(174av) Towards Identification of Critical Quality Attributes of Chondrogenic Microtissues-a Metabolomics Perspective

Tissue Engineering aims to replace lost or regenerate malfunctioning tissues by man-made biological substitutes, holding great promise to provide effective therapies for diseases that were untreatable until now. The use of cartilage intermediate templates that will ossify upon implantation following endochondral ossification is becoming a promising bone tissue engineering strategy for the healing of large defects.Recently, the use of 3D microtissues is becoming a standard for bone tissue engineering approaches, as this format allows cell-cell and cell-extracellular matrix interactions. However, this promising process is not fully characterized as single parameter measurements such as evaluation of a few genes cannot robustly predict the final product quality while no mechanistic insight is gained. Considering the role of metabolism as a key regulator of stem cell fate and the high sensitivity of metabolomics, this study aims to identify metabolic quality attributes indicative of a functional cartilage intermediate TE construct. In particular, we focus on the use of metabolomics for the comprehensive characterization of chondrogenic differentiation using spheroids, improving the current conventional set of markers for monitoring cell and tissue quality.

LC-MS (liquid chromatography-mass spectrometry) tracer analysis was conducted to investigate metabolic alterations during chondrogenic differentiation of spheroids of hPDCs (human periosteum derived stem cells). Subcultured cells were seeded in a 24 well plate containing microwell agarose insertsto create aggregates composed of 250 cells according to a previously established protocol that allowed high throughput production of microaggregates with controlled cell density per aggregate. To induce chondrogenic differentiation, the microwells were filled with 2 ml of an in-house developed chemically defined chondrogenic differentiation medium. Twice a week 50% of the medium was refreshed. ¹³C labeled glucose, glutamine but also serine and aspartate have been used, as these metabolites showed significant differences between the time points of interest in a prior exometabolomics study. Samples were analyzed at day 0, day 14 and day 21, as these time points capture the transition of hPDCs-derived chondrocyte phenotypes (proliferating, prehypertrophic, hypertrophic state).

Our results showed that hPDCs spheroids after the first week of culture presented a highly glycolytic metabolism, with high glucose consumption and lactate production rates. Additionally, progressive ¹³C glucose enrichment in palmitate from 0% at day 0 to 8% at day 14 and 22% at day 21 was observed, suggesting activation of fatty acid synthesis. Accordingly, whole-transcriptome analysis revealed upregulation of glycolytic genes and genes involved in fatty acid metabolism of the Day 21 and Day 14 when compared with Day 0. Furthermore, we observed ¹³C glutamine enrichment in proline from 0 % at day 0 to 20 % at day 14 and 42,5 % at day 21 and a similar trend of ¹³C glutamine contribution to hydroxyproline (from 0 % at day 0 to 35 % at day 14 and 38 % at day 21). These data indicate that glutamine contributes progressively more to proline biosynthesis, an important amino acid for collagen production. In agreement, we observed significant production of extracellular matrix (ECM) after Day 7. To the best of our knowledge, this is the first metabolomics analysis of chondrogenic differentiation of a human progenitor population cultured as spheroids. This study suggest that the consecutive stages of chondrogenic differentiation of hPDCs are characterized by specific metabolic adaptations and highlight the importance of quite unexplored metabolic pathways such as fatty acid and glutamine metabolism for the regulation of chondrogenic differentiation.