Injectable Bombyx mori (B. mori) silk fibroin/MXene conductive hydrogel for electrically stimulating neural stem cells into neurons for treating brain damage.

Brain damage is a common tissue damage caused by trauma or diseases, which can be life-threatening. Stem cell implantation is an emerging strategy treating brain damage. The stem cell is commonly embedded in a matrix material for implantation, which protects stem cell and induces cell differentiation. Cell differentiation induction by this material is decisive in the effectiveness of this treatment strategy. In this work, we present an injectable fibroin/MXene conductive hydrogel as stem cell carrier, which further enables in-vivo electrical stimulation upon stem cells implanted into damaged brain tissue. Cell differentiation characterization of stem cell showed high effectiveness of electrical stimulation in this system, which is comparable to pure conductive membrane. Axon growth density of the newly differentiated neurons increased by 290% and axon length by 320%. In addition, unfavored astrocyte differentiation is minimized. The therapeutic effect of this system is proved through traumatic brain injury model on rats. Combined with in vivo electrical stimulation, cavities formation is reduced after traumatic brain injury, and rat motor function recovery is significantly promoted.

Design of Bombyx mori (B. mori) Silk Fibroin Microspheres for Developing Biosafe Sunscreen.

Sunscreens play a crucial role in protecting the skin from ultraviolet (UV) damage. However, present commercial sunscreens have a tendency to generate free radicals in the UV window, resulting in serious inflammatory responses and health problems. In this study, we demonstrate that silk fibroin microspheres (SFMPs) assembled from regenerated silk fibroin (SF) could scavenge free radicals while preventing UV irradiation and thus present a promising sunscreen. The SFMP reflected more UV light than SF and presented a higher stability than that of organic commercial sunscreens. In vitro analysis proved that SFMP could more efficiently scavenge the hydroxy radical and reduce the intracellular reactive oxygen than titanium dioxide (TiO2). In vivo experiments exhibited that SFMP provided stronger skin protection against UV irradiation than commercial sunscreens and TiO2. Furthermore, SFMP treatment significantly inhibited the skin inflammatory response. This work suggests that the SFMP has great potential to be developed into a biosafe sunscreen.

Regenerated silk fibroin coating stable liquid metal nanoparticles enhance photothermal antimicrobial activity of hydrogel for wound infection repair.

The integration of liquid metal (LM) and regenerated silk fibroin (RSF) hydrogel holds great potential for achieving effective antibacterial wound treatment through the LM photothermal effect. However, the challenge of LM's uncontrollable shape-deformability hinders its stable application. To address this, we propose a straightforward and environmentally-friendly ice-bath ultrasonic treatment method to fabricate stable RSF-coated eutectic gallium indium (EGaIn) nanoparticles (RSF@EGaIn NPs). Additionally, a double-crosslinked hydrogel (RSF-P-EGaIn) is prepared by incorporating poly N-isopropyl acrylamide (PNIPAAm) and RSF@EGaIn NPs, leading to improved mechanical properties and temperature sensitivity. Our findings reveal that RSF@EGaIn NPs exhibit excellent stability, and the use of near-infrared (NIR) irradiation enhances the antibacterial behavior of RSF-P-EGaIn hydrogel in vivo. In fact, in vivo testing demonstrates that wounds treated with RSF-P-EGaIn hydrogel under NIR irradiation completely healed within 14 days post-trauma infection, with the formation of new skin and hair. Histological examination further indicates that RSF-P-EGaIn hydrogel promoted epithelialization and well-organized collagen deposition in the dermis. These promising results lay a solid foundation for the future development of drug release systems based on photothermal-responsive hydrogels utilizing RSF-P-EGaIn.

Ultrafast Bi-Directional Bending Moisture-Responsive Soft Actuators through Superfine Silk Rod Modified Bio-Mimicking Hierarchical Layered Structure.

