A Review of Structure Construction of Silk Fibroin Biomaterials from Single Structures to Multi-Level Structures

Abstract

The biological performance of artificial biomaterials is closely related to their structure characteristics. Cell adhesion, migration, proliferation, and differentiation are all strongly affected by the different scale structures of biomaterials. Silk fibroin (SF), extracted mainly from silkworms, has become a popular biomaterial due to its excellent biocompatibility, exceptional mechanical properties, tunable degradation, ease of processing, and sufficient supply. As a material with excellent processability, SF can be processed into various forms with different structures, including particulate, fiber, film, and three-dimensional (3D) porous scaffolds. This review discusses and summarizes the various constructions of SF-based materials, from single structures to multi-level structures, and their applications. In combination with single structures, new techniques for creating special multi-level structures of SF-based materials, such as micropatterning and 3D-printing, are also briefly addressed.

Keywords: biomaterials; silk fibroin; structure.

Figure 1

Schematic presentation of the silk fibroin (SF) structure; d represents the diameter of a single silkworm thread. Reproduced from [28].

Figure 2

Structural design of SF-based biomaterials from single structures to multi-level structures. Reproduced from [34,35].

Figure 3

Scanning electron microscope (SEM) images (A–C) of SF microparticles fabricated with different SF: ethanol ratios, (A) 2:1, (B) 3:1 and (C) 4:1, scale bar: 5 µm; (D) Release kinetics of bone morphogenetic protein (BMP)-2, BMP-9, and BMP-14 immobilized in SF particles, with 0.5 µg of BMP per mg of SF. Reproduced from [38]; (E) Mechanism of SF particles’ regular formation (F) with addition of poly vinyl alcohol (PVA) and irregular formation (G) without addition of PVA, scale bar: 10 µm. Reproduced from [39].

Figure 4

(A) Enzymatic degradation of SF films. (▲: SF films by water annealing in enzyme solution; ■: SF films by methanol treatment in enzyme solution; ×: SF films by water annealing in PBS; ●: SF films by methanol treatment in PBS), n = 5; Human bone-marrow stromal cells (hMSCs) attachment at 2 h on water-annealed SF film (B) and methanol-treated SF film (C). The cracks on the film in image (C) are induced by methanol treatment. Red arrows indicate fully attached cells and blue arrows indicate attaching cells. Reproduced from [56]; (D) SEM images of bone-marrow stromal cells (BMSCs) growing on polyethylene oxide (PEO) non-extracted and PEO extracted SF mats, respectively, after 1, 7, and 14 days. Scale bar: 500 µm; (E) Proliferation of BMSCs grown on SF mats. Seeding density: 2.5 E4 cells/cm², n = 4. Reproduced from [59].

Figure 5

Images of regenerated silk fibroin (RSF)/hydroxypropyl methyl cellulose 9 (HPMC9) hydrogels’ reaction to bending (A), knotting (B) and compressing (C); (D) Representative tensile curves of RSF/HPMC9 hydrogels with different solid contents; (E) Cytotoxicity test of mouse fibroblast cells cultivated with RSF hydrogels and RSF/HPMC9 hydrogels; RSF/HPMCP9: the ratio of RSF to HPMC was 9/1. Reproduced from [34].

Figure 6

(A) Scanning electron microscope images of SF scaffolds fabricated by freeze-drying technique using (a–c) 2 wt % SF at −20 °C; (d–f) 4 wt % SF at −80 °C; and (g–i) 6 wt % SF at −196 °C; scale bar: (a–f,h,i): 100 µm; (g): 50 µm. (B) Confocal laser micrographs of human dermal fibroblast cell migration on SF porous scaffolds fabricated at −196 °C at different time points. The cells are stained with Hoechst 33342 for nuclei (green) and Rhodamine–phalloidin for actin filaments (red); scale bar: 500 µm. The black dotted arrows indicated the region and direction corresponding to the red dotted arrows on the previous graphs; ROI: region of interest. Reproduced from [88].

Figure 7

(A) Schematic illustration of the production of patterned SF films: (a) Pre-designed polydimethylsiloxane (PDMS) stamp; (b) Spin-coating SF solution on the PDMS; (c) Extracting the ionic liquid solvent in a methanol bath; (d) Peeling the crystallized patterned SF film from the stamp; (B) Data of cell alignment on patterned SF films as compared to the unpatterned (collagen-coated) films: Optical micrographs of keratinocytes growing on (a) patterned SF films at 6 and 24 h and (b) unpatterned film at 24 h; Histograms of cell alignment on (c) patterned SF films and (d) unpatterned films. The x-axis represents cell angle; the y-axis represents cell counts. Adapted with permission from [101]. Copyright (2007) American Chemical Society.

Figure 8

(A) Scanning electron microscope images of micropatterned SF/gelatin methacrylate (GelMA) porous scaffolds; (B) Fluorescence images (2×, 10×, and 20×) of NIH-3T3 fibroblast cells stained with live/dead viability kit after cultured on the micropatterning scaffolds for one day. Reproduced from [108].

Figure 9

(A) Optical image of three-dimensional printing (3DP) silk/hydroxyapatite (HA) scaffold; (B) Scanning electron microscope image of individual silk/HA filaments at intersection. Scale bar: 100 µm; (C) Higher magnification image of the silk/HA filament surface. Scale bar: 10 µm; (D) Height profile of a representative silk/HA filament observed by atomic force microscopy (AFM). Reproduced from [113].