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β-Sheet rich silk nanofiber hydrogels are suitable scaffolds in tissue regeneration and carriers for various drugs. However, unsatisfactory mechanical performance limits its applications. Here, insight into the silk nanofibers stimulates the remodeling of previous solvent systems to actively regulate the assembly of silk nanofibers. Formic acid, a solvent of regenerated silk fibroin, is used to shield the charge repulsion of silk nanofibers to facilitate the nanofiber assembly under concentrated solutions. Formic acid was replaced with water to solidify the assembly, which induced the formation of a tough hydrogel. The hydrogels generated with this process possessed a modulus of 5.88 ± 0.82 MPa, ultimate stress of 1.55 ± 0.06 MPa, and toughness of 0.85 ± 0.03 MJ m -3 , superior to those of previous silk hydrogels prepared through complex cross-linking processes. Benefiting from the dense gel network and high β-sheet content, these silk nanofiber hydrogels had good stability and antiswelling ability. The modulus could be modulated via changing the silk nanofiber concentration to provide differentiation signals to stem cells. Improved mechanical and bioactive properties with these hydrogels suggest utility in biomedical and engineering fields. More importantly, our present study reveals that the in-depth understanding of silk nanofibers could infuse power into traditional fabrication systems to achieve more high performance biomaterials, which is seldom considered in silk material studies.

Scarless spinal cord regeneration remains a challenge due to the complicated microenvironment at lesion sites. In this study, the nerve growth factor (NGF) was immobilized in silk protein nanofiber hydrogels with hierarchical anisotropic microstructures to fabricate bioactive systems that provide multiple physical and biological cues to address spinal cord injury (SCI). The NGF maintained bioactivity inside the hydrogels and regulated the neuronal/astroglial differentiation of neural stem cells. The aligned microstructures facilitated the migration and orientation of cells, which further stimulated angiogenesis and neuron extensions both in vitro and in vivo . In a severe rat long-span hemisection SCI model, these hydrogel matrices reduced scar formation and achieved the scarless repair of the spinal cord and effective recovery of motor functions. Histological analysis confirmed the directional regenerated neuronal tissues, with a similar morphology to that of the normal spinal cord. The in vitro and in vivo results showed promising utility for these NGF-laden silk hydrogels for spinal cord regeneration while also demonstrating the feasibility of cell-free bioactive matrices with multiple cues to regulate endogenous cell responses.

Regenerated silk nanofibers are interesting as protein-based material building blocks due to their unique structure and biological origin. Here, a new strategy based on control of supramolecular assembly was developed to regulate interactions among silk nanofibers by changing the solvent, achieving tough mechanical features for silk films. Formic acid was used to replace water related to charge repulsion of silk nanofibers in solution, inducing interactions among the nanofibers. The films formed under these conditions had an elastic modulus of 3.4 ± 0.3 GPa, an ultimate tensile strength of 76.9 ± 1.6 MPa, and an elongation at break of 3.5 ± 0.1%, while the materials formed from aqueous solutions remained fragile. The mechanical performance of the formic acid-derived nanofiber films was further improved through post-stretching or via the addition of graphene. In addition, the silk nanofiber films could be functionalized with various bioactive ingredients such as curcumin. These new silk nanofiber films with a unique combination of mechanical properties and functions provide new biomaterials achieved using traditional solvents and processes through insight and control of their assembly mechanisms in solution.

Dysangiogenesis and chronic inflammation are two critical reasons for diabetic foot ulcers. Desferrioxamine (DFO) was used clinically in the treatment of diabetic foot ulcers by repeated injections because of its capacity to induce vascularization. Biocompatible carriers that release DFO slowly and facilitate healing simultaneously are preferable options to accelerate the healing of diabetic wounds. Here, DFO-laden silk nanofiber hydrogels that provided a sustained release of DFO for more than 40 days were used to treat diabetic wounds. The DFO-laden hydrogels stimulated the healing of diabetic wounds. In vitro cell studies revealed that the DFO-laden hydrogels modulated the migration and gene expression of endothelial cells, and they also tuned the inflammation behavior of macrophages. These results were confirmed in an in vivo diabetic wound model. The DFO-laden hydrogels alleviated dysangiogenesis and chronic inflammation in the diabetic wounds, resulting in a more rapid wound healing and increased collagen deposition. Both in vitro and in vivo studies suggested potential clinical applications of these DFO-laden hydrogels in the treatment of diabetic ulcers.

