oa Further Refinements in Collagen Mimetic Peptide Scaffolds for Tissue Engineering Heart Valves

Collagen is the essential protein in the extracellular matrix, which maintains the structural and mechanical integrity of tissues while providing key signals to regulate cell functions. Although animal-based collagens can be used as biomaterial for tissue engineering heart valves, they cause infections and lack flexibility. These limitations have stimulated the exploration of collagen mimetic peptides (CMPs) through a bottom-up approach using computational modeling followed by experiments to enzymatically cross-link the CMPs and produce hydrogels. The X-ray structure of triple-helices of CMP was used in software FIRST and in mutational code to identify its structural stability and hotspots. These data assisted to introduce charged residues by mutations to cross-link and to add binding motif (GFOGER) for integrin in the structure. The helical stability and self-association of the mutated CMP has been validated using molecular dynamics (MD) simulation. Experimentally, the peptide was synthesized by solid phase Fmoc chemistry and characterized by HPLC and mass spectrometry. Enzymatic cross-linking on primary amine and gel formation were obtained by incubating peptide and plasma amine oxydase (PAO) solutions in PBS at 37 and 58 °C. Peptide assembly and aggregation was monitored by turbidity (optical density at 314 nm) and morphology was analysed by transmission electron microscopy (TEM). The modelling analyses indicated the CMP to have the desired structural properties for self-assembly and high affinity towards integrin binding. The modification of the key positions with charged residues increased the possibilities for helical cross-link (gelation). In addition to cell signalling, the charged residues at the cell binding motif could further enhance the inter-helical association of the CMPs. The structural properties of the modelled CMP were reproduced in experimental conditions. Addition of PAO significantly improved turbidity of peptide solutions and lead to hydrogel formation. The peptides assembled in branched fibrillar structures around 25 nm in diameter as it was confirmed by TEM analysis. The proposed peptide promises to show some inherent structural properties of native collagen in silico and in vitro. These properties are required to produce functional scaffolds for tissue engineering heart valves.