Regenerated Silk Fibroin Research Articles

Highly stretchable porous regenerated silk fibroin film for enhanced wound healing.

Silk fibroin (SF) has received interest in tissue engineering owing to its biocompatibility, biodegradability, and favorable mechanical properties. However, the complex preparation, brittleness, and lack of pores in the structure of the silk fibroin film limit its application. Herein, we show that facile dissolution of SF in aqueous phosphoric acid followed by regeneration in aqueous ammonium sulfate ((NH4)2SO4) could afford highly stretchable films with nano-pores formed in the nonsolvent-induced phase separation process. The named phase separation, which determines the morphology and mechanical properties of the regeneration silk fibroin (RSF) films, is highly dependent on the (NH4)2SO4 concentration as well as the initial concentration of the SF solution. Therefore, the RSF films exhibit a tunable pore size ranging from 230 to 510 nm and excellent stretchability with tensile strain up to 143 ± 16%. Most interestingly, the RSF films were shown to support the proliferation of human skin fibroblasts in vitro as well as speed up full-thickness skin wound healing in a rat model. This work establishes an easy and feasible method to access porous RSF membranes that can be used for wound dressing in clinical settings.

Ingestible capsules carrying 3D printed springs: a possible future prospective for SBS treatment?

Background Short Bowel Syndrome (SBS) is a malabsorption syndrome characterised by a severe reduction of the absorbent surface of the intestinal mucosa. Treatment of this condition needs multi-professional teams and different therapies, which are not always enough to ensure enteral autonomy. New techniques are being explored, particularly distraction enterogenesis, which can allow the lengthening of the residual intestines of these patients. This study aims to demonstrate the possibility of using biodegradable materials to design ingestible capsules which carry 3D-printed springs capable of reaching the patient’s intestine. These devices could be used as a slightly invasive distraction enterogenesis technique, stimulating cell proliferation and intestinal elongation without surgery. Materials and methods Capsules were realised with gelatin from pigskin type A from Sigma-Aldrich mixed with regenerated silk fibroin (RS) obtained by reverse engineering on Bombyx Mori cocoons. Springs are composed of a structure of regenerated silk (RS) modified with graphene nanoplatelets (GNP) externally covered with a biodegradable polyhydroxybutyrate-valerate (PHBV) shell. Springs were realised with 3D printing, through which, with an extruder, PHBV and RS compounds are deposited simultaneously in a 3D structure. The springs’ capsules were then analysed with solvents simulating the gastric and intestinal environment to verify their resistance to degradation. PBS (Phosphate Buffered Saline), composed of calcium chloride and magnesium chloride (CaCl 2 + MgCl 2 ), with a pH value of 7.4, was used as a degradative agent; for the gastric tract, we chose the acetic acid, CH 3 COOH, at 12% with a pH value of 2.3. Results While the gelatin-only capsules showed poor resistance to degradation in PBS, the new compound based on gelatin and regenerated silk showed excellent resistance in gastric and intestinal environments, allowing the pills to reach the intestine without dissolving. In addition, the results show variability in the release times of the springs as a function of the pH values and the elastic constants of the springs used: the latter determined that in acetic acid, the release time is increased at an increase of the elastic constant. In contrast, in PBS, an opposite trend was observed. Conclusions Our results confirm the possibility of using gelatin, silk fibroin and PHBV to design devices capable of transporting implantable endoluminal 3D structures, drugs, or growth factors, laying the foundations for a new approach to distraction enterogenesis in SBS patients.

Sequential intervention of anti-inflammatory and osteogenesis with silk fibroin coated polyethylene terephthalate artificial ligaments for anterior cruciate ligament reconstruction.

