Orientation anisotropy of quantitative MRI parameters in degenerated human articular cartilage

Quantitative magnetic resonance (MR) relaxation parameters demonstrate varying sensitivity to the orientation of the ordered tissues in the magnetic field. In this study, the orientation dependence of multiple relaxation parameters was assessed in cadaveric human cartilage with varying degree of natural degeneration, and compared with biomechanical testing, histological scoring, and quantitative histology. Twelve patellar cartilage samples were imaged at 9.4 T MRI with multiple relaxation parameters, including T 1 , T 2 , CW − T 1 ρ , and adiabatic T 1 ρ , at three different orientations with respect to the main magnetic field. Anisotropy of the relaxation parameters was quantified, and the results were compared with the reference measurements and between samples of different histological Osteoarthritis Research Society International (OARSI) grades. T 2 and CW − T 1 ρ at 400 Hz spin ‐ lock demonstrated the clearest anisotropy patterns. Radial zone anisotropy for T 2 was significantly higher for samples with OARSI grade 2 than for grade 4. The proteoglycan content (measured as optical density) correlated with the radial zone MRI orientation anisotropy for T 2 ( r = 0.818) and CW − T 1 ρ with 400 Hz spin ‐ lock ( r = 0.650). Orientation anisotropy of MRI parameters altered with progressing cartilage degeneration. This is associated with differences in the integrity of the collagen fiber network, but it also seems to be related to the proteoglycan content of the cartilage. Samples with advanced OA had great variation in all biomechanical and histological properties and exhibited more variation in MRI orientation anisotropy than the less degenerated samples. Understanding the background of relaxation anisotropy on a molecular level would help to develop new MRI contrasts and improve the application of previously established quantitative relaxation contrasts.

most commonly prevalent in the weight-bearing joints such as the knee and hips. [2][3][4] Degenerating hyaline cartilage undergoes irreversible changes both in structure and function. 5 Besides being irreversible, there are no curative treatments for OA at the moment 6 .
X-ray imaging has traditionally been used for diagnosing OA, but magnetic resonance imaging (MRI) has been established as a good alternative to detect structural changes related to the disease. 7 MRI provides not only visual images but also quantitative parameters which could be used to describe macromolecules and their interactions. 8 Healthy cartilage consists of collagen matrix in which collagen fibers are oriented in a strict organization. 9 Earliest signs of OA are associated with disruptions of the collagen network and include tissue softening and surface fibrillation. 3,5,10 Based on the orientation of the collagen fibers, noncalcified cartilage is generally divided into three structural zones: superficial (SZ), transitional (TZ) and radial zone (RZ). [11][12][13] In the radial zone, the orientation of the fibers is perpendicular to the surface and the organization is the highest, causing the highest anisotropic properties. 9,14,15 With MRI it is possible to acquire high-resolution images from joints and distinguish between the cartilage layers. A layer-wise 16 or voxel-based analysis 17 is proposed to provide better analysis of cartilage quality than full thickness values.
Multiple quantitative MRI relaxation parameters have been shown to be sensitive to tissue orientation in the main magnetic field B 0 , and this directional dependence is called relaxation anisotropy. 18,19 The term "magic angle effect" has been used to describe the specific angle, that is, 54.7°, at which the residual dipolar interaction vanishes and the orientation dependent relaxation times reach their maximum value. 20 Generally, T 1 is not affected by the tissue orientation, 21 while T 2 has the strongest orientation dependence. 14,19,22-24 T 2 orientation dependence is generally explained to result from the dipolar interaction of water molecules, the arrangement and movement of which is restricted by the oriented collagen fibers. 20,25 Similarly, continuous wave (CW−)T 1ρ 26 and adiabatic T 1ρ , which have been studied as potential markers for cartilage properties or degeneration [27][28][29] , both demonstrate varying degree of orientation sensitivity. 19,24 In articular cartilage, the water molecules are either tightly bound to the proteoglycans and collagen macromolecules, or loosely bound, that is, exist as free water. 30 Degeneration of cartilage initiates with fibrillation of the superficial tissue and the loss of proteoglycans, 31 and thus the amount of free water increases, which can be generally observed as a prolongation of the relaxation times. 32 The magic angle effect may be seen as an artifact which can complicate the diagnosis of OA. 35,36 However, parameters sensitive to the orientation also seem to be sensitive to degenerative changes in cartilage. 19 The purpose of this study was to investigate how the orientation anisotropy of different quantitative MRI parameters depends on the degeneration of articular cartilage using cadaveric human tissue samples.

| Optical density and quantitative polarized light microscopy
After decalcification, the samples were cut along the same plane that was used in MR imaging. Subsequently, the samples were dehydrated, embedded in paraffin, and cut into 3 µm thick sections. After the cutting, the paraffin was removed and the sections for optical density

| Scoring
Histological scoring was used to evaluate the severity of OA in the samples and was conducted with the Osteoarthritis Research Society International (OARSI) grading system. 43 (Table S1). In the SZ, T 2 had a positive correlation with the equilibrium modulus. All calculated correlations are presented in the supplementary material (Tables S1 and S2).

