Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in Vitro Cell Culture: A Step toward Creation of Artificial ECM

Abstract

Presentation of immobilized growth factors with retained bioactivity remains a challenge in the field of tissue engineering. In the present study, we propose a strategy to covalently conjugate a pleiotropic growth factor, basic fibroblast growth factor (bFGF) to a partial spider silk protein at gene level. The resulting silk-bFGF fusion protein has the propensity to self-assemble into silk-like fibers, and also surface coatings, as confirmed by quartz crystal microbalance studies. Functionality of the silk-bFGF coating to bind its cognate receptor was confirmed with surface plasmon resonance studies. As a step toward the creation of an artificial ECM, the silk-bFGF protein was mixed with FN-silk, an engineered spider silk protein with enhanced cell adhesive properties. Bioactivity of the thereby obtained combined silk was confirmed by successful culture of primary human endothelial cells on coatings and integrated within fibers, even in culture medium without supplemented growth factors. Together, these findings show that silk materials bioactivated with growth factors can be used for in vitro cell culture studies, and have potential as a tissue engineering scaffold.

Recombinant spider silk coatings functionalized with enzymes targeting bacteria and biofilms

“…Likely, sterically hindrance by the added domain restricts the silk part from interacting with other silk proteins. In a previous study (Thatikonda, Nilebäck, Kempe, Widhe, & Hedhammar, 2018), similar adsorption pattern was observed for 4RepCT in fusion with another folded domain; the basic fibroblast growth factor (FGF2).…”

Bioactive Silk Coatings Reduce the Adhesion of Staphylococcus aureus while Supporting Growth of Osteoblast-like Cells

“…Another approach could be to combine the dual-functional silks used herein with other variants of bioactive silk proteins, for example, by mixing DspB-FN-silk with recombinant silk fused with growth factors 14 or substances that could regulate the polarization of macrophages to achieve better tissue integration and healing. It is indeed interesting to construct multifunctional coatings also with different types of antimicrobials.…”

“…We have previously described a method to prepare stable silk coatings from a recombinant spider silk protein called 4RepCT by utilizing its self-assembling properties . Under physiological-like conditions, these silk proteins assemble into thin nanofibrillar coatings, also after fusion with different functional peptides (i.e., cell-binding motifs and antimicrobial peptides) − and fold-dependent domains (i.e., growth factors and affinity domains) , using recombinant gene technology. To functionalize silk with larger proteins such as enzymes, we present a strategy for site-specific immobilization of enzymes with a short sortase recognition peptide motif (SrtTag) in mild buffer onto preassembled silk using an engineered variant of the transpeptidase Sortase A .…”

Immobilization of Growth Factors for Cell Therapy Manufacturing

“…In the latter approach, peptide sequences that have an affinity to certain materials or proteins are selected and used for fusion with GFs through recombinant technology. A wide variety of peptides that bind to different substrates including collagen, cellulose, hydroxyapatite, titanium, polystyrene, and beta-tri calcium phosphate, have been used to immobilize GFs (Doheny et al, 1999;Ishikawa et al, 2001;Kitajima et al, 2007;Kang et al, 2013b;Tada et al, 2014;Alvarez et al, 2015;Thatikonda et al, 2018). These peptides are derived from combinatorial screens of peptide libraries or based on naturally occurring binding domains.…”

“…However, the biotin derivative must be carefully selected to ensure preserved bioactivity of the GF (Hermanson, 2008). Common biotin derivatives for GF modification are the amine-reactive, such as sulfo-NHS-biotin (Shahal et al, 2012;Kim et al, 2016a; Ogiwara et al, 2005 GSH-functionalized nanopatterns Glutathione-s-transferase FGF2 Kolodziej et al, 2011 Gelatin and Fibrillar collagen sponges Fibronectin collagen-binding domain EGF Ishikawa et al, 2001 Beta Tricalcium Phosphate (βTCP) βTCP-binding peptide EGF Alvarez et al, 2015 Collagen Collagen binding domain HGF Kitajima et al, 2007 Cellulose Cellulose-binding domain SCF Doheny et al, 1999 Silk coated surfaces Spider silk protein bFGF Thatikonda et al, 2018 Fibrin Transglutaminase activity of factor XIIIa ?-NGF Sakiyama-Elbert et al, 2001 Fibrin Transglutaminase activity of factor XIIIa and plasmin substrate BMP-2 Schmoekel et al, 2004 Fibrin Transglutaminase activity of factor XIIIa VEGF Zisch et al, 2001 Artificial Gold-coated glass plate Hexahistidine residues EGF Kato et al, 2005 Polystyrene surfaces Maltose-binding protein VEGF Han et al, 2009 Titanium surfaces Titanium-binding peptides hEGF Tada et al, 2014 Hydroxypatite Statherin active site EGF Kang et al, 2013b Titanium surfaces Statherin active site EGF Kang et al, 2013b Hydroxypatite Diphosporylated serines from statherin hBMP4 Sakuragi et al, 2011Sakuragi et al, et al, 2017. The NHS ester of this compound reacts with the GF primary amines to form an amide bond and thus, couple with biotin.…”