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

Abstract

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.

Osteogenesis and mineralization of mesenchymal stem cells in collagen type I‐based recombinant peptide scaffolds

“…The use of amines in HMDIC crosslinking also meant that significantly fewer lysine residues remained after stabilization when compared to DHT, 28 and 42 mmol for HMDIC and DHT, respectively. Diminished numbers of lysine groups are also expected in genipin crosslinked samples, as genipin also relies on a reaction between two amines . In DHT samples, the loss of a lysine corresponds to a loss of a carboxyl group, which, in theory, can significantly reduce cell adhesion, as carboxyl groups are found in the RGD cell adhesion ligand .…”

“…In the case of HMDIC, a linear chain is added and genipin incorporates a ring shaped molecule . The addition of long chains into gelatin structures is known to increase water absorption and elastic response . Thus, of the three crosslinkers, HMDIC had the greatest overall hydration, or total amount of water which could be contained by the scaffold .…”

“…The addition of long chains into gelatin structures is known to increase water absorption and elastic response . Thus, of the three crosslinkers, HMDIC had the greatest overall hydration, or total amount of water which could be contained by the scaffold . While the overall hydration was low with DHT crosslinking, a large amount of the water was taken up by the scaffold within the pore walls, measured by water uptake.…”

Colloidal gelatin microgels with tunable elasticity support the viability and differentiation of mesenchymal stem cells under pro‐inflammatory conditions

“…Another advantage is that the characteristic of high surface‐to‐volume ratio of microgels enables a rapid mass transport between the interior and exterior of a microgel particle . However, many available methods for in situ gelation have also been limited by the use of cytotoxic initiators or radical oxidative reactions for crosslinking during the sol–gel transition …”

Collagen: a network for regenerative medicine

“…It was demonstrated on gas foamed scaffolds that cross-linkers with long chains, that are incorporated into the scaffold structure, for example HMDIC, increase water absorption and elastic response. 104 …”