Regenerated Silk Fibroin Materials Research Articles

Silk wastes and autoclaved degumming as an alternative for a sustainable silk process

Silk degumming is considered the first point in the preparation of silk-based materials since this process could modify the silk fiber and the properties of its related products. This study evaluated the differences in morphology, secondary structure, amino acid content, thermal stability, and mechanical properties of two types of raw materials, defective cocoons (DC) and silk fibrous waste (SW), degummed by chemical (C) and autoclaving (A) methods. Subsequently, silk fibroin films were prepared by dissolving each type of degummed fibers, and thermal and structural films properties were determined. The findings demonstrated that autoclaving is an efficient alternative to remove silk sericin, as the resulting fibers presented improved structural, thermal, and mechanical properties compared to those obtained by the chemical method. For films preparation, autoclave resulted in a good option, but dissolution parameters need to be adjusted for defective cocoons. Furthermore, similarities between the physicochemical properties of fibers and films from both fibrous wastes suggest that SW is a promising raw material for producing fibrous resources and regenerated silk fibroin materials. Overall, these findings suggest new recycling methods for fibrous waste and by-products generated in the silk textile production process.

Electrospun regenerated silk fibroin is a promising biomaterial for the maintenance of inner ear progenitors in vitro.

We sought to determine the biocompatibility of electrospun regenerated silk fibroin (RSF) mats with inner ear progenitors, especially their effect on the differentiation of inner ear progenitors into hair cells. Neonatal mouse cochleae (n = 20) were collected and digested and allowed to form spheres over several days. Cells digested from the spheres were then seeded onto aligned or random RSF mats, with laminin-coated coverslips serving as controls. The inner ear progenitor cell mortality was examined by TUNEL labeling, and the adhesion of cells to the RSF mats or coverslip was determined by scanning electron microscopy. Finally, the number of hair cells that differentiated from inner ear progenitors was determined by Myosin7a expression. Unpaired Student's t-tests and one-way ANOVA followed by a Dunnett's multiple comparisons test were used in this study (p < 0.05). After 5days of culture, the inner ear progenitors had good adhesion to both the aligned and random RSF mats and there was no significant difference in TUNEL+ cells between the mats compared to the coverslip (p > 0.05). After 7days of in vitro differentiation culture, the percentage of differentiated hair cells on the control, aligned, and random RSF mats was 2.5 ± 0.5%, 2.7 ± 0.4%, and 2.4 ± 0.2%, respectively, and there was no significant difference between Myosin7a+ cells on either RSF mat compared to controls (p > 0.05). The aligned and random RSF mats had excellent biocompatibility with inner ear progenitors and helped the inner ear progenitors maintain their stemness. Our results thus indicate that RSF mats represent a useful scaffold for the development of new strategies for inner ear tissue engineering research.

Silk fibroin/reduced graphene oxide composite mats with enhanced mechanical properties and conductivity for tissue engineering

In this work, we described a green and simple approach for fabricating regenerated silk fibroin (RSF)/reduced graphene oxide (RGO) fibrous mats by reducing GO in the RSF/GO mats through hydrothermal treatment. As a comparison, RSF mat incorporated with graphene (Gr) was fabricated by solution mixing method. Results showed that the reduction of GO in the mats did take place during hydrothermal treatment. An increase in the crystallinity of the mats was induced by hydrothermal treatment. The maximum Young’s modulus of RSF/RGO mats reached (122.7 ± 3.1) MPa, which was at least 9-fold higher than RSF mats. As the mass ratio of RGO/RSF was 1.5/100, the average sheet resistance of RSF/RGO mats reached (1.2 ± 0.1)×108 Ω/sq, which was better and more stable than that of RSF/Gr mats. Furthermore, cellular tests and antibacterial tests demonstrated that RSF/RGO mats possessed better biocompatibility and antibacterial property in comparison with RSF mats incorporated with GO or Gr. Thus, the RSF/RGO mats could be a good candidate for tissue engineering applications.

Laminin-Coated Electrospun Regenerated Silk Fibroin Mats Promote Neural Progenitor Cell Proliferation, Differentiation, and Survival in vitro.

Neural progenitor cell (NPC) transplantation is a promising technique for central nervous system (CNS) reconstruction and regeneration. Biomaterial scaffolds, frameworks, and platforms can support NPC proliferation and differentiation in vitro as well as serve as a temporary extracellular matrix after transplantation. However, further applications of biomaterials require improved biological attributes. Silk fibroin (SF), which is produced by Bombyx mori, is a widely used and studied protein polymer for biomaterial application. Here, we prepared aligned and random eletrospun regenerated SF (RSF) scaffolds, and evaluated their impact on the growth of NPCs. First, we isolated NPCs and then cultured them on either laminin-coated RSF mats or conventional laminin-coated coverslips for cell assays. We found that aligned and random RSF led to increases in NPC proliferation of 143.8 ± 13.3% and 156.3 ± 14.7%, respectively, compared to controls. Next, we investigated neuron differentiation and found that the aligned and the random RSF led to increases in increase in neuron differentiation of about 93.2 ± 6.4%, and 3167.1 ± 4.8%, respectively, compared to controls. Furthermore, we measured the survival of NPCs and found that RSF promoted NPC survival, and found there was no difference among those three groups. Finally, signaling pathways in cells cultured on RSF mats were studied for their contributions in neural cell differentiation. Our results indicate that RSF mats provide a functional microenvironment and represent a useful scaffold for the development of new strategies in neural engineering research.

