The effects of crosslinkers on physical, mechanical, and cytotoxic properties of gelatin sponge prepared via in-situ gas foaming method as a tissue engineering scaffold.

In this study porous gelatin scaffolds were prepared using in-situ gas foaming, and four crosslinking agents were used to determine a biocompatible and effective crosslinker that is suitable for such a method. Crosslinkers used in this study included: hexamethylene diisocyanate (HMDI), poly(ethylene glycol) diglycidyl ether (epoxy), glutaraldehyde (GTA), and genipin. The prepared porous structures were analyzed using Fourier Transform Infrared Spectroscopy (FT-IR), thermal and mechanical analysis as well as water absorption analysis. The microstructures of the prepared samples were analyzed using Scanning Electron Microscopy (SEM). The effects of the crosslinking agents were studied on the cytotoxicity of the porous structure indirectly using MTT analysis. The affinity of L929 mouse fibroblast cells for attachment on the scaffold surfaces was investigated by direct cell seeding and DAPI-staining technique. It was shown that while all of the studied crosslinking agents were capable of stabilizing prepared gelatin scaffolds, there are noticeable differences among physical and mechanical properties of samples based on the crosslinker type. Epoxy-crosslinked scaffolds showed a higher capacity for water absorption and more uniform microstructures than the rest of crosslinked samples, whereas genipin and GTA-crosslinked scaffolds demonstrated higher mechanical strength. Cytotoxicity analysis showed the superior biocompatibility of the naturally occurring genipin in comparison with other synthetic crosslinking agents, in particular relative to GTA-crosslinked samples.