A method was developed to fabricate light, water-insoluble silk fibroin nanofibrous aerogels (SNFAs) through solvent welding of lyophilized silk nanofibrous 3D networks at the junction points while converting silk structures from random-coils to β-sheets (water insoluble). Aromatic alcohols, especially phenethyl alcohol (PEA), supported robust solvent welding and the structural conversion of silk. PEA vapor treatment was a better approach than solvent infusion to retain volume, density, and mechanical strength of the SNFAs. The mechanical properties of highly orientated SNFAs were superior to randomly distributed fibers. The SNFAs had a low density (3.5 mg/cm 3 ), high hydrophobicity (140.9°), and a porous surface morphology on the individual nanofibers, resulting in high efficiency and selectivity for absorbing particulate matter and oils. Compared with commonly used inorganic aerogels, the SNFAs developed in this study are biocompatible, easily functionalized, environmentally friendly, and low-cost and therefore have potential for air and water purification, biosensors, drug delivery, and tissue engineering.

The hierarchical structure of the ECM provides specific niches for tissues to regulate cell behavior, yet the challenge remains to design biomaterial systems for tissue regeneration to recreate such features in vitro. Here, we achieved this goal through the use of aligned hierarchical structures of native silk fibers, generated through the integration of "bottom-up" and "top-down" strategies to generate regenerated silk fibers with aligned nano- to micro-hierarchical structures. To achieve these designs, we assembled and dispersed silk nanofibers (SNF) in formic acid and spun them into fibers using bioinspired microfluidic chips with a geometry mimicking the native silk gland. The fibers generated using this device exhibited aligned hierarchical structure with fiber mechanical properties superior to fibers derived from more traditional spinning approaches with regenerated silk solutions. Besides the improved mechanical properties, Raman spectroscopic results indicated similarly aligned structures to native fibers and active control of cell proliferation, migration, and aggregate orientation. The results indicate the feasibility of developing bioactive silk fiber materials with hierarchical structures to facilitate utility in a range of cell and tissue regeneration scenarios.

Multiple physical cues such as hierarchical microstructures, topography, and stiffness influence cell fate during tissue regeneration. Yet, introducing multiple physical cues to the same biomaterial remains a challenge. Here, a synergistic cross-linking strategy was developed to fabricate protein hydrogels with multiple physical cues based on combinations of two types of silk nanofibers. β-sheet-rich silk nanofibers (BSNFs) were blended with amorphous silk nanofibers (ASNFs) to form composite nanofiber systems. The composites were transformed into tough hydrogels through horseradish peroxidase (HRP) cross-linking in an electric field, where ASNFs were cross-linked with HRP, while BSNFs were aligned by the electrical field. Anisotropic morphologies and higher stiffness of 120 kPa were achieved. These anisotropic hydrogels induced osteogenic differentiation and the aligned aggregation of stem cells in vitro while also exhibiting osteoinductive capacity in vivo. Improved tissue outcomes with the hydrogels suggest promising applications in bone tissue engineering, as the processing strategy described here provides options to form hydrogels with multiple physical cues.

