FT-IR SPECTROSCOPY AS AN INNOVATIVE TOOL FOR CHONDROGENIC PROTEIN QUANTIFICATION IN OSTEOARTHRITIS

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage degradation, where the loss of extracellular matrix components such as collagen (Col) and glycosaminoglycans (GAGs) plays a central role [1]. Fourier-transform infrared (FT-IR) spectroscopy has emerged as a promising approach to quantify chondrogenic protein markers in both normal and pathological conditions, providing a non-destructive method for analyzing biochemical compositions critical to tissue regeneration studies. FT-IR has been shown to effectively monitor disease progression and evaluate tissue repair in cartilage degeneration [2]. Widely used to study materials, FT-IR is increasingly applied to biological materials, including cells, tissues, and extracellular vesicles [3, 4]. FT-IR has not been used for chondrocyte research, although it has been shown to detect lipids, GAGs, collagen, and aggrecan bands in various spectral regions to study cartilage matrix formation [5]. The aim of this study was to evaluate the potential of FT-IR spectroscopy to quantify key chondrogenic protein markers (Col II, GAGs) in chondrocyte micromass cultures. Micromasses of 250,000 OA chondrocytes were obtained and maintained in DMEM supplemented with 10% fetal bovine serum and 50 μg/ml ascorbic acid at 37°C for 7 days. At days 2 and 7, gene expression and proteins analysis (Col II, GAGs) were performed and FT-IR spectra were collected using a Perkin Elmer Spectrum 2 instrument with a diamond crystal ATR. Spectra were acquired over the full range (400–4000 cm −1 ) with all bands scaled relative to an external PBS band. Spectra for each sample type showed high reproducibility. To evaluate Col II and GAGs, a deconvolution process was applied to separate overlapping spectral peaks, and the area under the integral curve was calculated for Col II and GAGs at 1370–1270 cm −1 and 960–1185 cm −1 respectively. Western blot analysis showed an increase in Col II and GAGs at day 7 compared to day 2. FT-IR spectroscopy validated the protein expression results, detecting the peak of Col II at 1338 cm −1 . Also, gene expression analysis confirmed an increase of these proteins at day 7. These results suggest that FT-IR spectroscopy is an efficient and reliable method for quantifying chondrogenic protein markers, in agreement with traditional techniques like Western blot, thus represents a promising tool in the field of OA cartilage tissue evaluation.