Researchers and clinicians in the orthopedic fields will benefit from this study.A preliminary observational study to correlate quantitative T2, zonal, and biomechanical changes of hypertrophied articular cartilage using high-resolution microscopic magnetic resonance imaging (μMRI).Three canines underwent anterior cruciate ligament transection (ACLT) in one knee, and sacrificed 25 weeks post-operatively. ACLT medial tibiae were sectioned to obtain three blocks approximately 2.6mm x 5mm, then sealed in 5mm glass MR tubes with a 154mM saline solution containing 1% protease inhibitor and 1mM gadolinium contrast agent. Magnetization-prepared T2-weighted imaging was performed on a Bruker Ascend 300WB 7T/89mm vertical bore magnet with an attached Bruker imaging accessory. T2 imaging was performed with the cartilage articular surface oriented normal (0°) and at the magic angle (55°) with respect to the external magnetic field (B_o). T2-weighting was performed using five contrast echo times (TE_c­­) of 2, 10, 20, 40, and 80ms for 55° and 2, 8, 20, 40, and 70ms for 0°, and an imaging echo time (TE_i) of 7.2ms. The field of view was 4.5mm with an acquisition matrix of 256 x 128 (reconstructed to 256 x 256), which yielded a pixel resolution of 17.6μm. The samples were removed from the glass tubes and compressed to a strain of 18.5±4.6% in a homemade loading device, then resealed in the glass MR tubes and scanned using the identical protocol. Slice selection was taken as close as possible to the uncompressed slice selection (near the center of the tissue block). The T2 images were calculated using Matlab from the five weighted images by fitting an exponential curve for each pixel. T2 profiles were obtained by averaging 10 columns from the calculated T2 images, and plotted on a relative scale from 0 to 1, indicating the articular surface and bone, respectively. After μMRI, the blocks were cut in half at the slice location; one half was then compressed and one half remained uncompressed. The two halves were fixated in a cetylpyridinium chloride and 10% formol solution overnight, then frozen in ice and cryotomed to 6μm sections using a Leica CM1950 cryostat. The sections were laid on a glass slide and covered with a glass coverslide and fixed using Permount. Polarized light microscopy (PLM) was performed at 10x objective magnification on a Leica DMRX microscope with attached CRI Abrio imaging accessories. The retardation images were used in comparing μMRI and histological features.T2@0° (Fig. 3b,c) images qualitatively show the zonal changes in hypertrophied cartilage. PLM images (Fig. 3c,f) show the uncompressed hypertrophied cartilage (Fig. 3b) SZ is markedly increased in thickness compared to normal cartilage (Fig. 1a). After compression (Fig. 3e,f), the SZ was markedly reduced with minimal effect on the TZ, and nearly no effect on the RZ. T2@0° profiles (Fig. 4b) exhibit a double-peak shape for uncompressed, and a single-peak with near bell-shape curvature for compressed. The uncompressed PLM images (Fig. 3c) confirm the first peak is a hypertrophied region. The T2@55° (Fig. 3a,d) images show the SZ is the main part to be affected by compression. A significant drop in T2 (Fig. 4b) (compressed) was seen through approximately 0.4 relative depth (uncompressed), matching well with the hypertrophied zones, and little or no change for deeper tissue.The T2@55° is highly sensitive to water content within the tissue since the nuclear dipolar interaction is minimized, effectively eliminating a factor that influences T2 relaxation. Therefore, we can more accurately measure the regions affected by compression, since loading reduces the local concentration of water in the tissue. The only significant T2 change occurred in the upper hypertrophied regions, in contrast to healthy cartilage (Fig 2a), which exhibits whole-tissue reduction [3]. The increase in cell volume and number decreased the biomechanical stiffness of the hypertrophied region since cells have a much lower modulus than the extracellular matrix [4], which caused the sharp T2@55° change in the upper regions after compression. In contrast, healthy tissue (Fig. 2a) exhibits a decrease in all regions of the tissue [3]. The significant decrease in SZ thickness indicates a highly compliant hypertrophied upper region, in contrast to healthy AC, which changes less in the SZ due to compression. μMRI allows for studying the remarkable structural and biomechanical properties of hypertrophied articular cartilage. High-resolution T2-weighted imaging, in conjunction with standard optical imaging techniques, can link structural defects of cartilage with T2 images under uncompressed and compressed conditions. We found the biomechanical properties to be significantly altered due to hypertrophy. These types of studies can lead to advancements in diagnostics and the detection of early onset OA, and may even lead to studies for alternative treatment utilizing altered metabolic reactions.