Hydrophobic biomolecules realize their functions in vivo in aqueous environments, often through a delicate balance of amphiphilicity and chaperones. Introducing exogenous hydrophobic biomolecules into in vivo aqueous systems is a challenge in drug delivery and regenerative medicine, where labile linkers, carriers, and fusions or chimeric molecules are often designed to facilitate such aqueous interfaces. Here, we utilize naturally derived silk nanofiber shuttles with the capacity to transport hydrophobic cargos directly into aqueous solutions. These nanofibers disperse in organic solvents and in aqueous solutions because of their inherent amphiphilicity, with enriched hydrophobicity and strategically interspersed negatively charged groups. Hydrophobic molecules loaded on these shuttles in organic solvent-water systems separated from the solvent after centrifugation. These concentrated hydrophobic molecule-loaded nanofibers could then be dispersed into aqueous solution directly without modification. These shuttle systems were effective for different hydrophobic molecules such as drugs, vitamins, and dyes. Improved biological stability and functions of hydrophobic cargos after loading on these nanofibers suggest potential applications in drug delivery, cosmetology, medical diagnosis, and related health fields, with a relatively facile process.

Improved and more rapid healing of full-thickness skin wounds remains a major clinical need. Silk fibroin (SF) is a natural protein biomaterial that has been used in skin repair. However, there has been little effort aimed at improving skin healing through tuning the hierarchical microstructure of SF-based matrices and introducing multiple physical cues. Recently, enhanced vascularization was achieved with SF scaffolds with nanofibrous structures and tunable secondary conformation of the matrices. We hypothesized that anisotropic features in nanofibrous SF scaffolds would promote cell migration, neovascularization, and tissue regeneration in wounds. To address this hypothesis, SF nanofibers were aligned in an electric field to form anisotropic porous scaffolds after lyophilization. In vitro and in vivo studies indicated good cytocompatibility, and improved cell migration and vascularization than nanofibrous scaffolds without these anisotropic features. These improvements resulted in more rapid wound closure, tissue ingrowth, and the formation of new epidermis, as well as higher collagen deposition with a structure similar to the surrounding native tissue. The new epidermal layers and neovascularization were achieved by day 7, with wound healing complete by day 28. It was concluded that anisotropic SF scaffolds alone, without a need for growth factors and cells, promoted significant cell migration, vascularization, and skin regeneration and may have the potential to effectively treat dermal wounds.

Mass production of high-quality graphene dispersions under mild conditions impacts the utility of the material for biomedical applications. Various proteins have been used to prepare graphene dispersions, rare sources, and expensive prices for these proteins restrict their large-scale utility for the production of graphene. Here, inexpensive silk proteins as an abundant resource in nature were used for graphene exfoliation. The silk proteins were assembled into hydrophobic nanofibers with negative charge, and then optimized for the production of graphene. Significantly higher concentrations (>8 mg mL -1 ) and yields (>30%) of graphene dispersions under ambient aqueous conditions were achieved compared with previous protein-assisted exfoliation systems. The exfoliated graphene exhibited excellent stability in water and fetal bovine serum solution, cytocompatibility, and conductivity, suggesting a promising future in biomedical and bioengineering applications.

As a functional material, silk has been widely used in tissue engineering, drug release, and tissue regeneration. Increasing subtle control of silk hierarchical structures and thus specific functional performance is required for these applications but remains a challenge. Here, we report a novel silk nanofiber solution achieved through tuning solvent systems used to generate the material. Unlike the β-sheet rich silk nanofibers reported previously, these new silk nanofibers are mainly composed of amorphous structures and maintain a solution state in aqueous environments. The amorphous silk nanofibers are stable enough for storage and also metastable, making them easy to use in the further fabrication of materials through various processes. Silk scaffolds, hydrogels, and films were prepared from these silk nanofiber solutions. These silk materials from amorphous nanofiber solutions show different properties and tunable performance features. Therefore, these amorphous silk nanofibers are suitable units or building blocks for designing silk-based materials.

