Label-free resonance Raman spectral imaging reveals magnesium microsphere therapy attenuates oxidative damage in knee osteoarthritic rats

Knee osteoarthritis (KOA) is a prevalent chronic degenerative joint disorder hallmarked by progressive cartilage deterioration, localized inflammation, and declining joint mobility. Current approaches for evaluating therapeutic outcomes rely mainly on invasive methods that preclude longitudinal monitoring, or macroscopic imaging techniques that lack molecular-level biochemical specificity. To bridge this gap, we present a compact Resonance Scanning Confocal Raman Spectral Imaging (RSCRSI) system capable of non-invasive, real-time monitoring of biochemical tissue alterations throughout KOA treatment. This platform delivers a high spectral resolution of 0.29 nm alongside a lateral spatial resolution of 2.19 μm. We applied RSCRSI to monitor the efficacy of magnesium containing gelatin-methacryloyl microspheres (G-Mg) as a model therapeutic agent for KOA. Raman spectral biomarkers were quantified in vivo using characteristic vibrational bands, including cytochrome-related peaks at 1580 cm^-1 (mitochondrial redox state indicators), hydroxyapatite ν1-PO4^3- at 956 cm^-1 (mineralization), and collagen-associated peaks at ∼1452 and 1650 cm^-1. Raman spectral imaging of the gastrocnemius muscle and tibial cartilage in KOA rats demonstrated that G-Mg treatment successfully reduced mitochondrial oxidative stress, attenuated inflammatory signals, preserved collagen spectral features, and minimized subchondral mineral exposure. Therapeutic efficacy was further corroborated by microcomputed tomography (Micro-CT). This study establishes the compact RSCRSI system as a robust platform providing real-time, spatially resolved biochemical feedback for monitoring KOA therapies in vivo. Using novel G-Mg microspheres as a representative model, we directly visualized the treatment response, including reduced oxidative stress and cartilage preservation, providing a new paradigm for the precision management of KOA.