Understanding Secondary Structures of Silk Materials via Micro- and Nano-Infrared Spectroscopies.

The secondary structures (also termed conformations) of silk fibroin (SF) in animal silk fibers and regenerated SF materials are critical in determining mechanical performance and function of the materials. In order to understand the structure-mechanics-function relationships of silk materials, a variety of advanced infrared spectroscopic techniques, such as micro-infrared spectroscopies (micro-IR spectroscopies for short), synchrotron micro-IR spectroscopy, and nano-infrared spectroscopies (nano-IR spectroscopies for short), have been used to determine the conformations of SF in silk materials. These IR spectroscopic methods provide a useful toolkit to understand conformations and conformational transitions of SF in various silk materials with spatial resolution from the nano-scale to the micro-scale. In this Review, we first summarize progress in understanding the structure and structure-mechanics relationships of silk materials. We then discuss the state-of-the-art micro- and nano-IR spectroscopic techniques used for silk materials characterization. We also provide a systematic discussion of the strategies to collect high-quality spectra and the methods to analyze these spectra. Finally, we demonstrate the challenges and directions for future exploration of silk-based materials with IR spectroscopies.

Dielectric Breakdown Strength of Regenerated Silk Fibroin Films as a Function of Protein Conformation

Derived from Bombyx mori cocoons, regenerated silk fibroin (RSF) exhibits excellent biocompatibility, high toughness, and tailorable biodegradability. Additionally, RSF materials are flexible, optically clear, easily patterned with nanoscale features, and may be doped with a variety bioactive species. This unique combination of properties has led to increased interest in the use of RSF in sustainable and biocompatible electronic devices. In order to explore the applicability of this biopolymer to the development of future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a function of protein conformation. The application of processing conditions that increased β-sheet content (as determined by FTIR analysis) and produced films in the silk II structure resulted in RSF materials with improved Ebd with values reaching up to 400 V/μm.

Electrospun regenerated silk fibroin mats with enhanced mechanical properties

In this paper, a simple and effective method was applied to enhance regenerated silk fibroin (RSF) mats electrospun from aqueous solution. The mats were first mechanically drawn in 90vol.% ethanol aqueous solution and then immersed in the same solution for 30min. The morphology, structure, thermal and mechanical properties of the RSF mats with different draw rates and draw ratios were investigated by scanning electron microscope (SEM), Raman spectroscopy, wide angle X-ray diffraction (WAXD), differential scanning calorimetry (DSC) and tensile test. Results revealed that the content of β-sheet conformation, the crystallinity and the number of fibers aligned to the drawing direction increased evidently with the draw ratio. The breaking strength and breaking energy of the post-treated mats at 1.4× draw ratio and 0.1mm/s draw rate were 8.6MPa and 172.2J/kg, respectively. However, those of the as-spun mats were only 1.8MPa and 93.2J/kg, respectively. The enhanced RSF mats prepared from entirely aqueous solutions may have extensive applications for tissue engineering.

Fabrication of an alternative regenerated silk fibroin nanofiber and carbonated hydroxyapatite multilayered composite via layer-by-layer

A novel multilayered composite consisting of regenerated silk fibroin (RSF) nanofiber and carbonated hydroxyapatite (CHA) was fabricated with the combination of electrospinning of RSF aqueous solution and soaking in CaCl2 and Na2HPO4 solutions alternately. The chemical composition and morphologies of RSF/CHA composite were characterized by FT-IR, XRD, TGA, EDX, and SEM. The results showed that such an organic/inorganic composite had an alternate layered structure, while the CHA mineral partly penetrated into the porous RSF mats, which was similar to the structure of natural nacre. By tuning the CHA deposition procedure and RSF electrospinning condition independently, the thickness of each layer of CHA and RSF, as well as the layer numbers of composite, could be easily regulated. For example, the average thickness of CHA layers with 5 and 10 mineralization cycles were 1.63 and 3.19 μm, while 9.03 and 30.12 μm of porous RSF nanofiber layers could be formed with 7 and 24 h electrospinning process, respectively. Thus, it may provide an efficient and general approach to produce a series of inorganic/organic multilayered biomaterials for biomedical engineering.