Label-free analysis of grade-specific molecular alterations in osteoarthritic subchondral bone using Raman spectroscopy coupled with MCR-ALS.

Changes in subchondral bone metabolism are central to osteoarthritis (OA) progression, highlighting the need for sensitive, label-free analytical approaches capable of resolving molecular-level alterations in complex biological tissue matrices. Raman spectroscopy is a powerful, non-destructive tool for detecting molecular changes in intact pathological tissues; however, overlapping spectral bands obscure accurate interpretation. In this study, Raman spectroscopy combined with multivariate curve resolution-alternating least squares (MCR-ALS) was applied to human subchondral bone at different stages of osteoarthritic degeneration to identify grade-specific molecular changes. The MCR-ALS approach enabled decomposition of overlapping Raman spectra into distinct biochemical components attributed to mineral, lipid, and collagen. Comparative statistical analysis revealed grade-dependent biochemical alterations in osteoarthritic subchondral bone. No significant changes occurred in mineral or lipid content at the early stage; however, significant mineral reduction and enhanced lipid accumulation were observed in advanced-stage degeneration. Collagen content exhibited an initial increase in the early stage, followed by a substantial decline in the advanced stage, accompanied by progressive disruption of its structural organization. Deconvolution of the amide I band further enabled improved characterization of collagen structural changes in intact tissue. These findings demonstrate that the Raman-MCR approach effectively resolves complex biological spectra, sensitively detects stage-specific molecular alterations in osteoarthritic subchondral bone, and offers a robust analytical framework for the label-free characterization of intact tissues.