Fabrication of hydrogel scaffolds via photocrosslinking of methacrylated silk fibroin

Abstract

Silk fibroin is a promising biomaterial for tissue engineering due to its valuable mechanical and biological properties. However, being a natural product and a protein, it lacks the processability and uniform quality of an advanced synthetic material. Here we propose a way to overcome this contradiction using novel fibroin photocrosslinkable derivative (FBMA). FBMA was synthesized by methacrylation of native fibroin nucleophilic side groups. It was dissolved in either formic acid (FA) or hexafluoroisopropanol (HFIP), and the obtained solutions were photocrosslinked into hydrogel scaffolds of various structural forms including films, micropatterns, pads and macroporous sponges. UV-exposition of dry FBMA films through a photomask created complex microscaled patterns of the polymer. The nature of the solvent affected the properties of resulting hydrogels. When HFIP was used as the solvent, the resulting hydrogels had a storage modulus ∼4 times higher than that of hydrogels fabricated using FA and ∼20 times higher compared to the reference hydrogel obtained from pristine fibroin. Both FBMA-based hydrogels were biocompatible and supported fibroblast adhesion and growth in vitro. Cells cultivated on FBMA scaffolds produced with HFIP exhibited more spread phenotype at 4 and 24 h of cultivation, consistent with increased stiffness of the hydrogel. Hence, FBMA is an attractive material for fabrication of micropatterned scaffolds of centimeterscale size with minutely tunable physico-chemical properties via convenient and reproducible technological processes, applicable for rapid prototyping.

The Mechanical Properties, Secondary Structure, and Osteogenic Activity of Photopolymerized Fibroin

“…Methacrylated fibroin (FbMA) was synthesized according to a procedure described by us [18]. Briefly, 1 g of lyophilized Fb was placed in a round-bottom flask equipped with a magnetic stirrer and 20 mL of 0.1 M potassium phosphate buffer solution (pH 7.0) was added.…”

“…The experimental scheme of fibroin films preparation is shown in Figure 1. Pristine Fb as a lyophilisate was used for synthesis of its methacrylated derivative FbMA [18]. To fabricate the films, these proteins were dissolved in HFIP.…”

“…FbMA-F-et and Fb-F-et films were fabricated upon treatment of the corresponding water-treated precursor films with an excess of ethanol. Pristine Fb as a lyophilisate was used for synthesis of its methacrylated derivative FbMA [18]. To fabricate the films, these proteins were dissolved in HFIP.…”

“…Thus, the substrate's elasticity modulus (EM) can regulate cell morphology, motility, and the direction of differentiation. The EM of normal bone extracellular matrix (ECM) is 20-50 kPa [3], whereas for the mineralized bone, the values are as large as [10][11][12][13][14][15][16][17][18][19][20] GPa [4]. The respective values for typical polymer scaffolds are in the kPa range [5]; an increase in EM can be a factor of the osteogenic potency of the material [6][7][8].…”

“…The urethane methacrylic Fb derivative [17] has been synthesized using a highly reactive isocyanoethyl methacrylate that provided an exhaustive modification of abundant serine residues (~12% of total amino acid content) in the hydrophobic repeats (GAGAGS) that form the crystalline domains of beta sheets. We have proposed another synthetic route for Fb methacrylation, yielding the methacrylamide derivative FbMA [18]. In doing so, we introduced the methacrylic moieties under mild conditions selectively at the amino groups of Arg and Lys residues, whose total presence in Fb is only~0.5%.…”