Synopsis

gagCEST has been suggested as a method for in vivo evaluation of cartilage GAG content. The main type of GAG in cartilage is chondroitin sulfate (CS), most commonly CS-A and CS-C. Validation of gagCEST have mostly been performed using CS-A but the main type in mature human articular cartilage is CS-C. In this study we evaluate the gagCEST effect from GAG in different forms. Our results indicate that mainly CS-A is contributing to gagCEST effect in cartilage, while no or little effect is seen from CS-C. gagCEST may therefore not correctly reflect the GAG content of human articular cartilage.

Introduction

Proteoglycans, mainly aggrecan, are major components of articular cartilage and the negatively charged glycosaminoglycan (GAG) side chains are key elements for the tissue function¹. gagCEST has been suggested as a possible method for in vivo evaluation of osteoarthritis progression through assessment of cartilage GAG content.

The main GAG type in aggrecan, chondroitin sulfate (CS), exist in two common variations, sulfated at the 4^th (CS-A) and 6^th (CS-C) carbon of the amino sugar, respectively². The proportions of the two types changes with age and in mature human articular cartilage the 6-sulfated CS-C is dominating³. Attempts to validate the gagCEST method however, commonly includes phantoms with CS-A.

In this study we aim to investigate how well gagCEST reflects the actual GAG content of articular cartilage through gagCEST experiments with different types and forms of GAG.

Methods

Two series of phantoms were made using CS-A (85 % 4- and 15 % 6-sulfated, bovine trachea, Sigma-Aldrich) and CS-C/A (50 % 4- and 50 % 6-sulfated, shark cartilage, Sigma-Aldrich) dissolved in phosphate buffered saline (PBS) to seven different concentrations (0-30 mg/ml). Additionally, two phantoms were made with intact aggrecan (protein bound CS) from calf articular cartilage, extracted using guanidinium hydrochloride. The purified aggrecan was freeze dried and dissolved in PBS to concentrations of 10 and 20 mg/ml. pH of all phantom solutions were set to 7.3-7.4.

18 samples of ex vivo cartilage were collected from eight human femoral heads (harvested during hip replacement and stored at -20 °C). With the femoral heads immersed in PBS, gagCEST was measured in the cartilage before the samples were cut out. GAG content was subsequently measured in the cartilage samples using Alcian blue precipitation, a standard method for determination of GAG concentration⁴.

gagCEST measurements were conducted at 7 T (Philips Achieva) using a 32 channel head coil (NOVA). Saturation was achieved using a pulse train of 5 99 ms long RF pulses with 800° flip angle and 1 ms interpulse delay. 52 frequency offsets were imaged in an interval of ±2.5 ppm around the water frequency, using FFE readout with TE/TR=2.4/10 ms, FOV=120x120 mm², voxel size=1x1x3/0.7x0.7x3 (phantoms/ex vivo) mm³ and bandwidth=338 Hz/pixel. Correction for B₀ inhomogeneity was made using WASSR⁵.

Magnetization transfer ratio asymmetry (MTR_asym) was calculated for a frequency interval of 0.7-1 ppm from water, as a measure of gagCEST effect. Mean values of MTR_asym were calculated within regions of interest (ROIs) drawn within each phantom and cartilage sample.

Results

For both of the phantom series, MTR_asym resulted in a clear linear dependence on CS concentration. However, the gagCEST effect was markedly lower (on average 52 % lower in mean MTR_asym per mg/ml CS) for the CS-C/A phantoms than the CS-A phantoms (figure 1). When adjusting the scale to reflect 4-sulfated CS only, the gagCEST effect of the two phantom series are very similar (figure 2).

For the two phantoms containing intact aggrecan, the gagCEST effect was similar to the effect of the CS-A phantoms (figure 1).

For the human ex vivo cartilage samples, presumably consisting of mainly 6-sulfated CS, only a weak correlation (Pearsons rho = 0.55, 95% CI: 0.094, 0.81) was found with GAG concentration as assessed by Alcian blue (figure 3).

Discussion

Although there is a clear linear dependence of MTR_asym on CS concentration, there is also a clear difference in gagCEST effect between the CS-A and CS-C/A phantoms, suggesting that not all CS types contributes equally to the gagCEST effect. It seems that gagCEST works very well for 4-sulfated CS while there is no or little effect from 6-sulfated CS.

The immature calf articular cartilage contains a larger proportion of 4-sulfated CS which is also reflected in the results of the aggrecan phantoms. The similar result of CS bound in aggrecan and free CS (CS-A) suggests that the aggrecan structure does not have a large impact on the gagCEST effect.

GAG in mature human articular cartilage consists of mainly 6-sulfated CS. Our phantom experiments hence indicate that the gagCEST method may not correctly reflect the total GAG content in vivo. The fairly poor correlation between gagCEST effect and GAG content observed in our ex vivo experiments also supports this conclusion.

Conclusion

Acknowledgements

References