Fig 1: Schematic representation of the osteogenic differentiation timeline (days 0–21) showing the time points where medium exchanges (M) and sample collection (day numbers in pink boxes), in triplicate, were carried out. Cell pellets were used for metabolite extraction and subsequent NMR analysis of the polar phase. Cell lysates were used for the dsDNA and calcium evaluation, whereas medium samples were used for the osteogenic markers assessment (namely, OCN and OPN). M, medium exchange; †, OPN measurement was attempted, however, results were too low and variable to be discussed, possibly due to potential high retention of the protein in the extracellular matrix. Abbreviations: dsDNA, double-stranded deoxyribonucleic acid; OCN, osteocalcin; OPN, osteopontin.
Fig 2: Schematic representation of the main metabolic fluctuations observed in this work throughout the osteogenic differentiation of hAMSCs, along with a simplified depiction of the vesicle-mediated mineralization. Metabolite names in bold indicate compounds identified (additional detected metabolites may be found in Table S1), whereas those that were observed to vary carry a schematic scale as follows: unchanged metabolic levels are represented in yellow, increases are in red, and decreases are in blue. In some cases, an indication is given if the metabolite was seen to vary only between day 0 and day 21 or before and after day 7. Three-letter code is used for amino acids. Abbreviations: ALP, alkaline phosphatase; CDP, cytidine diphosphate; CoA, coenzyme A; DAG, diacylglycerol; DHAP, dihydroxyacetone phosphate; FA, fatty acid; FAD, flavin adenine dinucleotide (oxidized); FADH2, flavin adenine dinucleotide (reduced); GAP, glyceraldehyde 3-phosphate; Gla, γ-carboxyglutamate residues; HA, calcium hydroxyapatite; IMP, inosine monophosphate; NADH, nicotinamide adenine dinucleotide (reduced); PRPP, phosphoribosyl pyrophosphate; NPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; OCN, osteocalcin; OPN, ostepontin; PHOSPHO1, phosphoethanolamine/phosphocholine phosphatase; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine. Other metabolites are abbreviated as shown in Figure 4. Some elements of this picture were adapted from Servier Medical Art (smart.servier.com) and are licensed under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license.
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