Ultrasound Biomicroscopy Imaging for Monitoring Progressive Trypsin Digestion and Inhibition in Articular Cartilage

Abstract

This study reports an ultrasound biomicroscopy (UBM) imaging approach to monitor the progressive trypsin-induced depletion of proteoglycan (PG) and its inhibition in articular cartilage. Three fresh, normal bovine patellae were obtained and four full-thickness cartilage-bone specimens were prepared from the lower medial side of each patella. One sample was used as a control and the other three were divided into three groups: Groups A, B and C (n = 3 for each group). After a 40 min 0.25% trypsin digestion, samples from group A were continuously digested in trypsin solution, while those in groups B and C were immersed in physiologic saline and fetal bovine serum (FBS), respectively, for another 280 min. The trypsin penetration front was observed by UBM and M-mode images were acquired using 50 MHz focused ultrasound and custom-developed software. The results show that the 40 min trypsin digestion degraded nearly the whole surface layer of the cartilage tissue. Further digestion in trypsin or residual digestion in saline for 280 min depleted most of the PG content, as observed in groups A and B. The replacement of trypsin with a physiologic saline solution only slightly slowed the digestion process (group B), while trypsin inhibitors in FBS stopped the digestion in approximately 1.5 h (group C). The normalized digestion fractions of the digested tissues were calculated from ultrasound data and histology sections, and then compared between the groups. Without the use of FBS, 80% to 100% of the full thickness was digested, while this number was only approximately 50% when using FBS. Our findings indicate that the UBM imaging system could provide two-dimensional (2-D) visual information for monitoring progressive trypsin-induced PG depletion in articular cartilage. The system also potentially offers a useful tool for preparing cartilage degeneration models with precisely controlled PG depletion.