Development of stimulus-responsive materials is crucial for novel soft actuators. Among these actuators, the moisture-responsive actuators are known for their accessibility, eco-friendliness, and robust regenerative attributes. A major challenge of moisture-responsive soft actuators (MRSAs) is achieving significant bending curvature within short response times. Many plants naturally perform large deformation through a layered hierarchical structure in response to moisture stimuli. Drawing inspiration from the bionic structure of Delosperma nakurense (D. nakurense) seed capsule, here the fabrication of an ultrafast bi-directional bending MRSAs is reported. Combining a superfine silk fibroin rod (SFR) modified graphene oxide (GO) moisture-responsive layer with a moisture-inert layer of reduced graphene oxide (RGO), this actuator demonstrated large bi-directional bending deformation (-4.06 ± 0.09 to 10.44 ± 0.00 cm-1 ) and ultrafast bending rates (7.06 cm-1  s-1 ). The high deformation rate is achieved by incorporating the SFR into the moisture-responsive layers, facilitating rapid water transmission within the interlayer structure. The complex yet predictable deformations of this actuator are demonstrated that can be utilized in smart switch, robotic arms, and walking device. The proposed SFR modification method is simple and versatile, enhancing the functionality of hierarchical layered actuators. It holds the potential to advance intelligent soft robots for application in confined environments.

Hierarchically patterned protein scaffolds with nano-fibrillar and micro-lamellar structures modulate neural stem cell homing and promote neuronal differentiation.

Biophysical factors are essential in cell survival and behaviors, but constructing a suitable 3D microenvironment for the recruitment of stem cells and exerting their physiological functions remain a daunting challenge. Here, we present a novel silk fibroin (SF)-based fabrication strategy to develop hierarchical microchannel scaffolds for biomimetic nerve microenvironments in vitro. We first modulated the formation of SF nanofibers (SFNFs) that mimic the nanostructures of the native extracellular matrix (ECM) by using graphene oxide (GO) nanosheets as templates. Then, SFNF-GO systems were shaped into 3D porous scaffolds with aligned micro-lamellar structures by freeze-casting. The interconnected microchannels successfully induced cell infiltration and migration to the SFNF-GO scaffolds' interior. Meanwhile, the nano-fibrillar structures and the GO component significantly induced neural stem cells (NSCs) to differentiate into neurons within a short timeframe of 14 d. Importantly, these 3D hierarchical scaffolds induced a mild inflammatory response, extensive cell recruitment, and effective stimulation of NSC neuronal differentiation when implanted in vivo. Therefore, these SFNF-GO lamellar scaffolds with distinctive nano-/micro-topographies hold promise in the fields of nerve injury repair and regenerative medicine.

Silk Protein-Mediated Biomineralization: From Bioinspired Strategies and Advanced Functions to Biomedical Applications.

Biomineralization refers to the process through which minerals nucleate in a structured manner to form specific crystal structures by the regulating of biomacromolecules. Biomineralization occurs in bones and teeth within the human body, where collagen acts as a template for the nucleation of hydroxyapatite (HA) crystals. Similar to collagen, silk proteins spun by silkworms can also serve as templates for the nucleation and growth of inorganic substances at interfaces. By enabling the binding of silk proteins to inorganic minerals, the process of biomineralization enhances the properties of silk-based materials and broadens their potential applications, rendering them highly promising for use in biomedical applications. In recent years, the development of biomineralized materials using silk proteins has garnered considerable attention in the biomedical field. This comprehensive review outlines the mechanism of biomineral formation mediated by silk proteins, as well as various biomineralization methods used to prepare silk-based biomineralized materials (SBBMs). Additionally, we discuss the physicochemical properties and biological functions of SBBMs, and their potential applications in various fields such as bioimaging, cancer therapy, antibacterial treatments, tissue engineering, and drug delivery. In conclusion, this review highlights the significant role that SBBMs can play in the biomedical field.

Graphene Oxide-Coated Patterned Silk Fibroin Films Promote Cell Adhesion and Induce Cardiomyogenic Differentiation of Human Mesenchymal Stem Cells.

