-treated and untreated 3DOMMs, more intense expression of laminin and type zW collagen was seen in the XPP-treated one. Moreover, basal layer cells of the XPP treated 3DOMM strongly expressed integrin a6 and b1, ligands of laminin and type zW collagen, compared with untreated 3DOMM.

Wound Healing Effects of a Lipocalin-Derived Peptide

The role of lipocalins in wound healing is currently unknown, although the involvement of these proteins in injury response has been reported. This study aimed to investigate the effects of a peptide comprising the sequence of a previously described lipocalin conserved motif (pM2b), in an experimental model of skin lesion. Circular full-thickness wounds in the skin of rat dorsum were treated with pM2b or saline as control, and allowed to heal, keeping the wound occluded and moist. During wound healing, the following parameters were evaluated in the regenerating tissue: wound closure, collagen content, glycosaminoglycans (GAGs) and metalloproteinase (MMP) activity. In addition, tissue sections were subjected to histological analysis. Treatment with pM2b promoted an overall improvement in wound healing and tissue repair, with distinctly marked signals in the early stages of wound healing, such as the presence of histiocytes, fibroblasts and thick collagen bundles, as well as early reepithelization in the recovering tissue. In the latter stages, pM2b-treated wounds showed a better resolution of wound healing, with evidence of regeneration and reduced scars. The regenerating tissue showed collagen increase, no significant changes in the total amount of GAGs, and increased MMP-2 activity, in comparison with control lesions. The results suggest that lipocalins which share the sequence motif related to pM2b can play a role in wound healing. The lipocalin-derived peptide can serve as a tool to develop new pharmaceuticals and formulations to aid wound healing.

Fine structural characterization of chondroitin sulfate in urine of bladder pain syndrome subjects

Urothelial glycosaminoglycans (GAGs) are decreased in bladder pain syndrome (BPS), and urinary GAGs are thought to reflect this deficiency. In previous researches, urine GAG levels were found increased, decreased, or similar between BPS and controls. Additionally, no study is available on the structure characterization of urinary chondroitin sulfate (CS) in BPS patients. CS in the urine of BPS-affected patients and controls has been determined by specific electrophoresis, along with total GAGs and heparan sulfate (HS) percentage, and CS disaccharides have been quantified by high-performance liquid chromatography. No significant differences were obtained for total amount of GAGs, absolute content of CS and HS, and their relative percentages. Moreover, no differences were observed for CS structure confirming similar urine CS composition in BPS subjects and controls. This study found no significant differences of BPS and control urine GAG levels and CS structure to allow use of these parameters as diagnostic markers for BPS diagnosis.

Glycosaminoglycans in human normal lung tissues and lung cancer tissues

The quantitative changes of glycosaminoglycans in tumor tissue of human lung cancers (6 squamous cell carcinomas, 7 small cell carcinomas and 10 adenocarcinomas) were studied. Normal lung tissues contained of 3.38 mumol uronic acid/g dry weight glycosaminoglycans which consisted of hyaluronic acid, chondroitin sulfates, dermatan sulfate and heparan sulfate. The total amount of glycosaminoglycans in human lung cancer tissues increased 1.7 to 3.5 times in comparison with that in normal lung tissues. The increase in tissue content of glycosaminoglycans was accompanied by an increase in the chondroitin sulfate level in every histologic type of lung cancer, as well as marked increase in hyaluronic acid level in squamous cell carcinomas, and a moderate increase in small cell carcinomas. The concentrations of dermatan sulfate and heparan sulfate in lung cancer tissues did not show any significant changes compared with those in normal lung tissues. The increase in total amount and changes in the composition of glycosaminoglycans in human lung cancer tissue were closely related to the histologic type of the tumor. In adenocarcinomas, some acid glycoprotein with sialic acid was simultaneously detected during the separating course of glycosaminoglycans, which was considered to be derived from mucinous substances related to adenocarcinoma cells.

