Strategies involving mesenchymal stem cell (MSC) osteogenic differentiation have emerged as promising tools in regenerative medicine. These work by either promoting in situ regeneration, or through newly-fabricated tissue grafts, potentially overcoming clinical complications related to more conventional therapies. However, bioengineered bone tissue and cells do not yet achieve the required characteristics for optimal in vivo bone repair, resulting in weak or inadequate new tissue and/or biocompatibility issues. It is, therefore, crucial to further understand the differentiation of MSCs, to optimize their osteogenic commitment. The use of metabolites in this context was recently recognized, as some appear to have the potential to induce osteogenesis, while also ensuring the lack of (or residual) co-differentiation into other lineages; i.e. they can be both potent and specific. This project aims to optimize a bioreactor to drive osteogenic differentiation of human adipose MSCs (hAMSC) while allowing us to survey the cell metabolome. Based on detailed metabolomic screens, we will identify and exploit osteoinductive metabolites that will ensure the predominance of osteoblast-like cells, removing the need for use of osteogenic factors that can drive off-target , unwanted differentiation, such as dexamethasome. There is existing knowledge on the endo- and exometabolome adaptations of hAMSCs in 2D cultures and this has provided information on metabolic adaptations of osteogenesis and unveiled a set of preliminary osteoinductive metabolic candidates. The focus of this project is the translation of these approaches to 3D cultures, using an innovative encapsulation method to support hAMSC mono- or co-cultures (with endothelial cells, known to provide osteogenic factors) alongside mechanical cues provided by our bioreactor to promote osteogenesis. This non-chemical osteogenic differentiation protocol will allow detailed metabolomic analysis of the time-dependent intra- (endometabolome) and extracellular (exometabolome, including the secretome) and metabolite changes will help refine the previously proposed osteoinductive metabolic candidates, while unveiling new ones resulting from 3D cell-cell and cell-niche crosstalk. We will look for lead metabolites that can effectively and specifically stimulate osteogenic differentiation in 3D cultures in normal culture conditions with no co-differentiation into other lineages. The metabolic analysis thus generated will be confirmed by isotopic tracing studies and proteomics of intra- and extracellular environments including the vesicles secreted during MSC differentiation that are important for mineralization. Besides metabolomics and proteomics, synchrotron-based infrared microspectroscopy will provide sub-cellular scale information on the specific role of lipids and proteins in cell plasma membranes (and their fluidity), the cytoskeleton and the collagen extracellular matrix throughout osteogenesis, as a function of different 3D culture variables (mono-/co-cultures,absence/presence of mechanical force). To our knowledge, the combination of the above-mentioned biomechanical and analytical techniques represents an innovative approach to characterize the osteogenic differentiation of MSCs, providing a unique insight into the dynamic biochemical and cell-niche crosstalk adaptations, ultimately offering the means to optimize osteogenic lineage commitment using osteoinductive metabolite candidates. This project will culminate in the development of an optimized bioreactor (easily monitored overtime by exometabolomics), where osteogenesis can be enhanced with bioactive osteoinductive metabolites, ideally minimizing/excluding the addition of traditional osteoinductors. This will allow us to engineer high-quality bone tissue in a scientifically informed manner, to support a higher rate of success for biomedical applications.