Toward Standardized Ex Vivo Joint Models: Impact of Glucose and Oxygen Levels for Enhanced Tissue Maintenance

Ex vivo models bridge in vitro and in vivo systems by preserving native extracellular matrix architecture and multicellular interactions. In articular joint research, osteochondral–synovial co-cultures are particularly valuable for studying bone–cartilage crosstalk and synovial inflammatory regulation. However, a lack of standardized culture conditions regarding glucose and oxygen, two key regulators of cellular metabolism, limits reproducibility and translational relevance. This study aims to define how glucose and oxygen conditions influence joint tissues maintenance in an ex vivo model. Bovine osteochondral explants and synovium are harvested from the stifle joint and co-cultured using either high glucose DMEM (HG, 4.5 g/L) or low glucose DMEM (LG, 1 g/L) under hyperoxic (21% O 2 ) or physioxic (5% O 2 ) conditions. Cell viability, gene expression, and metabolomic profiles are evaluated across tissues. LG conditions increase cell death in the deep zone of cartilage and in subchondral bone. Gene expression and metabolomic analyses reveal tissue-specific effects of glucose and oxygen. In cartilage and bone, glucose-dependent gene regulation and metabolic changes occur under hyperoxia but are largely absent under physioxia, indicating buffering of glucose responses. Gene-specific sensitivity to glucose and oxygen is observed in bone and synovium; however, glucose-induced metabolic responses persist under physioxia only in synovium. Overall, these findings identify oxygen and glucose as critical modulators of joint tissue physiology and support the use of HG, physioxic culture conditions to improve cell viability and stabilize molecular outcomes in ex vivo joint models. This optimized ex vivo model provides platforms for investigating mechanisms relevant to joint-related diseases. Abstract Figure This study evaluates the effects of glucose concentration and oxygen tension in an ex vivo joint co-culture system to define optimal culture conditions. High glucose medium and physioxic conditions support tissue viability, preserve homeostasis, and enhance the physiological relevance of the ex vivo model.