Quantification of cartilage matrix components with nuclear magnetic resonance(NMR) has potential applications to the early diagnosis of osteoarthritis. MR parameters have been measured on cartilage explants digested with aqueous enzyme solutions to simulate osteoarthritic effects[1]. Results show increased T2, however it is not clear how sample handling and exposure to various aqueous and non-aqueous solutions impact observed MR parameters[2]. In this work we investigated the effects of storage in solutions commonly used in ex-vivo cartilage research, DPBS and Fluorinert, on T2 relaxation in bovine articular cartilage (BAC).

BAC samples were harvested from the load-bearing region of the patella of 6 adult animals. The patella was continuously washed during harvesting with Fluorinert® FC-770. Each extracted BAC plug was immediately weighed and placed in a glass NMR tube as shown in Fig.1. The remaining volume of the sample tube was filled with Fluorinert to match the susceptibility of the cartilage sample to improve shimming.

Experimental data were collected on a 9.4T Bruker Avance III NMR spectrometer equipped with a Micro2.5 micro-imaging probe and a 5 mm solenoidal coil. Signal from the subchondral region of the sample was eliminated using saturation slabs for the non-localized relaxometry experiments. Data were acquired at 4°C to minimize sample degradation and each sample was oriented so that B0 was approximately at the magic angle relative to a vector normal to the articular surface, maximizing SNR and eliminating laminar variations in image intensity. T2 measurements were conducted using a CPMG sequence with acquisition at echo maxima with echo time TE=100µs, number of echoes NE=4096 and repetition time TR=10s. Data were analyzed using the non-negative least squares algorithm as described in [3] and reported as the first moment, <T2>, as described in Fig.2.

The samples were randomly divided into three groups, each with N=2, with two groups exposed to Fluorinert only and one also exposed to DPBS. Samples from the first group(F1) were kept in the instrument undisturbed (at 4°C) and T2 relaxometry data were acquired at 1, 1.5, 2, 4, 6, 9, 12, 24, 48, 72 hours of Fluorinert exposure. The second group (D1) was used to observe the effect of conventional storage in DPBS. After each data acquisition, each D1 sample was taken out of its NMR tube and placed in DPBS (at 4°C), resulting in 0.5, 1, 2, 3, and 24 total hours of exposure to DPBS. Samples were weighed after each exposure time. The last group (F2) was used to evaluate the effects of the sample handling protocol followed for the D1 group. The same procedure for D1 was repeated in F2 but samples were placed in Fluorinert instead of DPBS during the soaking period.

Fig.3 shows <T2> values for F1, which varied little in the first 6 hours, and then stabilized after 24 hours. After 72 hours, <T2> increased by 7ms(15%). Figure 4a shows the <T2> values for D1 and F2. After the first thirty minutes of soaking in DPBS, the <T2> values increased by 55.15ms(130%). Conversely, the <T2> values of F2 decreased by 1.95ms(5.63%). Figure 4b displays the change in sample weight for D1 and F2. Throughout sample handling as described above, the average weight increased by 5.4mg(15.8%) and decreased by 4.95mg(14.3%) for D1 and F2 respectively.DPBS is often used to immerse cartilage tissue specimens during imaging, with the assumption that it prevents dehydration as it mimics the natural conditions of synovial fluid. However, the structure of the explanted cartilage tissue is compromised, exposing the transitional and deep zones directly to the aqueous storage solution, whereas cartilage tissue is exposed to synovial fluid only on the superficial surface under normal conditions. Water content of the cartilage tissue increased, as evidenced by increased sample weights, resulting in dramatically increased <T2>. One way to alleviate these effects is to use a hydrophobic solution that preserves water content of excised articular cartilage without infiltrating the tissue. As shown in Fig. 3, storing the BAC samples in Fluorinert resulted in a small increase in <T2> over 72 hours. On the other hand, the <T2> values of F2 did decrease gradually with each handling step, possibly due to the dehydration during sample manipulation, as evidenced by decreasing sample weights. Storing BAC tissue explants in DPBS can significantly increase the observed T2 relaxation time. The extent of this effect needs to be further explored for enzymatic degradation studies. An alternative storage medium to maintain T2 stability is Fluorinert.