Assessing collagen alterations in enzymatic degradation models of osteoarthritis via second harmonic generation microscopy

Abstract

Structural changes in the collagen II architecture of osteoarthritis (OA) are poorly understood, which is a large shortcoming in the early diagnosis of this disease. Though degradation can be simulated by enzymes including trypsin and bacterial collagenase, the specific structural features of each digestion and their relationship to naturally occurring OA remain unclear. We used collagen sensitive/specific Second Harmonic Generation (SHG) microscopy in conjunction with optical scattering measurements to probe the resulting architecture changes in bovine knee cartilage upon trypsin and collagenase degradation. Image features extracted from SHG images were used to train a linear discriminant (LD) model capable of classifying enzymatic degradation, which was then applied to human cartilage with varied modified Mankin histological scores. The treatment of cartilage with these enzymes resulted in more disorganized collagen structure, where this effect was greatest with collagenase treatment. Using the LD model, we classified the control and degraded tissues in the three zones with >92% accuracy, showing that these enzymes have distinct activity on the collagen assembly. Application of the LD model to human cartilage indicated that collagenase effects were more representative of in vivo degeneration and were also consistent with damage beginning at the articular surface and progressing into deeper zones. SHG and optical scattering measurements successfully delineate trypsin and collagenase degradation and suggest that collagen alterations in human OA are better simulated by the latter mechanism. These results lay the groundwork for using high-resolution SHG and optical scattering as an earlier diagnostic tool than is currently available.