Preservation of nano fiber morphology in EDC cross-linking of electrospun gelatin

Electrospun gelatin and collagen matrices are considered suitable substrate for regenerative medicine, due to their biocompatibility and biomimicry properties. To achieve the necessary stability and adjust scaffold degradation rate, cross-linking is generally required, but most protocols used are not capable to preserve the nanofiber morphology and the interconnected porosity. Among suitable cross-linking agents, carbodiimide, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is extremely interesting, because it is an effective zero-length cross-linker. EDC is generally dissolved in water or ethanol, but it causes an undesired fusion of fibers. To prevent this effect, in this study, the cross-linking protocol involves the use of ethyl acetate and acetonitrile as solvent instead of ethanol. Gelatin Type A (Sigma-Aldrich) was dissolved at a concentration of 10% w/v in a mixture of acetic acid and distilled water (9:1). The solution was electrospun in a standard laboratory system used to prepare homogeneous non-woven matrices. After determination of gelatin free amine groups by TNBSA assay, EDC was calculated added to obtain 5 to 1 and 10 to 1 stoichiometric ratios. Cross-linking was performed in ethyl acetate/ethanol (9:1) or acetonitrile for three different reaction times (2, 3 and 4 days). Cross-linked matrices were aged in PBS at 37 °C for 28 days to appraise degradation and evaluate the effectiveness of cross-linking protocol. Finally, in vitro cytotoxicity of cross-linked materials was assessed by culturing L929 mouse fibroblasts with scaffold eluates to investigate the effect of possible solvent or cross-linker residues on cell viability. As shown in Figure 1, the morphology and the porosity of electrospun matrices were much better preserved after cross-linking for all tested conditions compared with ethanol treatment. This effect is probably related to both polarity and protic character of solvents. While ethanol is a polar protic solvent, which causes fibers swelling due to the formation of hydrogen bonds, ethyl acetate and acetonitrile are not able to do that because the first one is a non polar solvent, promoting hydrophobic interactions, and the second one is a polar non protic, that prevents hydrogen bonding. The effectiveness of cross-linking methods was proved by a relatively reduced weight loss (30 - 50%) after 4 weeks ageing in PBS (Fig. 2). Moreover, no signs of cytotoxic effect deriving from cross-linker residues was observed in cell experiments performed (Fig. 3). Both solvents tested resulted in effective EDC cross-linking of electrospun gelatin scaffolds. After processing, gelatin scaffolds show improved stability and well preserve their nanofibers morphology.