The effect of charged synthetic polymers on proteoglycan synthesis and sequestration in chick embryo fibroblast cultures.

The polycation, poly(L-lysine), repressed the synthesis of glycosaminoglycans in secondary cultures of chick embryo skin fibroblasts and caused sequestration of glycosaminoglycans around the cells. The synthesis of chondroitin sulphate, dermatan sulphate, hyaluronic acid and a fourth component, thought to be heparan sulphate, were all inhibited to the same extent but the sequestration of the sulphated polymers were greater than that of the unsulphated. The sequestered material was retained around and not within the cells. Incubations with the polyanion, poly(L-glutamate), showed a slight stimulation of glycosaminoglycan synthesis and in these and control incubations (no additions to medium), most of the glycosaminoglycan synthesised appeared in the culture medium. The subsequent addition of poly(L-glutamate) to incubations containing poly(L-lysine) reversed the inhibitory and sequestering effect of the polycation. It was concluded that the inhibition of synthesis by poly(L-lysine) was either a direct effect of poly(L-lysine) on the cell membrane or a result of the high local pericellular concentration of sequestered proteoglycan.

A comparison of the glycosaminoglycans of weight-bearing and non-weight-bearing human dermis.

Twenty-six samples of human skin from three non-weight-bearing and 27 samples from three weight-bearing sites were obtained at autopsy from 11 subjects varying in age from 4 to 91 years. The dermis was idssected free of other tissues and after proteolytic digestion, the glycosaminoglycans were isolated quantitatively by ion-exchange chromatography. Analysis of the preparations showed that there was significantly more glycosaminoglycan in the dermis from weight-bearing sites (p less than 0.001). The major glycosaminoglycans were hyaluronic acid and dermatan sulfate with chondroitin sulfate as a minor component, but the relative proportions of these components were not significantly different in the two sets of samples (p greater than 0.1). It is suggested that the quantity of glycosaminoglycan in the dermis is influenced by the functional requirements of the dermis at that site.

A role for glycosaminoglycans in the development of collagen fibrils.

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.

Carrageenin-induced arthritis. I. The effect of intraarticular carrageenin on the chemical composition of articular cartilage.

A single intraarticular injection of carrageenin into rabbit knee joints initiated a localized synovial inflammatory response. This response was accompanied by a 20% loss of proteoglycan from the articular cartilage within 24 hours and by a further 30-60% loss within 5-7 days. The chondrocytes replaced the lost proteoglycan within 42 days. More than two injections caused only a further small decrease in proteoglycan content; the cartilage was then unable to replace the lost proteoglycan. The absence of recovery coincided with the appearance of erosion of the cartilage surface.

Hyaluronic acid in elastic cartilage.

Bovine ear cartilage contains more hyaluronic acid than do hyaline cartilages of the same animal. Most of it is in the elastin-rich residue not extractable by 4 M guanidinium chloride where it is associated with chondroitin sulphate in low relative concentration and of lower molecular weight than in non-elastic cartilage residue.

Carrageenin-induced arthritis. II. Effect of intraarticular injection of carrageenin on the synthesis of proteoglycan in articular cartilage.

An inflammation of the periarticular tissue induced by the intraarticular injection of carrageenin caused a 40-75% decrease in the rate of proteoglycan synthesis in the articular cartilage. This decrease was determined both in vitro and in vivo. Provided that the collagen network of the cartilage was not disrupted, the chondrocytes were able to recover this synthetic ability and exhibited rates of synthesis above those of the controls. This ability resulted in a net replacement of the proteoglycan lost during the initial period of inflammation.

The synthesis of hyaluronic acid by sheep and rabbit articular cartilage in vitro.

Standard methods of glycosaminoglycan separation were used to confirm the presence of hyaluronic acid in sheep and rabbit articular cartilage. During incubation of carilage in vitro in the presence of [1-14 C]acetate, cartilage cells synthesize this macromolecule and, under the conditions used, it appears to have a shorter turnover time than chondroitin suplhate in the same tissue. The possible functions of hyaluronic acid in cartilage are discussed.

The glycosaminoglycans of Dupuytren's disease.

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution. Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues. In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue. The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytren's process is discussed.

Carrageenin-induced arthritis. III. Proteolytic enzymes present in rabbit knee joints after a single intraarticular injection of carrageenin.

A single intraarticular injection of carrageenin into the rabbit knee joint initiates an inflammatory reaction in the synovial tissues. the exudate from the joint was able to degrade proteoglycan at pH 5.2 and pH 7.2. Further characterization of proteolytic enzymes in the inflamed synovial tissues showed the presence of cathepsin D, a neutral protease, and cathepsin B1. Maximum activities of two lysosomal enzymes, acid phosphatase and cathepsin D, were observed within 7 days of injection. Most of this activity was found to be associated with cells in the synovial fluid.

The proteoglycan content and the axial periodicity of collagen in tendon.

The glycosaminoglycan content and the axial periodicity of collagen was determined in various regions of the rabbit flexor digitorum profundus tendon. This tendon, which passes from the calf to the toes round the inner side of the ankle, contains a thickened sesamoid-like pad where it is subjected to friction and pressure. Other regions of the tendon are subject only to longitudinal tension. In tensional areas the axial periodicity of collagen was of the order of 62 nm and the tissue contained less than 0.2% proteoglycan on a dry weight basis. In the sesamoid-like region, however, the axial periodicity was a significant 13-15% less, and the proteoglycan constituted about 3.5% of the dry weight. Also, in the tensional areas the predominant glycosaminoglycan was dermatan sulphate, whereas in the sesamoid the predominant glycosaminoglycan was chondroitin sulphate. The possible interrelationships between collagen axial peroidicity and proteoglycan content in this tissue are discussed.

Carrageenin-induced arthritis. IV. Rate changes in cartilage matrix proteoglycan synthesis.

A localized inflammatory response was initiated by both single and repeated injections of carrageenin into femorotibial joints. Histologic changes were observed 24 hours after a single intraarticular injection, and an inhibition in the in vitro rate of proteoglycan synthesis was detected 72 hours after the injection. This inhibition was relieved in vitro by the addition of beta-D-xyloside, an exogenous initiator of glycosaminoglycan biosynthesis. Following repeated carrageenin injections, most cells appeared to be dead on histologic examination and no in vitro proteoglycan synthesis could be detected; nor could any stimulation be achieved by adding exyloside.

Collagen fibril diameters and glycosaminoglycan content of skins--indices of tissue maturity and function.

Samples of anatomically and functionally distinct regions of the skin of a variety of altricial and precocial animals were taken at various stages of development from birth to beyond maturity. The glycosaminoglycan (GAG) content and composition of the tissues were determined by chemical analysis and the collagen fibril diameters measured by transmission electron microscopy. The fibril diameters of the skins of two fish and a bird were also assessed for comparison. Analysis and comparison of the data collected show that there was a significant correlation between collagen fibril diameter distribution, GAG type and amount, and functional load-bearing of the various skins, and that the variations in the biochemical and physical composition of the tissues at different stages of development could be related to both their post-conceptual maturity and their prospective functional loading.