Silk is useful as a drug carrier due to its biocompatibility, tunable degradation, and outstanding capacity in maintaining the function of drugs. Injectable silk hydrogels could deliver doxorubicin (DOX) for localized chemotherapy for breast cancer. To improve hydrogel properties, thixotropic silk nanofiber hydrogels in an all-aqueous solution were prepared and used to locally deliver DOX. The silk hydrogels displayed thixotropic capacity, allowing for easy injectability followed by solidification in situ. The hydrogels were loaded with DOX and released the drug over eight weeks with pH- and concentration-dependent release kinetics. In vitro and in vivo studies demonstrated that DOX-loaded silk hydrogels had good antitumor response, outperforming the equivalent dose of free DOX administered intravenously. Thixotropic silk hydrogels provide improved injectability to support sustained release, suggesting promising applications for localized chemotherapy.

We report a new ultrathin filtration membrane prepared from silk nanofibrils (SNFs), directly exfoliated from natural Bombyx mori silk fibers to retain structure and physical properties. These membranes can be prepared with a thickness down to 40 nm with a narrow distribution of pore sizes ranging from 8 to 12 nm. Typically, 40 nm thick membranes prepared from SNFs have pure water fluxes of 13 000 L h(-1) m(-2) bar(-1), more than 1000 times higher than most commercial ultrathin filtration membranes and comparable with the highest water flux reported previously. The commercial membranes are commonly prepared from polysulfone, poly(ether sulfone), and polyamide. The SNF-based ultrathin membranes exhibit efficient separation for dyes, proteins, and colloids of nanoparticles with at least a 64% rejection of Rhodamine B. This broad-spectrum filtration membrane would have potential utility in applications such as wastewater treatment, nanotechnology, food industry, and life sciences in part due to the protein-based membrane polymer (silk), combined with the robust mechanical and separation performance features.

Concentration gradients of guidance molecules influence cell behavior and growth in biological tissues and are therefore of interest for the design of biomedical scaffolds for regenerative medicine. We developed an electrospining method to generate a dual-gradient of bioactive molecules and fiber density along electrospun nanofibers without any post spinning treatment. Functionalization with fluorescent molecules demonstrated the efficiency of the method to generate a discontinuous concentration gradient along the aligned fibers. As a proof of concept for tissue engineering, the silk nanofibers were functionalized with increasing concentrations of nerve growth factor (NGF) and the biological activity was assessed and quantified with rat dorsal root ganglion (DRG) neurons cultures. Protein assays showed the absence of passive release of NGF from the functionalized fibers. The results demonstrated that the NGF concentration gradient led to an oriented and increased growth of DRG neurons (417.6 ± 55.7 μm) compared to a single uniform NGF concentration (264.5 ± 37.6 μm). The easy-to-use electrospinning technique combined with the multiple molecules that can be used for fiber functionalization makes this technique versatile for a broad range of applications from biosensors to regenerative medicine.

Neutron reflectivity (NR) measurements of ultrathin surface films (below 30 nm) composed of Bombyx mori silk fibroin protein in combination with atomic force microscopy and ellipsometry were used to reveal the internal structural organization in both dry and swollen states. Reconstituted aqueous silk solution deposited on a silicon substrate using the spin-assisted layer-by-layer (SA-LbL) technique resulted in a monolayer silk film composed of random nanofibrils with constant scattering length density (SLD). However, a vertically segregated ordering with two different regions has been observed in dry, thicker, seven-layer SA-LbL silk films. The vertical segregation of silk multilayer films indicates the presence of a different secondary structure of silk in direct contact with the silicon oxide surface (first 6 nm). The layered structure can be attributed to interfacial β-sheet crystallization and the formation of well-developed nanofibrillar nanoporous morphology for the initially deposited silk surface layers with the preservation of less dense, random coil secondary structure for the layers that follow. This segregated structure of solid silk films defines their complex nonuniform behavior in the D(2)O environment with thicker silk films undergoing delamination during swelling. For a silk monolayer with an initial thickness of 6 nm, we observed the increase in the effective thickness by 60% combined with surprising decrease in density. Considering the nanoporous morphology of the hydrophobic silk layer, we suggested that the apparent increase in its thickness in liquid environment is caused by the air nanobubble trapping phenomenon at the liquid-solid interface.

