In this study, we describe the production of hybrid gelatin–poly-L-lactide electrospun scaffolds whose hydrophilicity was controlled by binding increasing concentrations of hyaluronic acid (HA). We show that cross-linking has advantages over coating when aiming to functionalize the scaffolds with HA. The here described scaffolds structurely mimicked the complexity of the extracellular matrix, and when excited by second harmonic generation, they produced a signal that is typical of collagen-containing biological fibers. Fluorescence lifetime imaging microscopy (FLIM) was used to marker-independently monitor the growth of human dermal fibroblasts on the electrospun scaffolds using reduced (phosphorylated) nicotinamide adenine dinucleotide as target. Benefitting from the different fluorescence lifetimes of the polymer and the endogenous cellular fluorophore, we were able to distinguish and separate the signals produced by the cells from the signals generated by the electrospun scaffolds. FLIM further allowed the detection of metabolic differences in the cells seeded on the HA-functionalized scaffolds compared with cells that were cultured on nonfunctionalized control scaffolds.
Impact statement
The study describes the production of hybrid gelatin–poly-L-lactide (GE:PLLA) electrospun scaffolds whose hydrophilicity was modified by cross-linking specific amounts of hyaluronic acid (HA) to the free amine residues of GE. Utilizing endogenous second harmonic generation signal and natural autofluorescence patterns as well as fluorescence lifetime imaging microscopy, we were able to marker independently and noninvasively monitor the seeded and unseeded electrospun scaffolds. The here presented data are highly relevant when aiming for the simultaneous in vitro imaging and metabolic analysis of cultured cells in an environment close to the physiological one, which is crucial for the in vitro testing of drugs, metabolites, and other bioactive molecules.
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