Stepwise micro-CT analysis of sample processing effects on the 3D morphology of human intervertebral discs.

Objective: Structural alterations of the intervertebral disc (IVD) contribute to low back pain and affect spinal biomechanics. Three-dimensional imaging of disc soft tissues requires sample preparation steps that may introduce artifacts and alter native morphology. This study presents a stepwise contrast-enhanced micro-CT evaluation of how sample processing affects the 3D morphology of human IVDs, aiming to quantify changes in volumetric parameters and radiodensity across preparation stages. Design: Human IVDs were imaged using micro-CT at four sequential stages: (i) frozen, (ii) thawed, (iii) after formalin fixation, and (iv) after Lugol iodine contrast staining. Qualitative and quantitative analyses assessed changes in tissue volume, lesions, rim tears, calcifications, and radiodensity across stages. Thawed samples served as the volumetric reference. Results: Frozen discs preserved annulus fibrosus lamellar architecture but showed reduced volume relative to the thawed state and low radiodensity. Formalin fixation induced the largest morphological changes, increasing mean disc volume by 21.5% and causing deformation associated with nucleus pulposus swelling. Subsequent staining caused partial shrinkage relative to the fixed condition but markedly increased X-ray attenuation (148.6% vs frozen), enabling improved visualization of internal disc microstructures. Internal lesions remained detectable across preparation stages, whereas calcifications appeared reduced after staining due to decreased contrast with surrounding soft tissue. Conclusions: Each preparation step introduces distinct and quantifiable effects on IVD morphology and imaging contrast. These findings provide a methodological reference for optimizing contrast-enhanced micro-CT workflows and for distinguishing biological features from preparation-induced artifacts in IVD research.