Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage.

ABSTRACT

OBJECTIVE: Chondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in hypoxia-inducible factor 1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. METHODS: Primary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4-16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). RESULTS: Increasing media volumes resulted in higher accumulation of cartilaginous ECM (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the tricarboxylic acid (TCA) cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. CONCLUSIONS: Elevated media availability upregulates ECM synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in ECM accumulation.