Biomass accumulation in chondrocyte metabolic modelling: Incorporating extracellular matrix proxies to predict tissue engineering outcomes

Metabolic modeling in chondrocytes plays a pivotal role in advancing our understanding of cellular function. These techniques have been used to study degenerative joint diseases (e.g. osteoarthritis), mechanotransduction, and more recently to optimize strategies for cartilage tissue engineering. Incorporating tissue formation into metabolic flux analysis is inherently challenging due to the complexity of linking metabolic activity to extracellular matrix (ECM) accumulation. Many ECM macromolecules are synthesized using metabolites derived from central carbon metabolism, but direct modeling of their accumulation remains complex. This study establishes a novel methodology for incorporating ECM synthesis into metabolic flux analysis (MFA). By utilizing chondroitin sulfate and hydroxyproline as measurable metabolic proxies for proteoglycan and collagen production, we demonstrate a framework for linking metabolic inputs with tissue formation. Extracellular flux data for glucose, lactate, carbon dioxide, glutamine, and glutamate, along with mass isotopomer distributions, were sourced from previous studies involving three-dimensional high-density cultures of articular cartilage tissue constructs. Additionally, the conditioned culture media used in these studies was used to quantify the production rates of chondroitin sulfate and hydroxyproline. Using a modular network model, proteoglycan and collagen metabolism were assessed independently, and in combination, with sensitivity analyses on ECM retention assumptions. Predicted proteoglycan production aligned well with previously observed trends; however, predicted collagen production was less consistent. These findings offer a novel approach for linking metabolic inputs with ECM production, advancing our ability to predict tissue formation and address key challenges in cartilage tissue engineering.