For FTIR microspectroscopy, -thick sections were cut with a microtome. For dewaxing, the sections were immersed in series of xylene baths () and subsequently washed with ethanol. The sections were placed onto 2-mm-thick ZnSe windows and were let dry in room air. The sections were measured using the Perkin Elmer Spotlight 300 FTIR imaging system (Perkin Elmer, Shelton, Colorado) with spectral resolution of , pixel size of , and using four scans per pixel. A rectangular region of interest (ROI) extending from cartilage surface to cartilage–bone junction was imaged from each section (Fig. 1). The ROI width was set to , whereas the ROI height, which varied depending on the cartilage thickness, was on average . On average, 1088 spectra were collected per sample. A dry air purge (Parker Balston, Haverhill, Massachusetts) was used to minimize the variation in the measurement conditions. The spectra of each section were first averaged to obtain one mean spectrum. Thereafter, resonant Mie scattering correction (RMieSC) algorithm18,19 was used to remove scattering-related effects from the spectra. After RMieSC, the spectra were vector normalized. Second derivative spectra were calculated using Savitzky–Golay algorithm with 11 smoothing points. In this paper, we use the following definitions for spectral regions: the amide I (1590 to ), the amide II (1500 to ), the mixed region (1200 to ), and the carbohydrate region (800 to ).20 All data analysis was done in MATLAB (R2015a, MathWorks, Inc., Natick, Massachusetts).