Cardiac tissue engineering is a promising strategy for the treatment of myocardial damage. Mesenchymal stem cells (MSCs) are extensively used in tissue engineering. However, transformation of MSCs into cardiac myocytes is still a challenge. Furthermore, weak adhesion of MSCs to substrates often results in poor cell viability. Here, we designed a composite matrix based on silk fibroin (SF) and graphene oxide (GO) for improving the cell adhesion and directing the differentiation of MSCs into cardiac myocytes. Specifically, patterned SF films were first produced by soft lithographic. After being treated by air plasma, GO nanosheets could be successfully coated on the patterned SF films to construct the desired matrix (P-GSF). The resultant P-GSF films presented a nano-topographic surface characterized by linear grooves interlaced with GO ridges. The P-GSF films exhibited high protein absorption and suitable mechanical strength. Furthermore, the P-GSF films accelerated the early cell adhesion and directed the growth orientation of MSCs. RT-PCR results and immunofluorescence imaging demonstrated that the P-GSF films significantly improved the cardiomyogenic differentiation of MSCs. This work indicates that patterned SF films coated with GO are promising matrix in the field of myocardial repair tissue engineering.

Efficient Tumor Immunotherapy through a Single Injection of Injectable Antigen/Adjuvant-Loaded Macroporous Silk Fibroin Microspheres.

Synthetic or natural materials have been used as vaccines in cancer immunotherapy. However, using them as vaccines necessitates multiple injections or surgical implantations. To tackle such daunting challenges, we develop an injectable macroporous Bombyx mori (B. mori) silk fibroin (SF) microsphere loaded with antigens and immune adjuvants to suppress established tumors with only a single injection. SF microspheres can serve as a scaffold by injection and avoid surgical injury as seen in traditional scaffold vaccines. The macroporous structure of the vaccine facilitates the recruitment of immune cells and promotes the activation of dendritic cells (DCs), resulting in a favorable immune microenvironment that further induces strong humoral and cellular immunity. We have also modified the vaccine into a booster version by simply allowing the antigens to be adsorbed onto the SF microspheres. The booster vaccine highly efficiently suppresses tumor growth by improving the cytotoxic T lymphocyte (CTL) response. In general, these results demonstrate that the macroporous SF microspheres can serve as a facile platform for tumor vaccine therapy in the future. Since the SF microspheres are also potential scaffolds for tissue regeneration, their use as a vaccine platform will enable their applications in eradicating tumors while regenerating healthy tissue to heal the tumor-site cavity.

A sustainable single-component "Silk nacre".

Synthetic composite materials constructed by hybridizing multiple components are typically unsustainable due to inadequate recyclability and incomplete degradation. In contrast, biological materials like silk and bamboo assemble pure polymeric components into sophisticated multiscale architectures, achieving both excellent performance and full degradability. Learning from these natural examples of bio-based "single-component" composites will stimulate the development of sustainable materials. Here, we report a single-component "Silk nacre," where nacre's typical "brick-and-mortar" structure has been replicated with silk fibroin only and by a facile procedure combining bidirectional freezing, water vapor annealing, and densification. The biomimetic design endows the Silk nacre with mechanical properties superior to those of homogeneous silk material, as well as to many frequently used polymers. In addition, the Silk nacre shows controllable plasticity and complete biodegradability, representing an alternative substitute to conventional composite materials.

Silk-based bioinspired structural and functional materials.

Natural biological materials provide a rich source of inspiration for building high-performance materials with extensive applications. By mimicking their chemical compositions and hierarchical architectures, the past decades have witnessed the rapid development of bioinspired materials. As a very promising biosourced raw material, silk is drawing increasing attention due to excellent mechanical properties, favorable versatility, and good biocompatibility. In this review, we provide an overview of the recent progress in silk-based bioinspired structural and functional materials. We first give a brief introduction of silk, covering its sources, features, extraction, and forms. We then summarize the preparation and application of silk-based materials mimicking four typical biological materials including bone, nacre, skin, and polar bear hair. Finally, we discuss the current challenges and future prospects of this field.

