resumo
Strategies involving mesenchymal stem cell (MSC) osteogenic differentiation are currently promising tools in regenerative medicine. However, there is a need to further understand the biochemical processes underlying differentiation, in order to optimize their osteogenic commitment. Several intra- and extracellular events, typically occurring over a period of 21 days of osteogenic differentiation, are known to determine osteogenic efficiency – namely protein synthesis and transport to the extracellular matrix (ECM), and mineralization in the ECM (both processes possibly mediated by extracellular vesicles), in tandem with several intracellular metabolic adaptations [1,2]. The use of vibrational spectroscopic techniques with high sensitivity and specificity is known to unveil exquisite detail on subcellular events triggered by different stimuli, e.g. carcinogenesis or drug treatment [3,4]. However, such techniques have been underexplored to follow osteogenic differentiation [5]. This work presents, for the first time to our knowledge, a systematic study by complementary vibrational spectroscopy (FTIR and Raman) of human adipose-tissue MSC (hAMSC) across multiple timepoints of osteodifferentiation, with particular focus on biomolecular changes taking place at the intra- and extracellular levels. Along a 21-day period, hAMSC under osteogenic differentiation were compared to the corresponding control samples (absence of osteogenetic stimuli, only cell proliferation occurring). Figure 1 shows the micro-Raman and micro-FTIR spectra of hAMSC after 6 days of differentiation. It is expected that hydroxyapatite features start to appear at the early stages of osteoblastic formation, namely the intense 1(PO4) band at ca. 960 cm-1 which is a biomarker of osteogenesis [5]. Additionally, cellular adaptation during osteogenesis will be monitored through changes in membrane fluidity and cytoskeleton/ECM structural characteristics. Overall, our results demonstrate the promise of subcellular insights of osteogenesis as provided by vibrational spectroscopy in tandem with microscopy. This knowledge should support the definition of informed strategies towards optimization of MSC osteodifferentiation, in biomedical applications.
autores
Marques, MPM; Martins, CB; Carvalho, ALMB; Nolasco, MN; Rodrigues, JA; Bispo, DSC; Nogueira, HIS; Ribeiro-Claro, PJA;, Oliveira, MB; Mano, JF; Gil, AM
Grupos
Projectos
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
Metabolite-activated 3D stem cell differentiation into bone (BetterBone)
agradecimentos
Acknowledgments: Portuguese Foundation for Science and Technology – SFRH/BD/150655/2020 (DSB); UIDB/00070/2020 (https://doi.org/10.54499/UIDB/00070/2020), UIDP/00070/2020 (https://doi.org/10.54499/UIDP/00070/2020); UIDB/50011/2020 (https://doi.org/10.54499/UIDB/50011/2020), UIDP/50011/2020 (https://doi.org/ 10.54499/UIDP/50011/2020), LA/P/0006/2020 (https://doi.org/ 10.54499/LA/P/0006/2020); BetterBone (2022.04286.PTDC, DOI 10.54499/2022.04286.PTDC), UIDB/00070/2020 (https://doi.org/10.54499/UIDB/00070/2020), UIDP/00070/2020 (https://doi.org/10.54499/UIDP/00070/2020). Portuguese National NMR Network (RNRMN, Project N-022161).