Supplementary file 1_Glucose concentration modulates bovine intervertebral disc response under physiological loading.docx

IntroductionEx vivo organ culture is a widely accepted model in intervertebral disc (IVD) research, providing valuable insights into pathology and potential new treatments. Many ex vivo culture protocols utilize high concentrations of glucose (25 mM), which do not reflect IVD physiology in vivo. In this study, we investigated effects of varying glucose concentrations on bovine IVDs in a bioreactor-loaded organ culture. In addition, given the growing body of literature showing differences in male vs. female in connective tissue diseases, we verified whether sex affected the responses.
MethodsWhole IVDs were cultured under physiological loading (0.02–0.20 MPa, 0.2 Hz, for 2 h per day) with different glucose supplements (25 mM, 11 mM, and 5.5 mM) for 1 week. The effects of glucose supplementation on IVD cell viability, metabolism, disc height, glycosaminoglycan (GAG) release, and the expression of key anabolic and catabolic markers were analyzed.
ResultsDecreasing glucose concentration had a dramatic influence on the viability of bovine IVD cells. Under 25 mM glucose, high viability was maintained after 7 days, reaching approximately 80% in the annulus fibrosus (AF) and 72% in the nucleus pulposus (NP). At the lower glucose concentrations (11 and 5.5 mM), cell viability decreased significantly to approximately 31% and 7.5% in the inner AF, and 27% and 11% in the NP. Metabolomic analysis showed reduced levels of metabolites related to β-oxidation, antioxidant protection, and osmoregulation from day 1 to day 7, suggesting an adaptive response to the ex vivo environment. Although no significant sex differences were observed in cell viability, the expression of apoptotic markers in NP was significantly higher in the male cohort for the 5 mM glucose groups. Additionally, anabolic and glucose transporter markers were significantly elevated in the male compared to the female cohort in a region- and glucose-dependent manner, underscoring the sex-dependent responses of disc cells to varying glucose concentrations.
ConclusionThis study enhances our understanding of bovine disc cell metabolism during organ culturing. Furthermore, the findings emphasize the need to optimize nutrient and loading conditions in ex vivo IVD models to more closely mimic in vivo physiology.