Metabolic phenotype and patient characteristics associate with inter-donor variability of osteogenic differentiation in human adipose stem cells

Adipose-derived stem/stromal cells (ASCs) are promising candidates for bone tissue engineering due to their abundance, accessibility, and osteogenic differentiation capacity. However, substantial inter-donor variability limits their standardized clinical application. This study investigated whether metabolic characteristics and donor-related parameters are associated with ASC osteogenic differentiation potential. ASCs were isolated from human subcutaneous adipose tissue (n = 26) and cultured under osteogenic conditions. Cell viability was evaluated at days 2 and 14, including cell proliferation (crystal violet staining), overall metabolic activity (resazurin conversion), as well as glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE). Osteogenesis was quantified by alkaline phosphatase activity, calcium deposition, and secretion of SPARC and osteocalcin (BGLAP). Osteogenic induction significantly enhanced osteogenic markers and mineralization across donors and was accompanied by a distinct metabolic phenotype characterized by reduced per-cell metabolic activity and glucose uptake, and elevated mitochondrial membrane potential. Early (day 2) metabolic parameters did not predict subsequent mineralization capacity, whereas metabolic activity at day 14 correlated inversely with calcium deposition. Donor age, body mass index, and blood glucose levels were not associated with osteogenic outcomes. These findings indicated that inter-donor variability in ASC osteogenesis was accompanied by a characteristic late-stage metabolic signature and suggested that metabolic profiling may support functional characterization of osteogenic differentiation potential.