RGD-Functionalized Bioengineered Spider Dragline Silk Biomaterial

Abstract

Spider silk fibers have remarkable mechanical properties that suggest the component proteins could be useful biopolymers for fabricating biomaterial scaffolds for tissue formation. Two bioengineered protein variants from the consensus sequence of the major component of dragline silk from Nephila clavipes were cloned and expressed to include RGD cell-binding domains. The engineered silks were characterized by CD and FTIR and showed structural transitions from random coil to insoluble beta-sheet upon treatment with methanol. The recombinant proteins were processed into films and fibers and successfully used as biomaterial matrixes to culture human bone marrow stromal cells induced to differentiate into bone-like tissue upon addition of osteogenic stimulants. The recombinant spider silk and the recombinant spider silk with RGD encoded into the protein both supported enhanced the differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) to osteogenic outcomes when compared to tissue culture plastic. The recombinant spider silk protein without the RGD displayed enhanced bone related outcomes, measured by calcium deposition, when compared to the same protein with RGD. Based on comparisons to our prior studies with silkworm silks and RGD modifications, the current results illustrate the potential to bioengineer spider silk proteins into new biomaterial matrixes, while also highlighting the importance of subtle differences in silk sources and modes of presentation of RGD to cells in terms of tissue-specific outcomes.

Biomimetic materials for tissue engineering

“…The hydrophobic blocks tend to form β-sheets or crystals through hydrogen bonding and hydrophobilc interactions, resulting in the tensile strength of silk fibroins [39]. Genetic engineering techniques have been utilized to synthesize recombinant spider silk fibroin-mimetic polymers, possessing excellent mechanical properties [40] and improved cell adhesion capacity [41].…”

High‐resolution NMR characterization of a spider‐silk mimetic composed of 15 tandem repeats and a CRGD motif

“…1 Therefore, the design of new and robust materials with a wide range of applications might be realized from duplicating the molecular design of silk proteins. [3][4][5][6][7] Native silk fibroins from silkworms and spiders are block copolymers, consisting of semicrystalline blocks covalently linked by amorphous modules. [8][9][10][11] Additional Supporting Information may be found in the online version of this article.…”

“…The Cystine-Arginine-Glycine-Aspartate (CRGD) silk mimetic's design is based on the consensus sequence of Nephila clavipes dragline silk combined with an extracellular matrix protein sequence (integrin recognition motif) covalently linked at the termini. 5,7 The RGD motif is an epitope for osteoblast binding. The primary structure of the engineered CRGD-silk mimetic consists of a semicrystalline block forming a 33-amino acid sequence repeated 15 times, covalently linked to an amorphous sequence and the CRGD motif spliced between these sequences (see Fig.…”

Bioengineered collagens

“…In addition, non-triple-helical binding motifs, such as RGD, could be added to the N-or C-terminal of the CL domain recombinantly (or throughout chemically), as has been done with other proteins. 42 Similar non-triple helical sequence additions also allow the development of yet other new molecules with defined, specific biological functions. Recently, the addition of a silk-like motif, (GAGAGS) n , to the C-terminal of the S. pyogenes CL domain has been shown to enable binding to a silk substrate, providing the opportunity to develop more complex composite structures.…”