Silk fibroin (SF) nanofibers, formed through electrospinning, have attractive utility in regenerative medicine due to the biocompatibility, mechanical properties, and tailorable degradability. The mechanism of SF electrospun nanofiber formation was studied to gain new insight into the formation and control of nanofibers. SF electrospinning solutions with different nanostructures (nanospheres or nanofilaments) were prepared by controlling the drying process during the preparation of regenerated SF films. Compared to SF nanospheres in solution, SF nanofilaments had better spinnability with lower viscosity when the concentration of silk protein was below 10%, indicating a critical role for SF morphology, and in particular, nanostructures, for the formation of electrospun fibers. More interesting, the diameter of electrospun fibers gradually increased from 50 to 300 nm as the concentration of SF nanofilaments in the solution increased from 6 to 12%, implying size control by simply adjusting SF nanostructure and concentration. Aside from process parameters investigated in previous studies, such as SF concentration, viscosity, and electrical potential, the present mechanism emphasizes significant influence of SF nanostructure on spinnability and diameter control of SF electrospun fibers, providing a controllable option for the preparation of silk-based electrospun scaffolds for biomaterials, drug delivery, and tissue engineering needs.

Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce β-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced ß-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk. [ABSTRACT FROM AUTHOR]

As a functional material, silk has been widely used in tissue engineering, drug release, and tissue regeneration. Increasing subtle control of silk hierarchical structures and thus specific functional performance is required for these applications but remains a challenge. Here, we report a novel silk nanofiber solution achieved through tuning solvent systems used to generate the material. Unlike the β-sheet rich silk nanofibers reported previously, these new silk nanofibers are mainly composed of amorphous structures and maintain a solution state in aqueous environments. The amorphous silk nanofibers are stable enough for storage and also metastable, making them easy to use in the further fabrication of materials through various processes. Silk scaffolds, hydrogels, and films were prepared from these silk nanofiber solutions. These silk materials from amorphous nanofiber solutions show different properties and tunable performance features. Therefore, these amorphous silk nanofibers are suitable units or building blocks for designing silk-based materials. [ABSTRACT FROM AUTHOR]

Silkwormsilk has been widely used as a textile fiber, as biomaterialsand in optically functional materials due to its extraordinary properties.The β-sheet-rich natural nanofiber units of about 10–50nm in diameter are often considered the origin of these properties,yet it remains unclear how silk self-assembles into these hierarchicalstructures. A new system composed of β-sheet-rich silk nanofibersabout 10–20 nm in diameter is reported here, where these nanofibersformed into “flowing hydrogels” at 0.5–2% solutionsand could be transformed back into the solution state at lower concentrations,even with a high β-sheet content. This is in contrast with othersilk processed materials, where significant β-sheet contentnegates reversibility between solution and solid states. These fibersare formed by regulating the self-assembly process of silk in aqueoussolution, which changes the distribution of negative charges whilestill supporting β-sheet formation in the structures. Mechanistically,there appears to be a shift toward negative charges along the outsideof the silk nanofibers in our present study, resulting in a higherzeta potential (above −50 mV) than previous silk materialswhich tend to be below −30 mV. The higher negative charge onsilk nanofibers resulted in electrostatic repulsion strong enoughto negate further assembly of the nanofibers. Changing silk concentrationchanged the balance between hydrophobic interactions and electrostaticrepulsion of β-sheet-rich silk nanofibers, resulting in reversiblehydrogel–solution transitions. Furthermore, the silk nanofiberscould be disassembled into shorter fibers and even nanoparticles uponultrasonic treatment following the transition from hydrogel to solutiondue to the increased dispersion of hydrophobic smaller particles,without the loss of β-sheet content, and with retention of theability to transition between hydrogel and solution states throughreversion to longer nanofibers during self-assembly. These reversiblesolution-hydrogel transitions were tunable with ultrasonic intensity,time, or temperature. [ABSTRACT FROM AUTHOR]