Objective. To investigate the correlation between joint disease and the composition of chondroitin sulfate in the joint fluid, unsaturated disaccharide isomers of chondroitin 4-sulfate (.1di-4S) and chondroitin 6-sulfate (.1di·6S) were measured in joint fluids obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA), or traumatic arthritis (TA).Methods. These pathologic joint fluids were digested with chondroitinase ABC, and the .1di·4S and .1di·6S produced were determined by high performance liquid chromatography combined with fluorometry.Results. Total content of .1di-4S plus .1di·6S was 71.8 ± 30.0 nmoles/ml (mean ± SD) in OA, 55.4 ± 29.3 nmoles/ml in RA, and 211 ± 149 nmoles/ml in TA joint fluids. The ratio of .1di·6S to .1di·4S was 3.81 ± 0.992 in OA, 1.13 ± 0.527 in RA, and 5.75 ± 2.46 in TA joint fluids. Differences between groups were statistically significant.Conclusion. These results strongly suggest that the levels of chondroitin sulfate isomers and the .1di-6S: .1di·4S ratio in joint fluid reflect the proteoglycan metabolism of joint tissues, particularly of articular cartilage; hence, they could be used to diagnose joint diseases

To demonstrate localization of hyaluronic acid (HA) in articular cartilage of the human femur, biotinylated HA-binding region, which specifically binds HA molecules, was applied to the tissue. In sections fixed by 2% paraformaldehyde-2% glutaraldehyde, HA staining was detected in lamina splendens and chondrocytes in the middle zone. By pretreatment with trypsin, intense HA staining appeared in the extracellular matrix of the deep zone and weak staining in the superficial and middle zones. Moreover, pre-treatment with chondroitinase ABC (CHase ABC) intensely enhanced the stainability for HA in the superficial and middle zones and weakly in the deeper zone. Combined pre-treatment of trypsin with CHase ABC abolished intra- and extracellular staining for HA in all zones. By microbiochemical study, the concentrations of HA and dermatan sulfate were high in the middle zone, whereas those of chondroitin sulfate and keratan sulfate were high in the deep zone. These results suggest that HA is abundantly synthesized in and secreted from the chondrocytes, particularly in the middle zone, whereas it is largely masked by proteoglycan constituents in the extracellular matrix.

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.

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The concentrations of hyaluronan (HA) and chondroitin sulfate (CS) in synovial fluids from patients with traumatic arthritis (TA) with and without hydrarthrosis were measured. The CS in synovial fluids was determined as a marker of cartilage destruction by high performance liquid chromotography. The concentration of HA in synovial fluids was lower in patients with hydrarthrosis than in healthy volunteers and patients with TA without hydrarthrosis, whereas the total amounts of HA and CS and the concentration of CS were higher in patients with hydrarthrosis. To investigate the relation between hydrarthrosis and production of HA in synovial tissues, TA synovial tissue biopsies were stained for HA with biotinylated HA binding region. The intensity of HA staining was higher in specimens from patients with hydrarthrosis than in normal and TA without hydrarthrosis specimens. Thus, there may be a correlation between hyperproduction of HA, cartilage destruction and increase in fluid volume in TA.

In order to investigate the biochemical alteration of proteoglycan (PG) synthesis during cartilage repair, reversible destruction was induced by injecting papain into the knee joint cavity of rabbits. The PG synthesis in the cartilage was examined using Na2 35SO4 and high performance liquid chromatography (HPLC). PGs labeled with 35SO4(2-) (35S-PGs) were extracted from normal and papain-treated cartilage, and the amount of synthesis, ability to aggregate with hyaluronan (HA), and the composition of glycosaminoglycan and chondroitin sulfate isomer labeled with 35SO4(2-) (35S-GAG and 35S-CS isomer) were analyzed. Synthesis of 35S-PGs, especially those that were unable to aggregate with HA (nonaggregating 35S-PGs), increased in papain-treated cartilage compared with that in normal cartilage. The acceleration and qualitative change in PG synthesis in the papain-treated cartilage are considered to be responses to the supplementation of the loss of cartilage PGs induced by papain. The compositions of 35S-GAG and 35S-CS isomer of the nonaggregating 35S-PGs differed from those of 35S-PGs which were able to aggregate with HA (aggregating 35S-PGs) in the papain-treated cartilage as well as in the normal cartilage. However, the compositions of both nonaggregating and aggregating 35S-PGs in the papain-treated and normal cartilage were similar. These results indicate that most of the nonaggregating 35S-PGs in papain-treated cartilage have properties similar to those in normal cartilage and are not simple degradation products of aggregating 35S-PGs; they also suggest that the supplementary reaction for PG content in the cartilage during its repair process is not simple acceleration in PG turn-over but the enhancement of PG synthesis accompanied by alterations in aggregating ability and the compositions of GAG and CS isomer.

The effect of sodium hyaluronate (Na-HA) on the migration of corneal epithelium was investigated in vitro using corneo-scleral sections of rabbits. The effect on migration was evaluated by the length of migrating epithelium on