Arginine induces protein self-assembly into nanofibers for triggering osteogenic differentiation of stem cells.

Although silk proteins are considered promising in building a scaffold for tissue engineering, one of the silk proteins, Bombyx mori silk sericin (BS), has limited processability in producing nanofibrous scaffolds because its surface charge anisotropy promotes gelation instead. To overcome this daunting challenge, we developed a mild and simple procedure for assembling BS into nanofibers and nanofibrous scaffolds. Briefly, arginine was added to the aqueous BS solution to reduce the negative charge of BS, thereby inducing BS to self-assemble into nanofibers in the solution. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra showed that arginine promoted the formation of ß-sheet conformation in BS and increased its thermal stability. Furthermore, the arginine-induced BS nanofiber solution could be casted into scaffolds made of abundant network-like nanofibrous structures. The BS scaffolds promoted cell adhesion and growth and stimulated osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) in the absence of differentiation inducers in culture media. Our study presents a new strategy for assembling proteins into osteogenic nanofibrous scaffolds for inducing stem cell differentiation in regenerative medicine.

Bioinspired Multifunctional Cellular Plastics with a Negative Poisson's Ratio for High Energy Dissipation.

Cellular plastics have been widely used in transportation, aerospace, and personal safety applications owing to their excellent mechanical, thermal, and acoustic properties. It is highly desirable to impart them with a complex porous structure and composition distribution to obtain specific functionality for various engineering applications, which is challenging with conventional foaming technologies. Herein, it is demonstrated that this can be achieved through the controlled freezing process of a monomer/water emulsion, followed by cryopolymerization and room temperature thawing. As ice is used as a template, this method is environmentally friendly and capable of producing cellular plastics with various microstructures by harnessing the numerous morphologies of ice crystals. In particular, a cellular plastic with a radially aligned structure shows a negative Poisson's ratio under compression. The rigid plastic shows a much higher energy dissipation capability compared to other materials with similar negative Poisson's ratios. Additionally, the simplicity and scalability of this approach provides new possibilities for fabricating high-performance cellular plastics with well-defined porous structures and composition distributions.

A Transparent, Skin-Inspired Composite Film with Outstanding Tear Resistance Based on Flat Silk Cocoon.

Flexible and transparent substrates play a fundamental role as a mechanical support in advanced electronic devices. However, commonly used polymer films, such as polydimethylsiloxane, show low tear resistance because of their crack sensitivity. Herein, inspired by the excellent mechanical robustness of the skin and its fibrous structure, an epoxy-resin-based composite with a flat silk cocoon as a reinforcing fiber network is fabricated. With only 1 wt% of silk fiber, the tensile strength and modulus of the as-prepared composite film are considerably increased by 300% and 612% compared to those of pure resin, while still maintaining flexibility and transparency. More importantly, the composite shows remarkable tear resistance: without fracture after ≈30 000 tensile cycles. The potential application of such transparent composite films as mechanically robust substrates for flexible electronics is also demonstrated. In addition, this study represents a bioinspired strategy to construct high-performance functional composite materials.

Preparation and biomedical applications of silk fibroin-nanoparticles composites with enhanced properties - A review.

Polymer-nanoparticles composites have attracted considerable attention over the years, due to the superior combination performance from both materials. As a versatile natural polymer, silk fibroin has been combined with various nanoparticles. The introduction of nanoparticles brings many optimized and new functionalities to silk fibroin, such as mechanical, biological, thermal, electrical, florescent and magnetical properties. This review focuses on the preparation methods and enhanced properties of silk fibroin-nanoparticle composites, especially their biomedical applications.

Self-stabilized silk sericin-based nanoparticles: In vivo biocompatibility and reduced doxorubicin-induced toxicity.