Graft-host integration after the anterior cruciate ligament (ACL) reconstruction sequentially follows the prognosis from the inflammation period to the regeneration period. However, due to insufficient bioactivity, polyethylene terephthalate (PET) artificial ligaments often require a long period for graft-host integration. To improve graft-host integration, sequential therapy targeting multifactor is widely advocated. In this study, a multilayer regenerated silk fibroin (RSF) coating loaded with heparin and bone morphogenetic protein binding peptide (BBP) for differentiated release was introduced on the surface of the PET artificial ligament by a stepwise deposition method. The drug release profiles of heparin and BBP on the coated PET artificial ligament indicated the features of differential drug release, i.e., with heparin in the outermost layer releasing a significant amount (more than 60%) during the first 5 days while BBP in the inner layer only releasing a small amount (ca. 30%) within 1 week without burst release. Based on the isometric ACL reconstruction model of rabbits, such drug-loaded RSF coating was verified to be able to modulate the early inflammatory response and promote the maturation of the graft in the articular cavity, meanwhile, it provided a continuous and stable signal of osteogenic induction to improve graft-bone integration. Thus, sequential intervention with heparin and BBP proved to be a reliable combination, and multifunctional RSF-coated PET artificial ligaments hold great potential for improving the clinical efficacy of ACL reconstruction.

Encapsulation of Volatile Monoterpene Fragrances in Mesoporous Organosilica Nanoparticles and Potential Application in Fruit Preservation.

In this work, we synthesized mesoporous silica nanoparticles (MSNs) and periodic mesoporous organosilica nanoparticles containing bridging groups of ethylene (E-PMO) and phenylene (P-PMO) and compared their adsorption properties using D-limonene (Lim), myrcene (Myr), and cymene (Cym) as model guest molecules. For the selected nanoparticles of ~100 nm in diameter, the loading capacity to the volatile fragrances was in the order of P-PMO < E-PMO < MSN, consistent with the trend of increasing total pore volume. For example, P-PMO, E-PMO, and MSN had a Lim uptake of 42.2 wt%, 47.3 wt%, and 62.7 wt%, respectively, which was close to their theoretical adsorption capacity. Under isothermal thermogravimetric analysis conditions (30 °C, a N2 flow of 1 mL min−1), the lowest fragrance release of ~56% over 24 h was observed for P-PMO, followed by E-PMO (74−80%), and MSN (~89%). The release kinetics of the fragrant molecules from MSN and PMO materials can be well described by first-order and Weibull models, respectively. Moreover, the incorporation of Lim-loaded P-PMO NPs in an aqueous solution of regenerated silk fibroin provided a composite coating material suitable for perishable fruit preservation. The active layer deposited on fruit peels using dip coating showed good preservation efficacy, enabling the shelf-life of mangoes in a highly humid and hot atmosphere (30−35 °C, 75−85% RH) to be extended to 6 days.

Fabrication and characterization of hydrogel films using A. mylitta regenerated silk fibroin with Poly vinyl alcohol for biomedical uses

Hydrogels are water-absorbing three-dimensional structures or materials that contain more water content. In recent years, hydrogels have been used in many fields like tissue engineering where they can imitate several body tissues that are present in the body. Hydrogel films are fabricated by blending PVA and RSF at different ratios without affecting their natural properties. The hydrogel films containing PVA and RSF in various ratios are fabricated which can be further used for tissue engineering and wound healing purposes with novel properties. In this study, the gelation time was observed for 1-2 days by the vial inversion test. The SEM images showed different pore sizes and surface morphology for all the different ratios of hydrogel films. According to FTIR-ATR spectroscopy, both the RSF and PVA molecules interacted well with each other and RSF showed amorphous as well as crystalline silk I structures i.e. β-sheet in the spectrum whereas DSC and TGA are used to determine the thermal stability of the blended hydrogel films. The mechanical properties of these hydrogel films were controlled by RSF. All the blended hydrogel films showed minimal or no toxicity against NIH3T3 fibroblast cells through the cell viability test, making them promising materials for biomedical uses.