Structural characterization of the skin glycosaminoglycans in patients with pseudoxanthoma elasticum

Complex polysaccharides, glycosaminoglycans (GAGs), their amount, and fine structure were determined in the skin (epidermis + dermis) of pseudoxanthoma elasticum (PXE)-affected patients in comparison with healthy subjects. Nonlesional skin GAGs were extracted and specifically determined by enzymatic treatment and high-performance liquid chromatography separation. Dermatan sulfate (DS) and hyaluronic acid (HA) were found to be the major GAG species in normal subjects, with contents of approximately 20% for DS and 58% for HA. The chondroitin sulfate (CS) content (unsaturated six-sulfated disaccharide) was approximately 21%. Skin from patients with PXE showed similar HA (61%), DS (22%), and CS (16.7%) contents. No change in the total charge density or nonsulfated/sulfated GAG ratio was noted in PXE-affected subjects, and no modification of the position of the sulfate groups (4s/6s) on the CS/DS backbone. A significant increase (approximately 88%; P < 0.01) in the total amount of GAGs (HA + DS + CS) was found in the PXE group vs. normal subjects, however. In the skin of PXE-affected patients, the altered metabolic processes produce an increase in the total amount of GAGs able to accumulate salts, in particular calcium ions, within the elastic fibers, producing ion precipitates that affect the organization of the matrix fiber.

Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease

The levels and fine structure of complex polysaccharides, glycosaminoglycans (GAGs), were determined in segments of the posterior mitral valve leaflet (MVL) taken from 15 patients affected by mitral regurgitation and degenerative disease and were compared with segments from 15 multiorgan donors. MVL GAGs were analyzed by agarose gel electrophoresis, and by HPLC and fluorophore-assisted carbohydrate electrophoresis to evaluate disaccharide patterns after treatment with chondroitinase ABC. GAGs from the control group were composed of approximately 37% hyaluronic acid and 63% chondroitin sulfate/dermatan sulfate with a charge density of approximately 0.61. Chondroitin sulfate/dermatan sulfate polymers contained approximately 23% of the disaccharide sulfated in position 6 on N-acetyl-galactosamine, approximately 38% of the 4-sulfated disaccharide and approximately 2% of the non-sulfated disaccharide (with a 4-sulfated/6-sulfated ratio of 1.7). The total amount of GAGs was 0.66 microg/mg tissue. The total amount of GAGs in patients suffering from mitral regurgitation and degenerative disease was approximately 51.5% higher (although the difference was not significant, probably because of the low number of subjects enrolled in the study). However, significantly higher hyaluronic acid content (approx. +38%, p<0.05) and lower sulfated GAG content (approx. -21%, p<0.005) were demonstrated. As a consequence, the total charge density decreased by approximately 23% (p<0.005). This macro-modification of GAG composition was also followed by a micro-alteration of the structure of the sulfated polysaccharides, in particular with a significant decrease in the 4-sulfated disaccharide (and a parallel increase in hyaluronic acid content) with no modification of the percentage of the 6-sulfated and non-sulfated disaccharides (with a significant decrease in the 4-/6-sulfated ratio). We assume that changes in the relative amount and distribution of GAGs in posterior MVL in subjects suffering from mitral regurgitation and degenerative disease are consistent with a decrease in the tension to which these tissues are subjected and with an abnormal matrix microstructure capable of influencing the hydration and of conditioning the mechanical weakness of these pathological tissues.

