Poster No. 1395 • 54th Annual Meeting of the Orthopaedic Research Society Microscopic MRI Analysis of Rat Intervertebral Disc – A Basis for Disc Tissue Engineering Applications Yoram Zilberman 1 , Galit Saar 2 , Hadassah Shinar 2 , Keren Keinan-Adamsky 2 , Gadi Pelled 1 , Gil Navon 2 , Dan Gazit 1,3 1 Skeletal Biotech Laboratory, Hebrew University–Hadassah Medical Campus, Jerusalem, Israel; 2 School of Chemistry, Tel Aviv University, Tel Aviv, Israel; 3 Department of Surgery – International Stem Cell Institute, Cedars Sinai Medical Center, Los Angeles, CA [email protected] Introduction: Low back pain is a common cause of disability and has enor- mous socioeconomic consequences (1). In cases of severe pain caused by the pres- sure of bulging nucleus pulposus (NP) on surrounding nerves, disectomy or nucle- us ablation are used. These surgical procedures do not repair the disc problem. Repair is an ideal therapeutic approach, which restores the normal structure and function of the disc (2). Tissue engineering could regenerate a damaged disc by the introduction of cells, like Mesenchymal Stem Cells (MSCs). One of the major goals in tissue regeneration research is the establishment of valid imaging modali- ties for quantitative analytical evaluation of the repair processes. We have previ- ously shown that Double Quantum Filtered (DQF) MRI can be used for the analysis of tendon repair using MSCs (3). In this study we hypothesized that advanced MRI methods including DQF-MRI could be a useful tool for the eval- uation of the collagen fibers in a rat model of IVD degeneration after NP ablation. The ability of non invasive monitoring of the IVD changes at the early stage post- nucleus ablation will provide an important base line for future attempt in disc repair. Materials and Methods: Using Fluoroscopy and Micromanipulator two nee- dles were inserted into the NP through the postero-lateral aspect of WISTAR rats coccyges spine (C3 or C4). NP was removed by wash with PBS till the leakage of NP substance through the opposite needle was completed. To analyze the early changes in the IVD after NP removal, animals were sac- rificed on day 5, C3-C4 segments were immersed in fluorinate oil for the MRI measurements. T 2 relaxation times were acquired using the multi slice multi echo s sequence (128x128, TR/TE=3000/3 ms, 128 echoes, 8.45T). The contribution of the residual dipolar interaction to T 2 was refocused by dipolar echo refocusing: 90 o – [τ cp – 90 o – τ cp ] n – Imaging (4,5), where τ cp is short (50 μs) and n = 20-5000. The relaxation time obtained by this method is denoted T DE . 1 H and 2 H double quan- tum filtered (DQF) images were acquired with the basic DQF pulse sequence: 90 – τ/2 – 180 – τ/2 – 90 – t DQ – 90 – Imaging (6), where t DQ is the double quan- tum evolution time and τ is the creation time of the 2 nd rank tensors. Results: T 2 -weighted images of intact and ablated IVDs are given in Fig. 1. For the intact disc there is a clear distinction between the nucleus and the annulus. After removal of the NP the width of the disc is significantly decreased, the image is relatively dark, and the distinction between the AF and the NP is lost T 2 values of intact NP and AF were 83.5±8.6 ms and 20.3±2.9 ms (n=11) respectively and for the ablated disc 15.0±1.5 ms (n=2). Dipolar echo refocusing gave relaxation times, T DE , of 171.5±32.5 ms and 31.0±6.2 (n=11) for the intact NP and AF, respectively, and 32.6±5.6 ms (n=2) for the ablated disc. Magnetization transfer experiments followed the same trend: high magnetization transfer ratio (MTR) for the intact AF, almost negligible MTR for the intact NP and a relatively high MTR for the ablated disk. The intact AF is clearly depicted by both 1 H and 2 H DQF (Fig. 2). In contrast to other MRI modalities, this compartment is also partially highlighted in the ablated disc. In the 2 H DQF image, most of the area of the ablated disc is high- lighted. Discussion: Our results revealed higher concentration and better ordering of the collagen fibers in the AF of the control disc relative to the NP.The shortening of T 2 and T DE , the larger MTR and the 1 H and 2 H DQF results obtained in the ablated disc suggest an increase in the concentration of the collagen fibers in the inner part of the disc. This indicates a collapse of the collagen fibers of the annu- lus into the void left after the NP removal and the decrease of the disc width. T DE results are in accordance to T1ρ obtained in human intact and degenerated sam- ples (7,8). As was previously pointed out (9), 1 H DQF signal is obtained only when there is a substantial residual dipolar interaction of water molecules as a result of high degree of order of the tissue. In contrast, 2 H DQF can be seen in relatively disor- dered systems (10). This is the reason why in the ablated disc the 1 H DQF of the inner part appears as a dark spot while in the 2 H DQF one can see the effect of the relatively disordered collagen fibers. The conclusions concerning the morphologi- cal changes of the ablated disc are highly supported by the histology (Fig. 3), in which shortly after NP removal the collagen fibers of the annulus spread into the inner part of the disc. These results demonstrate our ability to monitor very early changes in the IVD post-nucleus ablation. They also provide us with a baseline and a detection method for disc regeneration. References: 1. Biering, SRF. Dan Med Bull, 29, 1982; 2. Alini, M et al, Spine, 28, 2003; 3. Hoffmann et al, J Clin Invest, 116, 2006; 4. Muller, K et al, J. Magn. Reson, 90, 1990; 5. Eliav, U et al, Proc. ISMRM 6th Mtg, 602, 1998; 6. Tsoref, L et al, Magn Reson Med, 40, 1998; 7. Johannessen, W et al, Spine, 31, 2006; 8. Majumdar, S, NMR Biomed, 19, 2006; 9. Eliav, U et al, J Magn Reson, 137, 1998; 10. Sharf, Y et al, J Magn Reson, 107, 1995. Fig 1.T 2 -weighted images of intact (A,B) and ablated (A,C) IVD 5 days after NP removal. A – Sagittal sections, TE=9ms; B,C – Axial sections, TE=21ms. Fig 2. 1 H and 2 H DQF images of intact and ablated IVD 5 days after NP removal. Fig 3. Histology (Masson Trichrom) of intact and ablated IVD 5 days after NP removal. a - Intact; b -Ablated