An in vitro model for the pathological degradation of articular cartilage in osteoarthritis

Abstract

The objective of this study was to develop an in vitro cartilage degradation model that emulates the damage seen in early-stage osteoarthritis. To this end, cartilage explants were collagenase-treated to induce enzymatic degradation of collagen fibers and proteoglycans at the articular surface. To assess changes in mechanical properties, intact and degraded cartilage explants were subjected to a series of confined compression creep tests. Changes in extracellular matrix structure and composition were determined using biochemical and histological approaches. Our results show that collagenase-induced degradation increased the amount of deformation experienced by the cartilage explants under compression. An increase in apparent permeability as well as a decrease in instantaneous and aggregate moduli was measured following collagenase treatment. Histological analysis of degraded explants revealed the presence of surface fibrillation, proteoglycan depletion in the superficial and intermediate zones and loss of the lamina splendens. Collagen cleavage was confirmed by the Col II-3/4Cshort antibody. Degraded specimens experienced a significant decrease in proteoglycan content but maintained total collagen content. Repetitive testing of degraded samples resulted in the gradual collapse of the articular surface and the compaction of the superficial zone. Taken together, our data demonstrates that enzymatic degradation with collagenase can be used to emulate changes seen in early-stage osteoarthritis. Further, our in vitro model provides information on cartilage mechanics and insights on how matrix changes can affect cartilage's functional properties. More importantly, our model can be applied to develop and test treatment options for tissue repair.

Keywords: Articular cartilage; Collagenase; Mechanical properties; Osteoarthritis.

Figure 1

Schematic representation of the functional response of cartilage showing the loading phase from time 0 to t_o and the creep phase from t_o to t_f.

Figure 2

Comparison between experimental creep responses and theoretical fits after A) 45 min and B) 100 sec of confined creep compression.

Figure 3

Experimental values for permeability, instantaneous strain, and aggregate modulus were determined using the biphasic theory. Predicted theoretical creep deformations for intact and degraded cartilage explants were generated from these average experimental values.

Figure 4

Normalised cartilage explant properties (y-axis) versus series of consecutive creep tests (x-axis). The instantaneous modulus was calculated by fitting an exponential function to the stress-strain response during the loading phase and the slope calculated at a strain of 5%. The instantaneous strain was determined at the end of the loading phase. The aggregate modulus is a measure of the compressive resistance of the solid phase of the ECM at equilibrium and was approximated at the end of the 45 min creep test data (tests 4 and 8). The apparent permeability and apparent modulus were both measured after 100 sec for each creep test (tests 1–8). Two-way repeated measures ANOVA with Tukey post-hoc multiple comparisons were used for statistical analysis (α=0.05, N=10). Statistically significant differences were not observed between intact values (tests 1–4) for every graph, therefore the degraded values (tests 5–8) were compared to the average of tests 1–4 (average intact value). * indicates statistically significant difference with the average intact value. + indicates statistically significant difference with test 5. ^o indicates statistically significant difference with test 6. ^v indicates statistically significant difference with test 7.

Figure 5

Representative micrographs (10X) of Safranin-O, Picrosirius red, & Col II–¾Cshort staining of intact, 45, 90 and 120 min-degraded cartilage explants.

Figure 6

Normalised (A) sulfated glycosaminoglycans and (B) collagen content for intact and degraded cartilage specimens. Two-way repeated measures ANOVA with Tukey post-hoc multiple comparisons were used for statistical analysis (α=0.05, mean ± SEM).