A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis

Crucial events in myogenesis rely on the highly regulated spatiotemporal distribution of cell surface heparan sulfate proteoglycans to which are associated growth factors, thus creating a specific microenvironment around muscle cells. Most growth factors involved in control of myoblast growth and differentiation are stored in the extracellular matrix through interaction with specific sequences of glycosaminoglycan oligosaccharides, mainly heparan sulfate (HS). Different HS subspecies revealed by specific antibodies, have been shown to provide spatiotemporal regulation during muscle development. We have previously shown that glycosaminoglycan (GAG) mimetics called RGTA (ReGeneraTing Agent), stimulate muscle precursor cell growth and differentiation. These data suggest an important role of GAGs during myogenesis; however, little is yet known about the different species of GAGs synthesized during myogenesis and their metabolic regulation. We therefore quantified GAGs during myogenesis of C2.7 cells and show that the composition of GAG species was modified during myogenic differentiation. In particular, HS levels were increased during this process. In addition, the GAG mimetic RGTA, which stimulated both growth and differentiation of C2.7 cells, increased the total amount of GAG produced by these cells without significantly altering their rate of sulfation. RGTA treatment further enhanced HS levels and changed its sub-species composition. Although mRNA levels of the enzymes involved in HS biosynthesis were almost unchanged during myogenic differentiation, heparanase mRNA levels decreased. RGTA did not markedly alter these levels. Here we show that the effects of RGTA on myoblast growth and differentiation are in part mediated through an alteration of GAG species and provide an important insight into the role of these molecules in normal or pathologic myogenic processes.

Isolation, Identification, and Quantitation of Urinary Glycosaminoglycans

Background: Substantial amounts of glycosaminoglycans (GAGs) are present in the urine of healthy individuals, but the concentration in the serum is very low. This finding suggests that urinary GAGs come from the glomerulus and may reflect the turnover of GAGs in the glomerulus. Hypothesis: However, little is known about the physiologic regulation of the urinary GAGs in humans, and so investigations are needed to evaluate the effects of age and sex on urinary GAGs in normal individuals. Methods: Eighty-seven healthy subjects were included in this study. Urinary GAGs were isolated and quantified at the nanogram level by combined azure A-silver staining in agarose gels. Results: The level of urinary GAGs peaked at 10–19 years in both sexes. The proportion of chondroitin sulfate decreased with age, but the proportion of heparan sulfate increased with age. Conclusion: The total amount of GAGs and the proportions of chondroitin sulfate, heparan sulfate, and dermatan sulfate appear to change with age. Therefore, investigations in which urinary GAG is used as a parameter of glomerular GAG turnover should ensure that control groups are precisely matched for age. Changes in the proportions of each GAG may be more informative than their absolute levels.

Sulfated glycosaminoglycans and collagen in two bovine muscles (M. Semitendinosus and M. Psoas major) differing in texture.

M. semitendinosus (ST) and M. psoas major (PM) were used as models for tough and tender meat to study a possible role of sulfated glycosaminoglycans (GAGs) for muscle tenderness. The difference in texture was confirmed by Warner Bratzler shear force measurements. No significant difference in total amount of GAGs in the muscles was found. In contrast, a significant difference in the ratio of GAG/collagen was found between the two muscles. After separation of the GAGs by density gradient ultracentrifugation and ion-exchange chromatography, dermatan sulfate (DS), keratan sulfate (KS), chondroitin sulfate (CS), and heparan sulfate (HS) were identified by cellulose acetate electrophoresis after use of specific enzymes and chemical methods. The content of DS was higher in the tougher muscle (ST) than in PM, and the difference in DS content was statistically significant. Furthermore, a significant difference in the GAG composition pattern of the two muscles was found. The yield of GAGs extracted from the muscles was 77% for ST and 87% for PM. The residue after extraction was further analyzed and found to contain mainly HS. Immunohistochemical studies using antibodies against CS/DS showed a staining pattern of the perimysium of ST different from that of PM.

Glycosaminoglycan biosynthesis during 5-fluoro-2-deoxyuridine-induced palatal clefts in the rat

The biosynthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of palatal shelf-elevating force(s) in mammals, although the nature of the palatal shelf extracellular matrices during cleft palate formation remains poorly understood. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various periods of palatogenesis where clefts were induced experimentally using 5-fluoro-2-deoxyuridine (FUDR). For both normal and cleft palatal shelves, hyaluronan, heparan sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. Throughout the period of cleft development studied, the total amount of GAG was significantly decreased (by approx. 30%) compared with normal development, this decrease being particularly marked at a time equivalent to post-elevation during normal development (approx. 75%). Furthermore, and unlike normal palatogenesis, no significant differences were recorded between the anterior and posterior parts of the palatal shelves during cleft formation. As for normal palatogenesis, however, the percentages of each GAG were not altered at any stage. The findings are consistent with the view that suppression of GAG biosynthesis is related to the development of cleft palate in FUDR-treated rat fetuses and can therefore be interpreted as providing evidence of a role for the mesenchymal glycoconjugates in shelf elevation during normal palatogenesis.

