Synthesis and Characterization of Amine-Containing Copolymers: New Green Crosslinging Agents to Reinforce Gelatine for Tissue Engineering Application

The support structure (scaffold) necessary for cell adhesion and proliferation plays a pivotal role in tissue engineering (TE). The protein and polysaccharide constituents of the extracellular matrix (ECM), such as collagen, elastin, or hyaluronic acid (HA), as well as gelatine are the most attracting materials as possible scaffolds. To obtained a 3D structure with mechanical properties suitable for supporting cells adhesion and regeneration, gelatine, a lower-cost derivative of collagen, is commonly reinforced by crosslinking reactions using different synthetic crosslinking agents, with possible side effects. In this study, a novel and safer successful protocol to crosslink gelatine has been developed, based on the formation of imine bonds or on aldolic condensation reactions occurring between gelatine and properly synthesized copolymers containing amine residues, via lysil oxidase (LO)-mediated oxidation, as it occurs in nature for crosslinking collagen. Particularly, we have synthesized and fully characterized by FTIR and NMR analyses, an amino butyl styrene monomer (5), its biocompatible copolymers with dimethylacrylamide (DMAA) and its terpolymer with DMAA and acrylic acid (AA). Then, three acryloyl amidoamine monomers (11a-c) and their copolymers with DMAA were likewise prepared and characterized. Oxidation tests on amine copolymers with LO were performed to identify its best substrates to be used in subsequent experiments of gelatine crosslinking. Copolymers containing 5, 11b, and 11c demonstrated to be excellent substrates for LO and, upon particles size distribution and Z-potentials investigation, were employed in crosslinking tests with gelatine. Copolymers of 5, which in cytocoxicity experiments did not significanly affect cell viability of two human cell lines, afforded materials with good degree of reticulation, thus being suitable for further structural and mechanical investigations, as well as for biodegradability and biological experiments.