Metabolomics als hulpmiddel voor het identificeren van kritieke eigenschappen van weefsel-geëngineerde kraakbeenachtige micro-weefsels

Delayed and non-union fractures represent a largely unmet medical need as current treatment options such as bone grafting are not fully efficient, especially for large bone defects. Skeletal tissue engineering, inspired by developmental processes, shows promise for bone and cartilage regeneration, potentially addressing this clinical gap. Despite progress in the field, challenges remain in identifying critical quality attributes that predict the functionality of tissue-engineered products, as well as in scaling up bioprocesses for clinical application. This PhD research investigates metabolomics as a tool for characterizing the differentiation of cartilaginous microtissues in static as well as dynamic culture (bioreactor) setups supporting future scale-up. The first phase of this PhD focused on metabolic profiling during chondrogenic differentiation of human periosteum-derived cells (hPDCs) into microtissues, a process that has demonstrated success in regenerating critical size tibial defects in small animal models. Stable isotope tracer analysis and exometabolomics were used to map the metabolic changes during differentiation, identifying pathways such as glutamine metabolism, which plays a role in early extracellular matrix (ECM) production and later matrix remodeling. These metabolic profiles align with existing knowledge of growth plate chondrocytes and support the recapitulation of native cartilage features. Notably, the project uncovered a previously underexplored metabolic pathway in this context—glucose-dependent de novo fatty acid synthesis—which increased progressively during microtissue differentiation. This pathway's role was further investigated through chemical inhibition experiments, which demonstrated its importance for the differentiation process and subsequent bone formation capacity of the microtissues in vivo. The second phase of the project involved the development and validation of a novel microbioreactor system for the dynamic culture of cartilaginous microtissues in suspension. Dynamic culture conditions, characterized by intermittent shear stress, were found to accelerate chondrogenic differentiation, promoting early commitment to hypertrophy, which is crucial for endochondral ossification. Computational modeling helped optimize the mechanical environment within the bioreactor to prevent shear damage while promoting efficient tissue formation. The dynamic culture also resulted in distinct metabolic signatures obtained from the culture medium, which may serve as non-destructive markers for monitoring tissue quality during scale-up processes. This work suggested that suspension culture in bioreactors could be a scalable platform for producing clinically relevant quantities of tissue-engineered constructs in the future.