Real-Time Ultrasonic Assessment of Progressive Proteoglycan Depletion in Articular Cartilage

Abstract

1The loss of proteoglycan (PG) is regarded as one of the early signs of osteoarthritis (OA), 2 thus observing the progress of PG loss would be useful for the early detection of OA. In 3 this study, high-frequency ultrasound was used to monitor and analyze the trypsin-4 induced progressive degeneration in articular cartilage. Full-thickness cartilage-bone 5 specimens (n = 10) prepared from normal bovine patellae were digested using 0.25% 6 trypsin solution for different periods of time to evaluate the dynamic of the digestion 7 process. The trypsin penetration front was observed in M-mode image, which was 8 acquired using a nominal 50 MHz focused transducer. The transient speed of the 9 digestion process was estimated from the image. The digestion fraction, which represents 10 the ratio of the digestion depth to the total cartilage thickness, was estimated from 11 ultrasound data and histology sections. With ultrasound, the digestion fraction observed 12 in the 10 specimens ranged from 64 to 99%, and was correlated to that measured by 13 histology (R 2  0.63, p<0.05). It was found that the digestion speed decreased nonlinearly 14 with depth from 0.61 ± 0.16 m/s (mean ± SD) in the superficial zone to 0.04 ± 0.02 15 m/s in a region located at 70% of the cartilage thickness in depth. The relationship 16 between the digestion depth and the exposure duration in trypsin could be described 17 using a 3 rd order polynomial function. The full-thickness of digested and undigested 18 tissues was also measured using caliper, estimated from ultrasound data and histology 19 sections, and compared. These findings indicate that ultrasound could provide useful 20 information about the trypsin-induced progressive PG depletion in articular cartilage. 21Therefore, ultrasound represents a useful tool to evaluate the dynamic of models of OA in 22 vitro in cartilage specimens in a research environment, and this would ultimately help the 23 in-vitro examination of articular cartilage for research related to model of OA from the 24 early stages of tissue degradation. 25

Ultrasound speed and attenuation in progressive trypsin digested articular cartilage

“…From Figure 5, the speed of digestion was observed to decrease as the depth of tissue increased. This tendency is related to the distribution of the PG content and it also appeared in studies using high frequency ultrasound by Wang et al [31,35]. This result implies that the digestion of trypsin depends on the concentration of PG at different depths in the tissue.…”

“…A 1 (f) and A 2 (f) were obtained by a fast Fourier transform of the ROI data. For a more detailed description of ultrasound attenuation, the reader is referred to a related reference [35].…”

Quantitative ultrasound biomicroscopy for the analysis of healthy and repair cartilage tissue

“…The cells were obtained from the chondral part of the 10 th rib, a cell source providing phenotypically stable chondrocytes that are characterized by a high proliferative capacity and transcriptional activity of cartilage-specific genes (Xu et al, 2004;Isogai et al, 2006). The harvested cartilage tissue was minced and treated with 0.2% trypsin (Gibco Life Technologies, Grand Island, NY, USA) for 20 minutes, followed by exposure to 0.02% clostridial collagenase dissolved in Dulbecco´s modified Eagle´s medium (DMEM; Gibco Life Technologies) with 10% fetal calf serum (FCS; Gibco Life Technologies) for 10 hours at 37°C.…”

“…To date, valid parameters for the determination of the proteoglycan content by ultrasound analysis have still to be established. For this purpose, the role of sound velocity or frequency dependent attenuation measurements have been discussed in recent studies, however with inconsistent results so far (Pellaumail et al, 2002;Toyras et al, 2003;Kiviranta et al, 2007;Wang et al, 2008).…”

“…Although several strategies have proven their clinical efficacy (Peterson et al, 2002;Dorotka et al, 2005;Knutsen et al, 2007;Saris et al, 2008), the superiority of either method has not finally been proven, and it is still a matter of debate, if the financial burden and complexity of autologous cell transplantation is justified (Wasiak et al, 2006). To determine the putative long-term success of cartilage repair approaches, histological examination of the structural quality of the repair tissues is currently considered the gold standard.…”