Deformation mechanism and mechanical model construction for recycled regenerated silk fibroin/polyvinyl alcohol blended fibers

The mechanical properties of regenerated silk fibroin-based fibers have attracted much attention, but the related theoretical research and the mechanical model are insufficient. Thus, recycled regenerated silk fibroin (RRSF)/polyvinyl alcohol (PVA) blended as-spun fibers were taken as an example. Transformations of the apparent morphology, microstructure, and two-phase distribution of the as-spun fibers were explored at different tensile stages. The appropriate mechanical models were established with model formulas. The results showed that the RRSF component in the fiber gradually presented an axial fibril-like dispersion and split fragmentary areas under uniform stretching. Then, the PVA component undertook the external force, and ductile fracture finally occurred. At the micro level, the β-sheet content of the RRSF component reached a maximum of 54.7% at 50% elongation. The value of I⊥/I∥ reached the highest point (1.25) when stretched to 100%. The tensile process was divided into low-, negative-, and high-viscoelasticity deformation stages. Nonlinear spring or sticky pot elements were introduced to establish a two-element parallel mechanical model, and the fitting degree of the model formula was higher than 0.993.

Bioinspired Tough and Strong Fibers with Hierarchical Core–Shell Structure

AbstractStrong and tough bio‐based fibers are attractive for both fundamental research and practical applications. In this work, strong and tough hierarchical core–shell fibers with cellulose nanofibrils (CNFs) in the core and regenerated silk fibroins (RSFs) in the shell are designed and prepared, mimicking natural spider silks. CNF/RSF core–shell fibers with precisely controlled morphology are continuously wet‐spun using a co‐axial microfluidic device. Highly‐dense non‐covalent interactions are introduced between negatively‐charged CNFs in the core and positively‐charged RSFs in the shell, diminishing the core/shell interface and forming an integral hierarchical fiber. Meanwhile, shearing by microfluidic channels and post‐stretching induce a better ordering of CNFs in the core and RSFs in the shell, while ordered CNFs and RSFs are more densely packed, thus facilitating the formation of non‐covalent interactions within the fiber matrix. Therefore, CNF/RSF core–shell fibers demonstrate excellent mechanical performances; especially after post‐stretching, their tensile strength, tensile strain, Young's modulus, and toughness are up to 635 MPa, 22.4%, 24.0 GPa, and 110 MJ m−3, respectively. In addition, their mechanical properties are barely compromised even at −40 and 60 °C. Static load and dynamic impact tests suggest that CNF/RSF core–shell fibers are strong and tough, making them suitable for advanced structural materials.

Biocompatible and Printable Ionotronic Sensing Materials Based on Silk Fibroin and Soluble Plant-Derived Polyphenols.

The emergence of ionotronic materials has been recently exploited for interfacing electronics and biological tissues, improving sensing with the surrounding environment. In this paper, we investigated the synergistic effect of regenerated silk fibroin (RS) with a plant-derived polyphenol (i.e., chestnut tannin) on ionic conductivity and how water molecules play critical roles in regulating ion mobility in these materials. In particular, we observed that adding tannin to RS increases the ionic conductivity, and this phenomenon is accentuated by increasing the hydration. We also demonstrated how silk-based hybrids could be used as building materials for scaffolds where human fibroblast and neural progenitor cells can highly proliferate. Finally, after proving their biocompatibility, RS hybrids demonstrate excellent three-dimensional (3D) printability via extrusion-based 3D printing to fabricate a soft sensor that can detect charged objects by sensing the electric fields that originate from them. These findings pave the way for a viable option for cell culture and novel sensors, with the potential base for tissue engineering and health monitoring.

Design of Volumetric Nanolayers via Rapid Proteolysis of Silk Fibroin for Tissue Engineering.

Various methods have been studied to make a regenerated silk fibroin solution. However, most of them take too much time and effort to liquefy. Here, we report that a regenerated silk fibroin solution could be prepared within seconds through acid proteolysis for the first time. The solubilized fibroin could be applied to advanced tissue engineering. Our method shortened the production time to one day (more than 10 times) compared to the general fibroin solution preparation method. It was confirmed that the initial protein affinity nearly doubled from 0.028 to 0.076 μg·mm-2 in FF(ac) compared to FF(aq). A fibroin nanofiber layer having a volumetric hierarchical structure was prepared by electrospinning an acid-proteolyzed fibroin solution, followed by gas foaming. In vitro results of cell adhesion and proliferation capacity of the gas-foamed scaffold were not significantly different compared to the two-dimensional (2D) fibroin nanofiber membrane, overcoming the limitations of volumetric nanofiber scaffolds. We are confident that our research will greatly contribute to the development of regenerative engineering using other proteins.