Analysis of glycosaminoglycans in human prostate by high-performance liquid chromatography.

To investigate changes in glycosaminoglycan (GAG) profiles in human prostatic tissue. Seventeen human prostatic samples were examined; five were normal, six hyperplastic and six were cancerous. Prostatic proteoglycans were extracted with 4 mol/L guanidine-HCl containing protease inhibitors. After digestion of the freeze-dried proteoglycan extract with papain, the prostatic GAGs were purified. Total GAGs were measured by a modified dimethylmethylene blue (DMB) method. High-performance liquid chromatography (HPLC) was used to quantify the extracted GAGs. Six types of GAGs, i.e. chondroitin 4-sulphate (Ch-4S), chondroitin 6-sulphate (Ch-6S), dermatan sulphate (DS), chondroitin, heparan sulphate and hyaluronic acid, were analysed qualitatively and quantitatively. The total amount of GAGs was increased in hyperplastic and cancerous prostates, with the predominant components being DS and Ch-6S in these specimens. The Ch-S:DS ratio in cancerous prostate was significantly higher than that in normal and BPH tissue (P < 0.05). Moreover, chondroitin was increased in hyperplastic prostatic tissue (P < 0.01). These results suggest that an increase in chondroitin levels may be associated with hyperplastic change and an increase in the Ch-S:DS ratio may be related to the development of malignancy.

Identification of Glycosaminoglycans in Age-Related Macular Deposits

We investigated the presence and localization of glycosaminoglycans in basal laminar deposit and drusen in age-related maculopathy. Conventional histological staining techniques and monoclonal antibodies specific for several glycosaminoglycans were used on paraffin-embedded human maculae. Furthermore, macular homogenates were analyzed with two-dimensional electrophoresis. Quantitative analysis of glycosaminoglycans was done spectrophotometrically using dimethylmethylene blue. Immunohistochemically, all basal laminar deposit stained positive for chondroitin 4-sulfate and focally positive for heparan sulfate proteoglycan. Drusen were not stained with any of the monoclonal antibodies. With two-dimensional electrophoresis, it was demonstrated that macular extracts with and without age-related maculopathy contained chondroitin sulfate. Heparan sulfate was only expressed in maculae with age-related maculopathy. The total amount of glycosaminoglycans was significantly higher in maculae with basal laminar deposit than in maculae without basal laminar deposit (P = .001). There were significant differences in the amount and composition of glycosaminoglycans between maculae with and without age-related maculopathy.

Synthesis of stromal glycosaminoglycans in response to injury.

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14, 42, and 84 days. The tissue and/or discs were removed and labeled with 35S-sulfate for 18 h; GAGs were extracted with 4 M guanidine-HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS-PAGE. The nonbound fractions from the chromatography were assayed for TGF-beta using Western blot analysis and for hyaluronic acid using an 125I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-beta that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-beta. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling.

Changes in the composition of glycosaminoglycans during normal palatogenesis in the rat

The synthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of a palatal shelf-elevating force in mammals. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various stages of palatogenesis. Hyaluronan, heparan sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. The distribution of the GAG differed in the anterior and posterior regions (i.e. the presumptive hard and soft palates) and with the stage of development. At the time of palatal-shelf reorientation, there were no significant differences for either the total amount of GAG or for the percentages of specific GAG types between anterior and posterior regions. Indeed, the most marked differences were detected at the stages of histogenesis after shelf elevation. Nevertheless, the results support the view that hyaluronan is involved in palatal-shelf reorientation, its percentage being initially high and decreasing after shelf elevation. No changes were detected for the other (sulphated) GAG during the time of elevation. The findings point to the need to correlate the events during histogenesis with changes in the ground substance of palatal-shelf mesenchyme and indicate that there are different developmental mechanisms within the presumptive hard and soft palates.

