Effect of depletion of glycosaminoglycans and non-collagenous proteins on interstitial hydraulic permeability in rabbit synovium.

1. The hydraulic resistance of synovial interstitium helps to retain a lubricating fluid within the joint cavity. The contributions of sulphated glycosaminoglycans to resistance were assessed by selective depletion by chondroitinase ABC, keratanase and heparinases I, II and III in vivo. Also, since glycosaminoglycans do not account fully for the resistance, the contribution of non-collagenous, structural proteins in interstitium was assessed by treatment with chymopapain, a collagen-sparing protease. 2. Ringer solution containing enzyme was injected into the synovial cavity of the knee in anaesthetized rabbits. After >= 30 min the intra-articular pressure was raised and the relation between pressure (Pj) and trans-synovial outflow (Qs) determined. The slope dQs/dPj at low pressures, i.e. below yield pressure, represents the hydraulic conductance of the lining, i.e. 1/resistance. The contralateral joint received Ringer solution without enzyme as a control. Action of enzymes on the tissue was confirmed by histochemical and immunohistochemical studies. 3. Treatment with chondroitinase ABC (5 joints) increased the hydraulic conductance of the lining by 2.3 times (control, 1.34 +/- 0.22 microliter l min-1 cmH2O-1; post-enzyme, 3.11 +/- 0.45 microliter l min-1 cmH2O-1). This was significantly less than the effects of leech, Streptomyces and testicular hyaluronidases, which caused an average 4.7 times increase (P < 0.001, ANOVA). Analogous findings were made above yield pressure. 4. Treatment with keratanase (3 joints) or heparinases I, II and III (3 joints) caused no significant increase in trans-synovial flows or conductance, even though the concentration of heparan sulphate in synovium is higher than that of chondroitin sulphates or hyaluronan. 5. Treatment with chymopapain (7 joints) caused the greatest increases in trans-synovial flow, which exceeded control flow by an order of magnitude in one case. After 0.1 U chymopapain the average conductance was 6.6 times the control conductance below yield pressure. Immunohistochemical studies confirmed that chymopapain treatment removed the synovial proteoglycans. 6. It is concluded that, despite their similar resistivities in vitro, the different glycosaminoglycans do not contribute equally, weight for weight, to interstitial resistance in vivo. Hyaluronan is the dominant glycosaminoglycan governing synovial interstitial resistance. In addition, non-collagenous structural proteins contribute significantly to interstitial resistance.

Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium.

1. The hydraulic resistance of the synovial lining to fluid outflow from a joint cavity (Qs) is important for the retention of intra-articular lubricant. The resistance has been attributed in part to extracellular glycosaminoglycans, including hyaluronan and chondroitin sulphates. Increased permeability in joints infused with testicular hyaluronidase, which digests both chondroitin sulphates and hyaluronan, supports this view. In this study the importance of interstitial hyaluronan per se was assessed using leech and Streptomyces hyaluronidases, which degrade only hyaluronan. 2. Ringer solution was infused into the knee joint cavity of anaesthetized rabbits for 30 min, with or without hyaluronidase, after which intra-articular pressure (Pj) was raised and the relation between pressure and outflow determined. 3. Treatment with Streptomyces, leech or testicular hyaluronidases increased the fluid escape rates by similar factors, namely 4- to 6-fold. After Streptomyces hyaluronidase treatment the slope d 8d s/dPj, which at low pressures represents synovial hydraulic conductance, increased from a control of 0.90 +/- 0.20 microl min-1 cmH2O-1 (mean +/- s.e.m. , n = 6) to 4.52 +/- 0.70 microl min-1 cmH2O-1. The slope d 8d s/dPj increased to a similar level after testicular hyaluronidase, namely to 4.14 +/- 1.06 microl min-1 cmH2O-1 (control, 0.54 +/- 0.24 microl min-1 cmH2O-1). Streptomyces and leech hyaluronidases were as effective as testicular hyaluronidase (no statistically significant differences) despite differences in substrate specificity. 4. It was shown using histochemical and immunohistochemical techniques that hyaluronan was removed from the synovium by leech, Streptomyces and testicular hyaluronidases. The binding of antibodies 2-B-6 and 3-B-3 showed that the core proteins of the chondroitin sulphate proteoglycans remained intact after treatment with hyaluronidases, and the binding of 5-D-4 showed that keratan sulphate was unaffected. An azocasein digestion assay confirmed that the hyaluronidase preparations had no significant proteolytic activity. 5. The effect of the hyaluronidases was four times greater than predicted from the low concentration of interstitial hyaluronan and its resistivity. Factors that might amplify the effect of hyaluronan depletion include the matrix-organizing role of hyaluronan, and/or non-uniformity of hyaluronan distribution. It is concluded that interstitial hyaluronan makes a major contribution to synovial hydraulic resistance, but the mechanisms are as yet poorly understood.