Preparation of Highly Crystalline Silk Nanofibrils and Their Use in the Improvement of the Mechanical Properties of Silk Films.

Due to their commendable biocompatibility, regenerated silk fibroin (RSF) films have attracted considerable research interest. However, the poor mechanical properties of RSF films have limited their use in various biomedical applications. In this study, a novel, highly crystalline silk fibril was successfully extracted from silk by combining degumming with ultrasonication. Ultrasonication accelerated the development of silk nanofibrils measuring 130–200 nm on the surface of the over-degummed silk fibers, which was confirmed via scanning electron microscopy. Additionally, the crystallinity index of silk fibril was found to be significantly higher (~68%) than that of conventionally degummed silk (~54%), as confirmed by the Fourier-transform infrared (FTIR) spectroscopy results. Furthermore, the breaking strength and elongation of the RSF film were increased 1.6 fold and 3.4 fold, respectively, following the addition of 15% silk nanofibrils. Thus, the mechanical properties of the RSF film were remarkably improved by the addition of the silk nanofibrils, implying that it can be used as an excellent reinforcing material for RSF films.

Piezoresistive MXene/Silk fibroin nanocomposite hydrogel for accelerating bone regeneration by Re-establishing electrical microenvironment.

The electrical microenvironment plays an important role in bone repair. However, the underlying mechanism by which electrical stimulation (ES) promotes bone regeneration remains unclear, limiting the design of bone microenvironment–specific electroactive materials. Herein, by simple co-incubation in aqueous suspensions at physiological temperatures, biocompatible regenerated silk fibroin (RSF) is found to assemble into nanofibrils with a β-sheet structure on MXene nanosheets, which has been reported to inhibit the restacking and oxidation of MXene. An electroactive hydrogel based on RSF and bioencapsulated MXene is thus prepared to promote efficient bone regeneration. This MXene/RSF hydrogel also acts as a piezoresistive pressure transducer, which can potentially be utilized to monitor the electrophysiological microenvironment. RNA sequencing is performed to explore the underlying mechanisms, which can activate Ca2+/CALM signaling in favor of the direct osteogenesis process. ES is found to facilitate indirect osteogenesis by promoting the polarization of M2 macrophages, as well as stimulating the neogenesis and migration of endotheliocytes. Consistent improvements in bone regeneration and angiogenesis are observed with MXene/RSF hydrogels under ES in vivo. Collectively, the MXene/RSF hydrogel provides a distinctive and promising strategy for promoting direct osteogenesis, regulating immune microenvironment and neovascularization under ES, leading to re-establish electrical microenvironment for bone regeneration.

Insignificant Difference in Biocompatibility of Regenerated Silk Fibroin Prepared with Ternary Reagent Compared with Regenerated Silk Fibroin Prepared with Lithium Bromide.

Bombyx mori silk fibroin (SF) is widely used in the field of biomaterials due to its excellent biocompatibility and mechanical properties. However, SF cannot be used directly in many applications and needs to be dissolved first. Lithium bromide (LiBr) is a traditional solvent which is usually used to dissolve SF. However, LiBr has several limitations, e.g., it is expensive, it is toxic to organisms, and it is environmentally unfriendly. Herein, we investigate the possibility of developing a ternary reagent system that is inexpensive, non-toxic to organisms, and environmentally friendly as an alternative for silk fibroin solubilization. The results confirm that regenerated silk fibroin (RSF) prepared using a ternary reagent has the same morphology and amino acid composition as that prepared using LiBr, but the RSF prepared using a ternary reagent still had a small amount of calcium residue even after long-term dialysis. Further research found that the residual calcium does not cause significant differences in the structure and biological performance of the RSF, such as its cytotoxicity, blood compatibility, and antibacterial properties. Therefore, we believe that ternary reagents are an ideal alternative solvent for dissolving SF.

