Collagenase-Induced Changes in Articular Cartilage as Detected by Electron-Microscopic Stereology, Quantitative Polarized Light Microscopy and Biochemical Assays

Abstract

The purpose of this study was to compare the ability of electron-microscopic (EM) stereology with quantitative polarized light microscopy (PLM) and biochemical collagen (hydroxyproline) and crosslink (pyridinoline) analyses to detect changes in the superficial collagen network of bovine articular cartilage after digestion in vitro with purified bacterial (Clostridium histolyticum) collagenase (30 U/ml) for 24 and 48 h. Collagen volume (V_V) and surface (S_V) densities of the uppermost third of the superficial zone were estimated indirectly from zonal isotropic uniform random sections using collagen length density (L_V) and average collagen fibril diameter, or its average second power. Collagenase digestion caused a significant decrease in fibril diameter (64 to 62%), V_V (89 to 95%) and S_V (64 to 86%) after incubation for 24 and 48 h. Collagen L_V remained unchanged after 24 h incubation but decreased 63% after 48 h. Collagen concentration per dry weight, assayed biochemically from the whole superficial zone, decreased also significantly (29 to 60%) after 24 and 48 h digestions, respectively. The pyridinoline concentration per dry weight of the superficial zone decreased (31 to 57%) whereas the pyridinoline concentration per collagen remained unchanged. PLM revealed that the birefringence of the uppermost third of the superficial zone was decreased by 36% after digestion for 24 h though the total birefringence of the whole zone was not reduced. However, after 48 h, the birefringence of the whole superficial zone was significantly reduced (76%). All of the techniques compared in this study could detect collagen network degradation in bovine articular cartilage but the EM stereological technique was more sensitive at detecting the changes than PLM or biochemical assays.