| DISCUSSION
In this study, cadaveric human articular cartilage with variable spontaneous degeneration was investigated with multiple quantitative relaxation time measurements. Orientation anisotropy of the MRI parameters was studied by rotating the samples in the main magnetic field. Orientation anisotropy was quantified and the findings were compared with those of the previous study on intact bovine cartilage samples. 19 Orientation anisotropy profiles of multiple MRI parameters were of similar shape as previously noted for bovine articular cartilage 19 : T 2 has the clearest orientation dependence pattern followed by CW − T 1ρ with low spin-lock amplitudes (eg, 400 Hz) (Figure 4). In this study, sample 1 with OARSI grade 2 showed a slower transition of relaxation anisotropy between the transitional and the radial zone as compared with the intact bovine samples in the previous study. 19 For sample 3 with an OARSI grade 4, the shape of the relaxation anisotropy profile was very similar with those of the intact bovine samples in the previous study, 19 with fast change from the transitional zone to the radial zone, even though according to histology the surface was already fissured. The degeneration of the superficial layer of OARSI 4 samples made it challenging to apply zonal analysis on the histological and MRI parameters. However, depth-wise variation was observed in all the parameters and thus zonal analysis was applied in addition to the bulk value analysis. The definition of the radial zone was considered the most reliable as it comprises most of the cartilage thickness, and thus the radial zone values and correlations were looked into at more detail ( Figure 6).
While T 1 is generally understood to be orientation independent, statistically significant differences were still observed between the different sample orientations. The differences were in the scale of 10 ms, which is relatively small compared with T 1 values of 1000 ms, so their clinical relevance remains a question ( Figure 3). However, for T 2 and CW − T 1ρ with a low spin-lock amplitude (400 Hz), changes were prominent for samples of both early and advanced degeneration. In addition to zonal averages, also bulk values over the whole depth of cartilage were calculated for relaxation parameters to reflect a lower resolution setting. In clinical MRI, the resolution is typically too low to distinguish between the different histological zones of cartilage, 46 but the orientation anisotropy is observed also in the bulk value and thus the effect cannot be neglected. 47 Orientation anisotropy of the relaxation parameters is the highest and relatively stable in the radial zone ( Figure 4). This is true also for PLM anisotropy (Figure 2). Cartilage thickness does not appear to be a defining factor for the relaxation anisotropy, as a thick sample can have low radial zone anisotropy (sample 4, Figure 4) and a thin sample high radial zone anisotropy (sample 2, Figure 4). On the other hand, the thickness is known to vary with degeneration, starting with initial thickening (swelling) followed by eventual degeneration. 48 Different samples seemed to be variably de- Overall, the anisotropy decreases as the quality of cartilage decreases and more degenerative signs appear, as described by the OARSI grading system ( Figure 5). Samples with less degeneration have higher anisotropy, congruent with the idea of a higher degree of order in less degenerated tissue and also demonstrate less variation in anisotropy than samples with advanced degeneration. Intact bovine cartilage has been reported to have T 2 orientation anisotropy of 80%, and CW − T 1ρ with 250 Hz spin-lock 65% and with 500 Hz spinlock 45%. 19 Assuming that CW − T1 ρ with 400 Hz falls between the latter two, both T 2 and CW − T 1ρ anisotropy values are higher for intact bovine cartilage samples compared with the degenerated human samples of the present study ( Figure 5). The effect is the most prominent in the superficial zone (Figures 4 and 5).
Biomechanical properties seem to be able to clearly distinguish only one or two of the grade 2 samples (Figure 6). No correlation was found between the biomechanical parameters and MRI orientation anisotropy. Of remarkable curiosity is the finding that MRI relaxation anisotropy did not positively correlate with the PLM anisotropy either, and for adiabatic T 1ρ there was even a statistically significant, though not strong, negative correlation. This is in contrary to the previously reported findings in intact bovine samples. 19 It seems probable that in severely degenerated cartilage, the PLM anisotropy does not vary in a systematic and linear manner. In general, the MRI anisotropy, especially of T 2 , was much greater than the PLM anisotropy, possibly reflecting the limitations of the sub-optimal assessment of PLM anisotropy via the entropy method. 15,19 However, MRI anisotropy of T 2 and CW − T 1ρ with 400 Hz spin-lock correlated with the optical density, which is proportional to the amount of proteoglycans. 42 PLM anisotropy reflects the properties of the collagen fiber network, not of the proteoglycans, especially given that the histological slices for PLM were carefully digested to remove the proteoglycans.
Generally, it is believed that the organization and alignment of collagen fibers create the orientation dependent properties of cartilage. 18 Xia 21 suggested in 1998 that proteoglycans might have an effect on orientation anisotropy, and later he phrases a possible explanation 49 that "although the T 2 -anisotropic characteristic of cartilage is linked to the collagen fibril orientation in the tissue, the water-proteoglycan interaction influences the mobility of most water and "amplifies" the prevailing directional orientation of collagen fibers to make it measurable by NMR." Trypsin digestion can be used to deplete proteoglycans from the samples, but in one study the trypsin treatment alone did not make the relaxation anisotropy disappear in the case of intact cartilage samples. 50 In conclusion, anisotropic properties of cartilage seem to be a property of the collagen matrix and proteoglycan content together, as they both affect the movement of water molecules. In further studies, collagen content should be separately measured to better understand the roles of both macromolecules.
The most prominent limitation of this study is the small sample size, and that only patellar cartilage was analyzed. Also, to measure anisotropy more reliably, more than three sample orientations should be measured. Therefore, further research with samples of different levels of degeneration and measurements at multiple orientations is needed to obtain a clear understanding of the MRI relaxation anisotropy in degeneration of cartilage. According to our results, the anisotropy of the MRI parameters changes as the cartilage degenerates.
The difference is observable at least for badly degenerated samples, but for understanding changes taking place early in the degeneration, samples with OARSI grades 0 to 1 should be included and analyzed.
Intact samples or samples with minimal degeneration were unfortunately not available in the studied cadaveric sample set,  (Table S1). Taken together, the effect of orientation anisotropy should not be neglected when making quantitative MRI measurements of cartilage or other highly ordered tissue types. In