Non-invasive characterization of hybrid gelatin:poly-l-lactide electrospun scaffolds using second harmonic generation and multiphoton imaging.

Hybrid scaffolds composed of synthetic polymers and naturally occurring components have become more relevant in the field of tissue engineering and regenerative medicine. Synthetic polymers are responsible for scaffold durability, strength and structural integrity; however, often do not provide biological signals. Introducing a biological component leads to more advanced and biocompatible scaffolds. In order to use these scaffolds as implants, a deeper knowledge of material characteristics and the impact of the biological component on the scaffold mechanical properties are required. Furthermore, it is necessary to implement fast, easy and non-invasive methods to determine material characteristics. In this work, we aimed to generate gelatin-poly-l-lactide (PLA) hybrids via electrospinning with defined, controllable and tunable scaffold characteristics. Using Raman microspectroscopy, we demonstrated the effectiveness of the cross-linking reaction and evaluated the increasing PLA content in the hybrid scaffolds with a non-invasive approach. Using multiphoton microscopy, we showed that gelatin fibers electrospun from a fluorinated solvent exhibit a second harmonic generation (SHG) signal typical for collagen-like structures. Compared to pure gelatin, where the SHG signal vanishes after cross-linking, the signal could be preserved in the hybrid scaffolds even after cross-linking. Furthermore, we non-invasively imaged cellular growth of human dermal fibroblasts on the hybrid electrospun scaffolds and performed fluorescence lifetime imaging microscopy on the cell-seeded hybrids, where we were able to discriminate between cells and scaffolds. Here, we successfully employed non-invasive methods to evaluate scaffold characteristics and investigate cell-material interactions.