Three-dimensional tailor-made collagen-like proteins hydrogel for tissue engineering applications

Despite the potential tunable properties of blank slate collagen-like proteins (CLP), an alternative to animal -originated collagen, assembling them into a stable 3D hydrogel to mimic extracellular matrix is a challenge. To address this constraint, the CLP (without hydroxyproline, CLPpro) and its variants encoding functional un-natural amino acids such as hydroxyproline (CLPhyp) and 3,4-dihydroxyphenylalanine (CLPdopa) were gener-ated through genetic code engineering for 3D hydrogel development. The CLPhyp and CLPdopa were chosen to enhance the intermolecular hydrogen bond interaction through additional hydroxyl moiety and thereby facilitate the self-assembly into a fibrillar network of the hydrogel. Hydrogelation was induced through genipin as a cross -linker, enabling intermolecular cross-linking to form a hydrogel. Spectroscopic and rheological analyses confirmed that CLPpro and its variants maintained native triple-helical structure, which is necessary for its function, and viscoelastic nature of the hydrogels, respectively. Unlike CLPpro, the varients (CLPhyp and CLPdopa) increased pore size formation in the hydrogel scaffold, facilitating 3T3 fibroblast cell interactions. DSC analysis indicated that the stability of the hydrogels got increased upon the genetic incorporation of hydroxy-proline (CLPhyp) and dopa (CLPdopa) in CLPpro. In addition, CLPdopa hydrogel was found to be relatively stable against collagenase enzyme compared to CLPpro and CLPhyp. It is the first report on 3D biocompatible hydrogel preparation by tailoring CLP sequence with non-natural amino acids. These next-generation tunable CLP hydrogels open a new venue to design synthetic protein-based biocompatible 3D biomaterials for tissue engi-neering applications.

Automated summary

Through genetic engineering, the researchers successfully developed tunable three-dimensional biocompatible hydrogel materials by modifying the sequence of collagen-like proteins (CLP) and incorporating non-natural amino acids. These hydrogels maintained the native triple-helical structure and viscoelastic properties, promoting cell interactions and pore formation. The hydrogels showed increased stability and biocompatibility compared to the original CLP. This study presents a new approach for designing synthetic protein-based biomaterials for tissue engineering applications.