Towards constructing extracellular matrix-mimetic hydrogels: An elastic hydrogel constructed from tandem modular proteins containing tenascin FnIII domains

Protein-based hydrogels have been developed for various biomedical applications where they provide artificial extracellular microenvironments that mimic the physical and biochemical characteristics of natural extracellular matrices (ECMs). In natural ECMs, a large number of proteins are tandem modular proteins consisting of many individually folded functional domains that confer structural and biological functionalities. Such tandem modular proteins are promising building blocks for constructing ECM-mimetic biomaterials. However, their use for such purposes has not been explored extensively. Tenascin-C (TNC) is an ECM tandem modular protein and plays an important role in mechanotransduction by regulating important cell-matrix interactions. The third FnIII domain of TNC (TNfn3) contains an RGD sequence and is known to bind integrins. Here we use the TNfn3 domain and resilin sequence-based tandem modular protein FRF4RF4R (F represents the TNfn3 domain and R represents the resilin sequence, respectively) as a building block to construct protein-based ECM-mimetic hydrogels. The tandem modular protein-based building block FRF4RF4R closely mimics the architecture of the naturally occurring tandem modular ECM protein TNC and incorporates intact RGD-containing FnIII domains. Our results demonstrate that tandem modular proteins containing TNfn3 can be readily photochemically crosslinked into elastic hydrogels, whose Young's modulus can be tuned by the concentration of the tandem modular protein solution. In vitro studies demonstrate that none of the photochemical crosslinking reaction components are cytotoxic at the level tested, and the hydrogel supports the spread of human lung fibroblast cells. Our results demonstrate that FRF4RF4R-based hydrogel is a novel ECM-mimetic hydrogel.