Nanofibrillar hydrogel scaffolds from recombinant protein‐based polymers with integrin‐ and proteoglycan‐binding domains

Abstract

This study describes the design, production, and testing of functionalized variants of a recombinant protein‐based polymer that forms nanofibrillar hydrogels with self‐healing properties. With a view to bone tissue engineering applications, we equipped these variants with N‐terminal extensions containing either (1) integrin‐binding (RGD) or (2) less commonly studied proteoglycan‐binding (KRSR) cell‐adhesive motifs. The polymers were efficiently produced as secreted proteins using the yeast Pichia pastoris and were essentially monodisperse. The pH‐responsive protein‐based polymers are soluble at low pH and self‐assemble into supramolecular fibrils and hydrogels at physiological pH. By mixing functionalized and nonfunctionalized proteins in different ratios, and adjusting pH, hydrogel scaffolds with the same protein concentration but varying content of the two types of cell‐adhesive motifs were readily obtained. The scaffolds were used for the two‐dimensional culture of MG‐63 osteoblastic cells. RGD domains had a slightly stronger effect than KRSR domains on adhesion, activity, and spreading. However, scaffolds featuring both functional domains revealed a clear synergistic effect on cell metabolic activity and spreading, and provided the highest final degree of cell confluency. The mixed functionalized hydrogels presented here thus allowed to tailor the osteoblastic cell response, offering prospects for their further development as scaffolds for bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3082–3092, 2016.

Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach

“…The design and production in P . pastoris of the genetically engineered protein-based polymers C 2 S H 48 C 2 and B RGD C 2 S H 48 C 2 was described recently by us [ 19 , 23 , 24 ].…”

Functional Polymeric Materials Inspired by Geckos, Mussels, and Spider Silk

“…Additionally, rheological analysis revealed that the gels exhibit strain stiffening and are able to self‐heal rapidly, even after repeated breaking cycles. Both effects (strain stiffening and self‐healing) resemble the behavior of natural collagen type I …”

“…At higher protein concentrations, a fibrous gel is formed, with stiffness depending on the polymer concentration. Reproduced with permission . Copyright 2016, Wiley Periodicals, Inc.…”

“…All together Włodarczyk‐Biegun et al concluded that inclusion of both types of functional domains was most beneficial for cells. Most importantly, it was shown that cell behavior can be controlled by relatively easy, yet very precise tailoring of recombinant material …”

“…The gels of the highest stiffness and fastest gelation kinetics are obtained when significant bundling took place, as observed by AFM. These findings suggest that the self‐assembly of the protein, the self‐assembly of the fiber, and the gel formation can be easily controlled in a biocompatible manner, i.e., by varying both the KRSR domain density and the heparin concentration . The results presented in this section show that the investigated C 2 S H 48 C 2 protein has potential for use as biomaterial for basic studies, for testing models, and, possibly in the future, for clinical applications.…”