Silk Fibroin-Based Biomaterials for Tissue Engineering Applications

Abstract

Tissue engineering (TE) involves the combination of cells with scaffolding materials and appropriate growth factors in order to regenerate or replace damaged and degenerated tissues and organs. The scaffold materials serve as templates for tissue formation and play a vital role in TE. Among scaffold materials, silk fibroin (SF), a naturally occurring protein, has attracted great attention in TE applications due to its excellent mechanical properties, biodegradability, biocompatibility, and bio-absorbability. SF is usually dissolved in an aqueous solution and can be easily reconstituted into different forms, including films, mats, hydrogels, and sponges, through various fabrication techniques, including spin coating, electrospinning, freeze drying, and supercritical CO₂-assisted drying. Furthermore, to facilitate the fabrication of more complex SF-based scaffolds, high-precision techniques such as micro-patterning and bio-printing have been explored in recent years. These processes contribute to the diversity of surface area, mean pore size, porosity, and mechanical properties of different silk fibroin scaffolds and can be used in various TE applications to provide appropriate morphological and mechanical properties. This review introduces the physicochemical and mechanical properties of SF and looks into a range of SF-based scaffolds that have recently been developed. The typical applications of SF-based scaffolds for TE of bone, cartilage, teeth and mandible tissue, cartilage, skeletal muscle, and vascular tissue are highlighted and discussed followed by a discussion of issues to be addressed in future studies.

Keywords: biomaterial; silk fibroin; tissue engineering.

Figure 1

SF-based scaffolds with different morphologies—film, mat, fiber, micro-patterning, sponge, nano-particle, 3D structure, and hydrogel.

Figure 2

SF-based biomaterials applied in TE, including hard tissues (bone and cartilage) and soft tissues (ligaments, skin, blood vessels, nerves, and muscles).

Figure 3

Schematic diagram of the silk source and structure. (A) Different species of 5th instar silkworm larvae, their cocoons, and the fibers obtained from the silk cocoons. (B) The heavy chain (i.e., the N-terminus, β-sheets, amorphous loops, and the C-terminus) and the light chain, which is linked via disulphide bonds. Reprinted with permission from Ref. [45]. Copyright 2019 MDPI.

Figure 4

(A) Field emission scanning electron microscope images of spin-coated silk films and electrospun silk fibers: (a), the as-spun coated film; (b–d), the film treated in a 70%, 80%, and 90% ethanol solution, respectively. (B) Changes in the crystallinity index (CI) as a function of ethanol treatment time. Here, the CI is the ratio between the peak intensities at 1626 cm⁻¹ and 1650 cm⁻¹ in the infrared spectrum. Reprinted with permission from ref. [61]. Copyright 2016 Elsevier. (C) Schematic representation of the film assembly process according to vertical deposition method. (D) Topographical atomic force microscope images at different scales of silk monolayer films (a,c) and silk multilayer films (b,d). Reprinted with permission from ref. [64]. Copyright 2012 American Chemical Society.

Figure 5

(A) 3D printing of spider silk hydrogel scaffolds by robotic dispensing. Reprinted with permission from ref. [75]. Copyright 2015 John Wiley and Sons. (B) Schematic representation of the SF preparation from raw silk cocoons through degumming process and solubilization. Electrospinning of SF solution leads to the formation of nanofibrous SF mats. Reprinted with permission from ref. [79]. Copyright 2020 American Chemical Society.

Figure 6

(A) Silk processing and (B) silk sponge scaffold fabrication flowchart. Scanning electron microscope images of the outer structure silk sponge scaffold formed by freeze-drying (C), salt leaching (D), and gas foaming (E) methods after methanol treatment. Reprinted with permission from ref. [92]. copyright 2004 American Chemical Society.

Figure 7

Schematic representation of the strategies used to modify, manufacture, and characterize silk materials. Reprinted with permission from ref. [97]. copyright 2018 John Wiley and Sons.

Figure 8

(A) Illustrations emphasizing the differences between bioplotting and bioprinting strategies. (B) Schematic of a bioinkjet style printing strategy. Reprinted with permission from ref. [102]. copyright 2016 American Chemical Society. (C) Representative sets of 3D DLP projections (CAD) and their corresponding printed specimens of alveolar structure and images of a transverse section of a human spinal cord. Reprinted with permission from ref. [105]. copyright 2020 American Chemical Society.

Figure 9

(A) Schematic showing the process used to fabricate the 3D radially aligned nanofiber scaffolds. (B) Top and side scanning electron microscope images of the radially aligned scaffold. (C) BMSCs proliferated on the radially aligned scaffolds and linearly aligned scaffolds. (D) Histological analysis of the scaffolds after implantation for 4 and 8 weeks. Reprinted with permission from ref. [26]. copyright 2021 IPC Science and Technology.

Figure 10

(A) Schematic presentation of methacrylation of SF with GMA (Silk-GMA) and bio-printing of chondrocytes with Silk-GMA by a digital light processing 3D printer. (B) Confocal microscopic images for the Live/Dead assay with Calcein-AM (live cells, green fluorescence) and ethidium homodimer-1 (dead cells, red fluorescence) staining showing that human chondrocytes proliferated well in 30% Silk-GMA hydrogel for up to 2 weeks of cultivation. (C) In vitro histological detection of human chondrocyte-laden Silk-GMA hydrogel for cartilage tissue formation. (D) Schematic summaries of chondrocyte-laden Silk-GMA hydrogel transplantation and endoscopic observation of rabbit trachea for 6 weeks after transplantation. Reprinted with permission from ref. [30]. copyright 2020 Elsevier.

Figure 11

(A) The schematic diagram of different electrospinning devices. (B) The ability to resist thrombosis. (C) Live cell staining photomicrograph of HUVECs and scanning electron microscope images of HUVECs grown on different materials at day 4. (D) The HE stains of different vascular scaffolds. Reprinted with permission from [128]. copyright 2019 Dove.