A facile and green approach toward precise fabrication of silk fibroin-based microfluidic devices using water as etchant

Regenerated silk fibroin (RSF) is considered as a superior substrate material for biocompatible and biodegradable microfluidic devices. However, the manufacture of RSF-based microfluidic chips in a simple, low-cost, and environmentally friendly manner remains a difficulty. We devised a tape mask-assisted approach for preparing RSF-based microfluidic chips for cell adhesion and growth utilizing water as an eco-friendly etchant. The RSF solution was pre-hydrolyzed to assure the RSF film's water-solubility. The UV–visible spectra reveal that the RSF film, as made, is easily dissolved in water. The film could be turned into a water-insoluble state after being treated with ethanol for 40 min. Concave and convex micro-patterns may be formed on the RSF film using tape mask-assisted water etching and ethanol-induced insolubilization. Controlling the etching period allowed the tape-mask designs to be carefully transferred to the RSF film. Unlike LiBr etching, water treatment creates channels that are covered by a smooth RSF hydrogel layer, which may aid cell adhesion and development. The creation of the hydrogel layer might be explained by the insoluble RSF chains exposed after water etching and the ethanol-induced conformation change. Human umbilical vein endothelial cells were seeded in a blood vessel-like RSF-based channel, demonstrating the quick attachment and growth tendency. After 7 days of growth, a micro-vessel-like structure was visible in the channel, suggesting the device's high biocompatibility and vast potential. This work introduces a low-cost, facile and eco-friendly approach to fabricating RSF-based microfluidic devices for cell- or tissue-based assays.

Polyacrylamide‐Conductive Hydrogel Modified with Regenerated Silk Fibroin Resulting in Low‐Temperature Resistance and Self‐Healing Properties for Flexible Electronic Skin

AbstractIn this work, we developed a novel method to prepare a hydrogel‐based electronic skin by self‐assembling cross‐linking. The conductive hydrogel (named PGRS) integrates multiple properties. The hydrogel has good moisture retention (70 °C, 14 days), adhesion, and high elongation (2799 %). When the hydrogel is damaged externally, it can repair itself without external stimulation. The hydrogel adheres to the skin surface and monitors large strain movements, such as finger joints, wrist joints, elbow joints, head lowering and head lifting. It can also detect small movements, such as drinking and talking. It is used as an adhesive in liquid nitrogen (−196 °C). PGRS conductive hydrogel with multiple synergistic functions. It has great potential in the direction of flexible electronic skin. It indicates that PGRS conductive hydrogels have highly prospective applications in wearable devices.

Degradation of Regenerated Silk Fibroin in Soil and Marine Environments

Degradation of Regenerated Silk Fibroin in Soil and Marine Environments

Bioinspired silk fibroin materials: From silk building blocks extraction and reconstruction to advanced biomedical applications

Silk fibroin has become a promising biomaterial owing to its remarkable mechanical property, biocompatibility, biodegradability, and sufficient supply. However, it is difficult to directly construct materials with other formats except for yarn, fabric and nonwoven based on natural silk. A promising bioinspired strategy is firstly extracting desired building blocks of silk, then reconstructing them into functional regenerated silk fibroin (RSF) materials with controllable formats and structures. This strategy could give it excellent processability and modifiability, thus well meet the diversified needs in biomedical applications. Recently, to engineer RSF materials with properties similar to or beyond the hierarchical structured natural silk, novel extraction and reconstruction strategies have been developed. In this review, we seek to describe varied building blocks of silk at different levels used in biomedical field and their effective extraction and reconstruction strategies. This review also present recent discoveries and research progresses on how these functional RSF biomaterials used in advanced biomedical applications, especially in the fields of cell-material interactions, soft tissue regeneration, and flexible bioelectronic devices. Finally, potential study and application for future opportunities, and current challenges for these bioinspired strategies and corresponding usage were also comprehensively discussed. In this way, it aims to provide valuable references for the design and modification of novel silk biomaterials, and further promote the high-quality-utilization of silk or other biopolymers.

