For each sample (or pair of cartilage punches) collected, HRMAS NMR spectra were obtained with a 11.7 T (500 MHz for 1H) Varian INOVA spectrometer (Varian Inc., Palo Alto, CA, USA) equipped with a 4 mm gHX nanoprobe at 1° C. This study followed the protocols established in previous studies that focused on optimizing the quality of HRMAS NMR spectra and maximizing tissue integrity (11 (link), 32 (link)). In brief, water pre-saturated 1-D spectra were acquired with 40,000 complex points over a 90° pulse, 20,000 Hz spectral width, a TE of 144 ms, and a TR of 4.2 s. In total, sample preparation, tuning, shimming, pulse width calibration, and spectral acquisition required approximately 1.5 hours per sample. Metabolite signals were quantified using the Electronic Reference to access In vivo Concentrations (ERETIC) method as established by Swanson, et al (33 (link)). The resulting spectra were referenced to 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) at 0 ppm.
For this study, the N-acetyl peak (2.04 ppm) and, when present, the Alanine peak (1.47 ppm) were quantified, as shown inFigure 2 . Previous work identified N-acetyl and Alanine as markers for total proteoglycan and collagen content in cartilage, respectively (11 (link)).
For this study, the N-acetyl peak (2.04 ppm) and, when present, the Alanine peak (1.47 ppm) were quantified, as shown in
