Preserving tissue integrity under the beam: high-energy, low-dose synchrotron CT for in situ imaging of bovine intervertebral discs

In situ tomography enables non-destructive, time-lapse imaging of biological tissues under load, offering insights into structural and mechanical changes. However, repeated scans can expose samples to high radiation doses, potentially altering tissue properties. This study evaluated the feasibility of low-dose synchrotron computed tomography (sCT) for high-resolution, in situ imaging of intact bovine intervertebral discs (IVDs), and assessed the effects of repeated x-ray exposure on mechanical, microstructural, and molecular integrity. Intact oxtail IVD segments were imaged using propagation-based phase contrast sCT at 54 keV. Scan parameters were optimised to achieve high image quality within 66 s per scan, resulting in a total absorbed dose of ∼30 kGy over six scans. Mechanical properties were assessed under cyclic loading, microstructural changes via digital volume correlation (DVC), and molecular alterations using Raman spectroscopy. High-resolution imaging of soft and calcified tissues was achieved. Changes in sample stiffness, hysteresis, or stress recovery between irradiated and control were not identified. DVC revealed no microstructural damage or strain accumulation in the calcified endplate. Raman spectroscopy indicated minimal changes in soft tissues, with bone showing slightly increased collagen crosslinking and reduced mineralisation. Overall, this study demonstrates that high-energy, low-dose sCT enables repeated imaging of musculoskeletal tissues without compromising integrity, supporting its application in dynamic, time-lapse imaging studies. Importantly, larger, intact samples, such as whole bovine IVDs, were imaged overcoming limitations of previous studies that relied on small animal models. This approach supports more physiologically relevant investigations of tissue mechanics and degeneration in complex systems. STATEMENT OF SIGNIFICANCE: This study demonstrates the feasibility of high-resolution, low-dose phase contrast synchrotron computed tomography (sCT) for in situ imaging of intact bovine intervertebral discs (IVDs), preserving mechanical, microstructural, and molecular integrity. By optimising scan parameters to achieve 66-second acquisitions at 54 keV, the total radiation dose was limited to ∼30 kGy - below the frequently cited threshold for tissue damage. Unlike previous studies constrained to small animal models, this work successfully applies these imaging capabilities to larger, more physiologically relevant samples, representing an important step toward models that better reflect native biomechanics. Importantly, no adverse effects were observed in tissue mechanics or microstructure, and only minimal molecular changes were detected. These findings support the use of high-energy, low-dose sCT for dynamic, multi-scan imaging of musculoskeletal tissues, enabling more accurate investigations of tissue biomechanics and degeneration.