Integrated nanoindentation-Raman spectroscopy reveals microscale biomechanical and molecular biomarkers of early intervertebral disc degeneration in a rat model.

Intervertebral disc degeneration (IVDD) is a major cause of chronic lower back pain, yet its early microscale mechanisms remain poorly understood, hindering timely intervention. This study employed an integrated multimodal approach-combining instrumented nanoindentation, Raman spectroscopy, MRI, and histology-in a controlled rat model (healthy, moderate, and severe IVDD induced via caudal puncture) to identify early, pre-structural biomarkers of degeneration. Nanoindentation uncovered significant, stage-dependent biomechanical alterations: progressive softening (decreased reduced modulus, E r ) in the nucleus pulposus (NP) and complex stiffening responses in the annulus fibrosus (AF) regions. Importantly, NP softening was detectable in the moderate stage, preceding overt structural changes. Raman spectroscopy revealed distinct molecular signatures: degenerated NP exhibited proteoglycan (PG) loss (revealed (C-O)νs/Phe, (COO-)νs/Phe), altered collagen, and increased cellular distress (elevated Lipid-to-Protein ratio), while AF regions showed collagen disorganization and PG depletion. Notably, a strong quantitative correlation was established between NP E r and the functional PG marker (COO-)νs/Phe (r = +0.76), confirming that mechanical softening is a direct consequence of osmotic failure. This integrated analysis delineates unique degeneration pathways for the NP, the transition zone or inner AF (IAF), and outer AF (OAF), linking molecular shifts to mechanical failure mechanisms. The identified microscale biomarkers, particularly early NP softening and specific Raman ratios, hold strong potential to revolutionize early IVDD diagnosis and guide the development of targeted, region-specific precision therapies.