title: Genetically engineered silk-collagen-like copolymer for biomedical applications: production, characterization and evaluation of cellular response.
abstract: 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.
authors.affiliation: Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, NL-6703 HB Wageningen, The Netherlands.
authors.forename: Małgorzata K
authors.initials: MK
authors.lastname: Włodarczyk-Biegun
authors.affiliation: Wageningen UR Food & Biobased Research, Bornse Weilanden 9, NL-6708 WG Wageningen, The Netherlands.
authors.forename: Marc W T
authors.initials: MW
authors.lastname: Werten
authors.affiliation: Wageningen UR Food & Biobased Research, Bornse Weilanden 9, NL-6708 WG Wageningen, The Netherlands.
authors.forename: Frits A
authors.initials: FA
authors.lastname: de Wolf
authors.affiliation: Department of Biomaterials, Radboud UMC, Philips van Leydenlaan 25, NL-6525 EX Nijmegen, The Netherlands.
authors.forename: Jeroen J J P
authors.initials: JJ
authors.lastname: van den Beucken
authors.affiliation: Department of Biomaterials, Radboud UMC, Philips van Leydenlaan 25, NL-6525 EX Nijmegen, The Netherlands.
authors.forename: Sander C G
authors.initials: SC
authors.lastname: Leeuwenburgh
authors.affiliation: Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, NL-6703 HB Wageningen, The Netherlands. Electronic address: marleen.kamperman@wur.nl.
authors.forename: Marleen
authors.initials: M
authors.lastname: Kamperman
authors.affiliation: Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, NL-6703 HB Wageningen, The Netherlands.
authors.forename: Martien A
authors.initials: MA
authors.lastname: Cohen Stuart
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry* / Q000502:physiology; D020100:Hydrogels; D006863:Hydrogen-Ion Concentration; D008422:Materials Testing; D010843:Pichia / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047011:Silk / Q000737:chemistry* / Q000502:physiology; D012995:Solubility
chemical_list: D001672:Biocompatible Materials; D020100:Hydrogels; D011994:Recombinant Proteins; D047011:Silk; D003094:Collagen
keywords: Cellular response; Genetically engineered protein polymers; Hydrogels; Pichia pastoris; Silk–collagen-like block copolymers