Charge‐Tunable Autoclaved Silk‐Tropoelastin Protein Alloys That Control Neuron Cell Responses

Abstract

Tunable protein composites are important for constructing extracellular matrix mimics of human tissues with control of biochemical, structural, and mechanical properties. Molecular interaction mechanisms between silk fibroin protein and recombinant human tropoelastin, based on charge, are utilized to generate a new group of multifunctional protein alloys (mixtures of silk and tropoelastin) with different net charges. These new biomaterials are then utilized as a biomaterial platform to control neuron cell response. With a +38 net charge in water, tropoelastin molecules provide extraordinary elasticity and selective interactions with cell surface integrins. In contrast, negatively charged silk fibroin protein (net charge −36) provides remarkable toughness and stiffness with morphologic stability in material formats via autoclaving-induced beta-sheet crystal physical crosslinks. The combination of these properties in alloy format extends the versatility of both structural proteins, providing a new biomaterial platform. The alloys with weak positive charges (silk/tropoelastin mass ratio 75/25, net charge around +16) significantly improved the formation of neuronal networks and maintained cell viability of rat cortical neurons after 10 days in vitro. The data point to these protein alloys as an alternative to commonly used poly-L-lysine (PLL) coatings or other charged synthetic polymers, particularly with regard to the versatility of material formats (e.g., gels, sponges, films, fibers). The results also provide a practical example of physically designed protein materials with control of net charge to direct biological outcomes, in this case for neuronal tissue engineering.

Electrodeposited Gels Prepared from Protein Alloys

“…Recombinant human tropoelastin, displaying a wide range of biological activities, is especially suitable for mediation of the cell responses to biomaterials [2,4–5,25–26]. Our results confirmed that the addition of tropoelastin constituents to silk-tropoelastin alloys promoted the attachment of HEFs and the endothelialization by HUVECs.…”

“…For example, optimized silk/tropoelastin ratios for promoting neurite outgrowth of primary cortical neuronal cells (E18) has been determined using a set of autoclave- gelled, compositionally graded protein alloy films. The physical interactions between silk and tropoelastin across hierarchical levels as well as the composition-dependent surface features, including surface charge, roughness and stiffness, have been investigated and linked to neural cell substrate responses [2]. …”

3D in vitro modeling of the central nervous system

“…Hydrogels can be of natural or synthetic origin and include agarose (Balgude et al, 2001; Bellamkonda et al, 1995a, 1995b; Cullen et al, 2007a; Dillon et al, 1998; O'Connor et al, 2001), collagen type I (Coates and Nathan, 1987; Coates et al, 1992; Krewson et al, 1994; O'Connor et al, 2000a; O'Shaughnessy et al, 2003), hyaluronic acid (Brännvall et al, 2007, 2007; Hou et al, 2006; Pedron et al, 2013; Tian et al, 2005), N -(2-hydroxypropyl) methacrylamide (HPMA) (Woerly et al, 1990, 1991), poly(ethylene glycol) (PEG) (Pedron et al, 2013; Wang et al, 2014; Zustiak et al, 2013), chitosan (Gao et al, 2014; Leipzig et al, 2011; McKay et al, 2014), alginate (Frampton et al, 2011; Kuo and Chang, 2013; Matyash et al, 2012; Mosahebi et al, 2003), silk fibroin (Benfenati et al, 2010, 2012; Hopkins et al, 2013; Hu et al, 2013; Kim et al, 2010; Tien et al, 2013; Zhang et al, 2012) and methylcellulose (Stabenfeldt and LaPlaca, 2011; Tate et al, 2001), Table 2 .…”

Elastin-based biomaterials and mesenchymal stem cells

“…A number of cross-linking options are available for the formation of hydrogel scaffolds including traditional chemical cross-linkers, such as glutaraldehyde 104, 114 and bis(sulfosuccinimidyl) suberate (BS3) 115 , to more novel chemical-free cross-linking strategies, such as pH instigated cross-linking 116 and photocrosslinking 117 . Subsequent to the variations in fabrication, tropoelastin, ELP and elastin-based hydrogel scaffolds have demonstrated great tunability in terms of microstructure 118, 119 , mechanical properties 117, 120 , and biological function 121, 122 .…”