The effect of low sulfate concentrations on the glycosaminoglycan synthesis in anatomically intact articular cartilage of the mouse

We have studied the effect of environmental sulfate concentration on the glycosaminoglycan synthesis of anatomically intact patellar cartilage of the mouse in vitro. Incubation of mouse patellae in medium with sulfate concentrations below 0.5 mM resulted in a diminished incorporation of sulfate but in unaltered incorporation of glucosamine. This suggested the synthesis of undersulfated glycosaminoglycans under these conditions. We characterized glycosaminoglycans synthesized at three different sulfate concentrations: a sulfate concentration physiological for the mouse (1.0 mM), a sulfate concentration in the range where sulfate incorporation was strongly diminished (0.1 mM), and an extremely low sulfate concentration (10 nM). Analysis of glycosaminoglycan disaccharides and DEAE anion chromatography of the glycosaminoglycans could not confirm the synthesis of undersulfated glycosaminoglycans at 0.1 mM. The chromatogram of glycosaminoglycans synthesized in medium containing 10 nM showed the presence of a very low sulfated glycosaminoglycan pool not observed at higher medium sulfate concentrations. Intermediately sulfated glycosaminoglycans were also synthesized during incubation with 10 nM sulfate. So, our data indicate that only very low sulfate concentrations in the medium lead to the synthesis of undersulfated glycosaminoglycans and that the sulfation mechanism of murine patellar cartilage chondrocytes does not seem to fit completely in an "all-or-nothing" pattern.     

The development of fibrocartilage in the rat intervertebral disc

The development of fibrocartilage in rat lumbar intervertebral discs has been correlated with an immunohistochemical analysis of the changing distribution of extracellular matrix components. Disc anlagen were first recognised by embryonic day 14 as segmental cell condensations. By E16, the notochord formed a series of bulges, each representing a future nucleus pulposus, and the annulus fibrosus had differentiated in the disc anlagen. The inner part of the annulus was composed of cartilage which linked that of adjacent vertebral bodies. The outer part was fibroblastic, with layers of parallel fibroblasts. The long axes of the cells in successive layers lay at an angle of approximately 90° to each other. This criss-cross orientation of cells preceded the oriented deposition of collagen fibres to form the lamellae. Disc anlagen were immunolabelled weakly for types I and III collagen, chondroitin 6-sulphate and dermatan sulphate. Later tissue differentiation was marked by the appearance of type II collagen, chondroitin 4-sulphate and keratan sulphate in the inner annulus. These components also appeared in the outer annulus, but only in adult animals, and indicated metaplastic change in the lamellar fibroblasts. Fibrocartilage in the nucleus pulposus was only seen in old animals, and the origin of the tissue was less clear. However, the fibrocartilage cells appeared to be derived from the cartilage end plate and/or from the inner annulus. We conclude that fibrocartilage in the intervertebral disc is derived from several sources and that the radial distribution patterns of extracellular matrix components in the adult disc are explained by the embryonic origins of its parts.     

Age-related changes in the cerebrospinal fluid outflow glycosaminoglycans

The glycosaminoglycan distribution patterns of the cerebrospinal fluid (CSF) outflow pathway, dura mater and cerebral cortex of young New Zealand red rabbits and 1-, 3- and 12-week-old C-57 mice were identified by analyses of the glycosaminoglycan moieties and by the use of zone electrophoresis. The glycosaminoglycans were identified by specific degradation procedures, i.e., hyaluronate lyase, chondroitin ABC lyase, endo-gb-D-galactosidase and nitrous acid treatment. The CSF outflow pathway and dura mater glycosaminoglycan components were primarily hyaluronic acid and chondroitin sulfatedermatan sulfate, whereas the cerebral cortex glycosaminoglycan components were hyaluronic acid, chondroitin sulfatedermatan sulfate, keratan sulfate and heparan sulfate. The glycosaminoglycan components of the dura mater and cerebral cortex decreased and those of the CSF outflow pathway increased as a function of age. These results demonstrate the feasibility of analyses of the CSF outflow pathway glycosaminoglycan components and suggest that topographical changes in the glycosaminoglycan distribution profiles may contribute to the pattern of cerebrospinal fluid outflow.     

An unidentified macromolecular inhibitory constituent of calcium oxalate crystal growth in human urine

Summary We have detected and isolated a macromolecular constituent in normal human urine possessing calcium crystal growth inhibitory activity. The purification procedure consisted of two anion exchange chromatographies and one affinity chromatography. The crystal growth inhibitor was found to be heterogeneous in net charge as well as in size. It has not been identified. It is not an uronic acid-containing glycosaminoglycan, hitherto presumed to be responsible for the inhibitory activity. Whether an urinary fragment of inter--trypsin inhibitor is responsible has yet to be resolved.     

