UC Berkeley

Lower back pain (LBP) is a leading global public health concern, affecting up to 80% of all adults at some point in their lives. While the pathophysiology of LBP is complex and multifaceted, approximately 40% of LBP cases arise from discogenic pain. The intervertebral discs are soft, cartilage-like tissues that exist between vertebrae of the spine, functioning to distribute load, dissipate energy, and facilitate motion. They are composed of distinct sub-tissues that work together to impart mechanical function to the disc; when these structures are damaged, the mechanical behavior of the disc joint is altered.Examining the effects of biological or environmental stimuli on intervertebral disc material properties can provide insight into potential sub-tissue mechanisms for LBP. Therefore, mechanical testing of disc joints is common in intervertebral disc research. However, there is no standard for such testing, making it difficult to compare studies or form cohesive conclusions. Therefore, this work aims to highlight the challenges of measuring the mechanical properties of disc joints, develop an experimental method for testing small animal disc joints, and apply this method to assess the effects of spaceflight on the murine lumbar spine. Through a collaboration with two labs in the United Kingdom (UK), this work aimed to establish cross-institution repeatability of measurements of material properties of bovine intervertebral disc joints. Despite using identical sample preparation and mechanical testing protocols, results were not repeatable; samples tested at the UC Berkeley lab exhibited significantly higher compressive stiffnesses. Further investigation revealed that due to the inclusion of a polymer plate in the load string, machine compliance contributed substantially to mechanical testing results. Therefore, load string compliance was comprehensively characterized and the test setup was modified accordingly. Additionally, cattle rearing practices were proposed as a source of variation, as hormonal growth promotants are commonly used in the American cattle industry but are banned in the UK. A brief preliminary investigation confirmed that caudal discs from organically raised cattle exhibit significantly different mechanical behavior than those from conventionally raised cattle. Thus, the collaboration with the two UK labs revealed that machine compliance and sample source may both complicate cross-institution repeatability of mechanical test results. Repeatability of results across institutions can also affect subsequent computational models. Attempts to validate a multiscale model of the disc identified disc geometry measurement methods were identified as a potential source of variation across studies. Mechanical testing results are commonly normalized to disc geometry, which is measured with a variety of methods, including computed tomography (CT), X-ray, manual measurement with calipers, and digital imaging. In an experiment to assess the differences between methods, compressive stiffness of bovine caudal disc joints was measured and normalized to geometries collected using these methods. In the worst-case scenario, differences in methods contributed to a difference of up to 65% in reported normalized compressive stiffness. This is likely due to alterations in sample geometry during experimental manipulation. We find that CT and X-ray imaging are the optimal geometry measurement methods. We developed a method for fixturing and testing murine intervertebral disc joints using CT. Mice are useful animal models because of their availability, fast growth, and ease of genetic manipulation; however, their small size renders experimental manipulation and mechanical testing challenging. Through an iterative process, the SAM Box V3 was developed to mount murine intervertebral disc joints to a universal tester with screw-side action grips. To maintain sample geometry between CT scanning and testing, a pilot study was conducted to determine the appropriate concentration of saline polyethylene glycol needed to maintain in situ disc geometry. Finally, a method for analyzing test data, previously published by Smit et al. 2011, was adapted and described in detail. These optimized methods were then applied to examine the effects of spaceflight on the murine lumbar spine. We measured the effects of microgravity on disc tissues collected from the 28-day Rodent Research-10 spaceflight mission, including disc biochemical content, disc joint mechanical behavior, and bone microarchitecture in the vertebrae and tibiae. Interestingly, spaceflight did not affect the tissues of the lumbar spine. Further examination revealed that increased physical activity of the mice during spaceflight, developed as a compensatory mechanism to microgravity, may reload the lumbar spine, mitigating the previously observed effects of mechanical unloading in spaceflight. This study points to yet another factor to consider when designing experiments focused on the lumbar spine: physical activity in a quadrupedal model organism may have different effects than in a bipedal organism and should be actively monitored during an experiment. In all, this work highlights important factors in the design of experiments for the assessment of intervertebral disc material properties, including machine compliance, sample source, geometry measurement methods, mounting protocol, and the use of animal models, providing guidance for future studies.