Composition of cell‐polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.

Syndecan--a cell surface proteoglycan that selectively binds extracellular effector molecules.

Syndecan is a cell surface proteoglycan (Saunders et al., 1989), which was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains (Rapraeger et al., 1985). The ratio and total amount of GAG bound to syndecan is known to vary in different tissues (Sanderson & Bernfield, 1988; Salmivirta et al., 1991) and cells (Rapraeger 1989). Syndecan binds several extracellular matrix molecules such as fibrillar collagens of interstitial matrix (Koda et al., 1985), fibronectin (Saunders & Bernfield, 1988) and thrombospondin (Sun et al., 1989) but shows no binding to some other heparin binding matrix molecules, like vitronectin and laminin (Koda et al., 1985; Elenius et al., 1990). Syndecan also binds growth factors, like bFGF (Kiefer et al., 1990), and could therefore play a major role in the regulation of cell adhesion, growth and differentiation (for a review see Jalkanen et al., 1991).

Possible Genetic Defects in Regulation of Glycosaminoglycans in Patients With Diabetic Nephropathy

The hypothesis of genetic defects in glycosaminoglycan (GAG) regulation among patients with insulin-dependent diabetes mellitus (IDDM) and nephropathy was assessed by studies in tissue cultures of fibroblasts obtained from 7 patients with normal urinary albumin excretion, 11 patients with diabetic nephropathy, and 6 nondiabetic control subjects. The incorporation of [2H] glucosamine and [35S] sulfate into hyaluronic acid (HA), chondroitin sulfate and dermatan sulfate (CS + DS), and heparan sulfate (HS) was measured in cells, matrix, and medium and related to micrograms of tissue protein. Large interindividual variations were seen in all three groups, and the incorporation of [3H] glucosamine into HA, CS + DS, and HS and [35S] sulfate into CS + DS and HS were not significantly different between the three groups. However, the fractional incorporation of [3H]glucosamine into HS was significantly reduced in diabetic patients with nephropathy compared with control subjects. This was the case not only when related to the total amount of GAGs (P = 0.014) but also when related to HA (P = 0.014). No significant difference was seen between control subjects and normoalbuminuric diabetic patients. The degree of N-sulfation of HS was not significantly different between the experimental groups. The results suggest that patients with diabetic nephropathy may suffer from deficiencies of coordinate regulation in the biosynthesis of GAG in fibroblasts, which may lead to a reduced density of HS in the extracellular matrix. If these changes reflect alterations in the biosynthesis of GAG from endothelial, myomedial, and mesangial cells, this observation may be relevant for the pathogenesis of severe diabetic complications.

Glycosaminoglycan composition of anguilliform and elopiform leptocephali

Whole‐body glycosaminoglycan (GAG) composition of leptocephalous larvae was studied in eight eel (Anguilliformes) species, representing five different families (Congridae, Moringuidae, Muraenidae, Nettastomatidae and Ophichthidae) and the bonefish (Elopiformes: Albulidae: Albula vulpes). The extracted GAGs were identified by cellulose acetate electrophoresis, using standard GAGs as a reference, and by their susceptibility to GAG‐degrading enzymes (keratanase, chondroitinase ABC, chondroitinase AC and testicular hyaluronidase). The principal GAG in bonefish larvae was keratan sulphate. However, keratan sulphate was not the main GAG in any of the eel leptocephali studied, althoughit was present in small amounts in most species. The identities of the principal GAGs in eel leptocephali are still unknown, but the most likely possibilities include hyaluronic acid, chondroitin, and chrondroitin sulphate. In addition, a high degree of variability existed in the total amount of GAG that could be extracted from the different eel species and in the electrophoretic migration patterns of the extracted GAGs. Although a basic similarity is thought to exist in the body plan and developmental strategy of all leptocephali, the present results indicate that the GAG composition of the gelatinous body matrix is variable.