Role of glycosaminoglycans in diabetic nephropathy

A number of abnormalities of glycosaminoglycan metabolism have been reported in diabetes mellitus. These include anomalous synthesis, catabolism and sulphation. In this review glycosaminoglycan metabolism in diabetes mellitus is discussed with reference to its possible impact on glomerular basement membrane permeability, and on the physiology and metabolism of glomerular resident cells, specifically of the mesangium. Experimental results demonstrating favourable therapeutic activities of some glycosaminoglycans in mesangioproliferative glomerulitis and diabetic nephropathy are reported. These pharmacological actions do not depend on the well-known anticoagulative activity of glycosaminoglycans, but most probably on their antiproliferative effect. A more complex role of glycosaminoglycans in the pathogenesis of diabetic nephropathy is suggested.     

Glycosaminoglycans and PDGF Signaling in Mesenchymal Cells

Platelet derived growth factor (PDGF) is involved in the autocrine growth stimulation of normal and malignant cells, the stimulation of angiogenesis, and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the extracellular microenvironment. The present review discusses the effects of glycosaminoglycans on the functions mediated by the PDGF on cells of mesenchymal origin. Recent studies have demonstrated that both soluble and surface bound glycosaminoglycan chains can modulate PDGF-BB isoform signaling depending on the cell type. These data demonstrated that the microenvironment rich in GAGs/PGs is able to significantly modify the cellular response to PDGF-BB signaling in a critical way for cell growth and differentiation.     

Glycosaminoglycan entrapment by fibrin in engineered heart valve tissues

Tissue engineered heart valves (TEHVs) may provide a permanent solution to congenital heart valve disease by permitting somatic valve growth in the pediatric patient. However, to date, TEHV studies have focused primarily on collagen, the dominant component of valve extracellular matrix (ECM). Temporal decreases in other ECM components, such as the glycosaminoglycans (GAGs), generally decrease as cells produce more collagen under mechanically loaded states; nevertheless, GAGs represent a key component of the valve ECM, providing structural stability and hydration to the leaflets. In an effort to retain GAGs within the engineered constructs, here we investigated the utility of the protein fibrin in combination with a valve-like, cyclic flexure and steady flow (flex–flow) mechanical conditioning culture process using adult human periodontal ligament cells (PLCs). We found both fibrin and flex–flow mechanical components to be independently significant (p < 0.05), and hence important in influencing the DNA, GAG and collagen contents of the engineered tissues. In addition, the interaction of fibrin with flex–flow was found to be significant in the case of collagen; specifically, the combination of these environments promoted PLC collagen production resulting in a significant difference compared to dynamic and statically cultured specimens without fibrin. Histological examination revealed that the GAGs were retained by fibrin entrapment and adhesion, which were subsequently confirmed by additional experiments on native valve tissues. We conclude that fibrin in the flex–flow culture of engineered heart valve tissues: (i) augments PLC-derived collagen production; and (ii) enhances retention of GAGs within the developing ECM.     

聚酸酐-氨基葡聚糖作为干细胞载体的研究

研究聚酸酐-氨基葡聚糖三维载体材料对胎儿肝干细胞黏附及增殖的影响,寻找胎儿肝干细胞新的载体材料。采用改进的两步胶原酶灌注消化法加Percoll液不连续密度梯度离心的方法,分离胎儿肝干细胞。选取胎儿肝干细胞的传代细胞,种植于聚酸酐-氨基葡聚糖三维载体材料上。倒置显微镜下观察细胞的黏附和生长状况;计算细胞贴壁率;MTT法检测各组细胞的吸光度值(OD值);收集载体支架上细胞并计数。取细胞载体进行组织学切片,HE染色光镜下观察。在细胞培养第7 d进行免疫荧光化学染色和流式细胞仪检测。结果表明,聚酸酐-氨基葡聚糖三维载体能促进肝干细胞在材料内黏附并保持其在机体内的形态;载体材料内的肝干细胞功能活跃;在材料表面和三维空间内部培养的肝干细胞均能持续增殖;经过连续40 d共同培养聚酸酐-氨基葡聚糖三维载体对干细胞无毒性,人胎儿肝干细胞可以很好的贴附于聚酸酐-氨基葡聚糖三维载体支架上,细胞增殖活力良好,标志物持续表达,培养7 d得到的细胞数量增多19.7%。聚酸酐-氨基葡聚糖三维载体能促进肝干细胞的增殖,可作为肝干细胞的载体应用于肝脏组织工程。     

氨基多糖的提取、分离和分析测定方法及其研究进展

氨基多糖(Glycosaminoglycan)又称粘多糖、酸性粘多糖、结缔组织多糖等,是动物中含氨基的一类多糖,是动物体内的一类重要大分子物质。氨基多糖在许多生物学、生理学和病理学过程中起着重要作用,已引起世界医药界的广泛关注。鉴于氨基多糖的特殊生物功能和生物活性,人们已经在利用外源性氨基多糖的生物活性研制有效药物防治某些疾病方面取得很大进展。近20～30十年来,人们从各种动物中提取分离氨基多糖,并进行筛选,试图找到有效的氨基多糖类药物。