Peptide-Functionalized Silk Fibers as a Platform to Stabilize Gelatin for Use in Ingestible Devices.

The combination of pharmacologic and endoscopic therapies is the gold standard for treating intestinal failures. The possibility of chemical solubility in water is mandatory for intelligent capsules. Functionalised silk fibroin with peptides and covalently linking different molecular entities to its structure make this protein a platform for preparing gels dissolving in the small and large intestine for drug delivery. In the present study, we linked a peptide containing the cell-adhesive motif Arginine–Glycine–Aspartic acid (RGD) to degummed silk fibres (DSF). Regenerated silk fibroin (RS) films obtained by dissolving functionalised DSF in formic acid were used to prepare composite gelatin. We show that such composite gelatin remains stable and elastic in the simulated gastric fluid (SGF) but can dissolve in the small and large intestines’ neutral-pH simulated intestine fluid (SIF). These findings open up the possibility of designing microfabricated and physically programmable scaffolds that locally promote tissue regeneration, thanks to bio-enabled materials based on functionalised regenerated silk.

Viscoelastic Liquid from Regenerated Silk Fibroin in the Silk I Conformation: A Writeable and Shapeable Material

Viscoelastic Liquid from Regenerated Silk Fibroin in the Silk I Conformation: A Writeable and Shapeable Material

Effect of post-treatment methods and nanoparticles on the conformation of silk fibroin and their impact on electrical properties

ABSTRACT This paper investigates the effect of different plasticizers (glycerol and dextrose), post-treatment methods, and metal oxide nanoparticles, i.e. zinc oxide and copper oxide on the structural properties of silk fibroin films and their effect on electrical properties, i.e. dielectric strength. Structural properties of the silk fibroin films have been evaluated using Fourier transform infrared spectroscopy and X-ray diffraction technique. It was found that plasticizers and post-treatment applied on the regenerated silk fibroin films have impacted the β-sheet conformation significantly. The dielectric strength of the silk fibroin films was measured using the parallel plate capacitor method and an interrelation with structural properties was observed. Nanoparticles have been introduced into the silk fibroin films as a progressive method to impart substantial effect on the dielectric strength of the silk fibroin films. However, an unexpected change in dielectric strength has been obtained. The mechanism behind the change in dielectric strength has been explored in this work.

Novel Dry Spinning Process of Natural Macromolecules for Sustainable Fiber Material -1- Proof of the Concept Using Silk Fibroin.

Researchers around the world are developing technologies to minimize carbon dioxide emissions or carbon neutrality in various fields. In this study, the dry spinning of regenerated silk fibroin (RSF) was achieved as a proof of concept for a process using ionic liquids as dissolution aids and plasticizers in developing natural polymeric materials. A dry spinning equipment system combining a stainless-steel syringe and a brushless motor was built to generate fiber compacts from a dope of silk fibroin obtained by degumming silkworm silk cocoons and ionic liquid 1-hexyl-3-methyl-imidazolium chloride ([HMIM][Cl]) according to a general method. The maximum stress and maximum elongation of the RSF fibers were 159.9 MPa and 31.5%, respectively. RSF fibers containing ionic liquids have a homogeneous internal structure according to morphological investigations. Elemental analysis of fiber cross sections revealed the homogeneous distribution of nonvolatile ionic liquid [HMIM][Cl] in RSF fibers. Furthermore, the removal of ionic liquids from RSF fibers through impregnation washing with organic solvents was verified to enhance industrial applications. Tensile testing showed that the fiber strength could be maintained even after removing the ionic liquid. Thermogravimetric analysis results show that the organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol is chemically coordinated to silk fibroin and, as a natural polymer, can withstand heat up to 250 °C.