To examine whether gagCEST imaging/CEST effect reflects the histopathological changes concerning glycosaminoglycans in human menisci using MR-micro-imaging on an ultra high field human MR-scanner in vitro. Histochemical staining was performed for correlation to imaging findings.

Meniscus degeneration is characterized by collagen fibre disorganization, increase of water content and increase of glycosaminoglycans¹. Glycosaminoglycan Chemical Exchange Saturation Transfer (gagCEST) imaging is a relatively novel quantitative imaging method. The gagCEST effect (MTR_asym) is related to glycosaminoglycan concentrations and has been shown to be a useful technique for direct estimation of GAG content in various cartilaginous tissues ^2,3,4, which makes this technique a promising approach for the early detection of pathological changes in menisci without contrast agents⁵.

Sample preparation: Five meniscal samples were obtained from a pair of human medial and lateral menisci from one osteoarthritic knee joint of one patient. The meniscal samples were fitted into a 5ml tube filled with PBS solution. Orientation of circumferential meniscal fibers parallel to B₀ and radial fibers perpendicular to B₀ was chosen to avoid magic angle artefacts. All MRI acquisitions were performed on 7T MRI (Magnetom Siemens Healthcare, Erlangen, Germany) with a MR-microimaging system⁶. For gagCEST imaging a 19mm 1H-NMR volume coil (Rapid Biomedical, Wuerzburg, Germany) was used. gagCEST was applied using a train of adiabatic RF pulses⁷ followed by signal readout with a 3D RF spoiled GRE sequence. Adiabatic pulses were used to ensure a constant flip angle. The following saturation parameters were used: B1-CWAE (continuous wave amplitude equivalent) = 1.5μT, number of CEST pulses = 6, pulse duration PD = 60 ms, interpulse delay IPD = 30 ms, number of slices = 28. The GRE imaging parameters were: FOV =16.8 x 20 mm², pixel size = 0.3 x 0.3 mm², slice thickness = 0.4 mm, TR/TE = 5 ms/2.1 ms, flip angle = 4°, Number of offsets = 60, acquisition duration =25:23 min.

The CEST curves were calculated for each pixel and were shifted for the water resonance to appear at 0 ppm of the CEST-spectrum. The magnetization transfer asymmetry rate (MTR_asym (δ) = MTR (+δ) – MTR(-δ)) was integrated over the offset range δ from 0.6 – 1.8 ppm, which corresponds to the resonance frequency range of GAG–hydroxyl protons, and was used as signal intensity for gagCEST images. After the MR measurement, histological analysis of the menisci specimens was performed. Meniscal specimens were fixed in formalin, decalcified, dehydrated and then embedded in paraffin. Subsequently, deparaffined sections (2.5µm) were stained with safranin-O for visualization of glycosaminoglycans, picrosirius red for analyzing of collagen distribution and H&E to evaluate the cellularity and cell morphology.

Figure 1 shows a representative in-vitro CEST map and the corresponding safranin-O stained image. Qualitatively, the gagCEST map and the corresponding safranin-O image show the same relative regional intensity of glycosaminoglycans. This match of gagCEST maps with histochemical finding was found for all 5 samples. Figure 2a shows a representative Z-spectra and Fig. 3 the MTR_asym curves from three regions of interest (ROI) of the same meniscal specimen. The ROIs were placed in the red (vascularized) zone, the red-white zone and the white (avascular) zone (Fig.4). The mean CEST effects of these regions are 4.5 % (red zone), 3.3 % (red-white zone) and 1.6 % (white zone).In vivo gagCEST imaging in cartilaginous tissues is very prone to errors due to partial volume effects and motion. The advantage of using gagCEST imaging in a 7T microimaging system in vitro is that it allows motion artefact free imaging with resolution far better than that of clinical imaging. Overall, this setup allows a very detailed look into the glycosaminoglycan distribution in the human meniscus.For the first time, gagCEST imaging of human meniscus was successfully performed on a 7 T human scanner at with high spatial resolution using a microimaging insertsystem. The results of this study highlight the potential of gagCEST imaging for early assessment of slight pathological changes in this tissue before morphological changes are seen.