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

P. J. Coleman, D. Scott, A. Abiona, D. E. Ashhurst *, R. M. Mason ¹ and J. R. Levick

1. The hydraulic resistance of the synovial lining to fluid outflow from a joint cavity (_s) 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 (P_j) 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_s/dP_j, which at low pressures represents synovial hydraulic conductance, increased from a control of 0·90 ± 0·20 µl min^-1 cmH₂O^-1 (mean ± s.e.m., n = 6) to 4·52 ± 0·70 µl min^-1 cmH₂O^-1. The slope d_s/dP_j increased to a similar level after testicular hyaluronidase, namely to 4·14 ± 1·06 µl min^-1 cmH₂O^-1 (control, 0·54 ± 0·24 µl 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.

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