A variety of colloid stabilizers and cryoprotectants confer improved nanoparticle (NP) colloidal stability and redisperability. However, discounted tumor targetability, delivery efficacy and possible side effects limit the application in vascular delivery of NPs. Here we present water-soluble silk sericin (SS) not only as a material for the preparation of NPs, but also both a dispersion stabilizer and a cryoprotectant. In the absence of any stabilizers, SS-based NPs (SSC@NPs) can resist the adsorption of serum proteins, preventing the formation of particle agglomerates. Following freeze-drying without addition of cryoprotectants, SSC@NPs powder can be easily resuspended into NP dispersion with a nearly monodispersed distribution. Additionally, SSC@NPs do not result in acute toxicity in mice at a dose of 400 mg/kg with a slow injection. Moreover, doxorubicin (DOX)-loaded SSC@NPs (DOX-SSC@NPs) diminish the biodistribution of DOX in the heart, mitigating DOX-induced cardiotoxicity of mice without compromising therapeutic efficacy. Our results suggest that the self-stabilized SSC@NPs could be a secure and effective drug carrier for intravenous administration when deprived of protective agents. STATEMENT OF SIGNIFICANCE: During manufacturing process such as freeze-drying, or interaction with complex fluids like blood, NPs for systemic drug delivery need to be highly dispersible and structurally intact. In this work, we have demonstrated the self-stability of SSC@NPs subjected to biological media and freeze-drying. This study represents the first work showing water-soluble SS could both act as a dispersion stabilizer and a cryoprotectant due to its hydrophilicity. Plus, good in vivo biocompatibility of SSC@NPs has been confirmed. Therefore, it may be promising that water-soluble SS can be generally used as a safe biomaterial against serum adsorption.

Preparation of a novel silk microfiber covered by AgCl nanoparticles with antimicrobial activity.

We prepared silk fibroin microfibers in which silver chloride (AgCl) nanoparticles were dispersed, by sequential dipping of microfibers obtained using alkaline hydrolysis in alternating solutions of silver nitrate and potassium chloride. Scanning and transmission electron microscopy showed an increase in nanoparticle size and quantity with increase in dipping cycles and solution concentration, but ultrasound irradiation did not affect nanoparticle formation. The presence of cubic AgCl crystals was confirmed by energy dispersive X-ray spectroscopy and X-ray diffractometry. Differential scanning calorimetry and Fourier transform infrared spectroscopy revealed that the nanoparticles do not affect the microfiber properties. The growth of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria was inhibited by microfiber covered with AgCl nanoparticles. This antimicrobial activity allows to use microfiber as a reinforced or surface additive biomaterial. Microsc. Res. Tech. 80:272-279, 2017. © 2016 Wiley Periodicals, Inc.

Fabrication of a novel blended membrane with chitosan and silk microfibers for wound healing: characterization, in vitro and in vivo studies.

Pure chitosan membranes present insufficient mechanical properties and a high swelling ratio, which limits their application in biomedical field. In this study, silk microfibers were obtained by chemical hydrolysis, and a novel type of chitosan/silk microfiber (CS/mSF) blended membrane was reported and its multiple physical properties were evaluated. The mechanical properties were significantly improved after blending silk microfibers with a chitosan matrix, while the swelling ratio was decreased. Observation of the surface microstructures of the blended membranes via scanning electron microscopy showed abundant embedding of mSF into the CS matrix, as well as connections among mSF. In vitro cytocompatibility was also investigated, and the blended membranes exhibited significant cytocompatibility, which was demonstrated by cell proliferation and cell morphology. Furthermore, the in vivo healing effects of the blended membrane as a wound dressing were determined on a full-thickness skin wound model of rats. Animal studies revealed that the membranes containing mSF exhibited increased wound healing efficiency compared with pure CS membranes and treatment without wound dressing. From an examination of histological changes, a higher level of epithelialization and collagen formation was observed with treatment of CS/mSF blended membranes after a 21 day repair period. In conclusion, our results indicated that the blended membranes with CS and mSF might be a potential candidate material for wound healing.