2.4.2. Crosslinking with EDC/NHS

The reaction between amine groups in the corresponding (EDC)/N-hydroxy succinimide (NHS) solution and the carbonyl groups of the available in the polymers of the scaffold layers can be described by several steps. The EDC reacts with carbonyl groups and forms an unstable intermediate which should react immediately with three possible compounds (Figure 1b):

The NH₂-terminal provided for natural polymers in the multilayer scaffold resulting, a stable amide bond;

The NHS, which is a result of a semi-stable amine NHS ester that forms with the terminal NH₂ provided by natural polymers a stable amide bond;

A small quantity of carbonyl groups are regenerated because the unstable intermediate reacts with water of the medium [32,33].

For chemical crosslinking, the samples were immersed in a solution of EDC/NHS (33 mM/6 mM) in ethanol (95%) for 4 h at room temperature. Then the crosslinking scaffolds were washed (5 times × 5 min), frozen at −80 °C, and lyophilized.