Marc Willem Theodoor Werten

Genetically engineered silk–collagen-like copolymer for biomedical applications: Production, characterization and evaluation of cellular response

Genetically engineered protein polymers (GEPP) are a class of multifunctional materials with precisely controlled molecular structure and property profile. Representing a promising alternative for currently used materials in biomedical applications, GEPP offer multiple benefits over natural and chemically synthesized polymers. However, producing them in sufficient quantities for preclinical research remains challenging. Here, we present results from an in vitro cellular response study of a recombinant protein polymer that is soluble at low pH but self-organizes into supramolecular fibers and physical hydrogels at neutral pH. It has a triblock structure denoted as C2S(H)48C2, which consists of hydrophilic collagen-inspired and histidine-rich silk-inspired blocks. The protein was successfully produced by the yeast Pichia pastoris in laboratory-scale bioreactors, and it was purified by selective precipitation. This efficient and inexpensive production method provided material of sufficient quantities, purity and sterility for cell culture study. Rheology and erosion studies showed that it forms hydrogels exhibiting long-term stability, self-healing behavior and tunable mechanical properties. Primary rat bone marrow cells cultured in direct contact with these hydrogels remained fully viable; however, proliferation and mineralization were relatively low compared to collagen hydrogel controls, probably because of the absence of cell-adhesive motifs. As biofunctional factors can be readily incorporated to improve material performance, our approach provides a promising route towards biomedical applications.

Multi-responsive physical gels formed by a biosynthetic asymmetric triblock protein polymer and a polyanion

We report the design, production and characterization of a biosynthetic asymmetric triblock copolymer which consists of one collagen-like and one cationic block spaced by a hydrophilic random coiled block. The polymer associates into micelles when a polyanion is added due to the electrostatic interaction between the cationic block and the polyanion. The collagen-like block self-assembles into thermo-responsive triple helices upon cooling. When both end blocks are induced to self-assemble, a physical gel is formed via thermo-responsive association of the charge-driven micelles. The self-assembly of both end blocks and the effects of salt and temperature thereon were characterized by light scattering and rheology.