Silk Fibroin Substrates Research Articles

Flexible Neural Interface From Non-Transient Silk Fibroin With Outstanding Conformality, Biocompatibility, and Bioelectric Conductivity.

Silk fibroin (SF) with good biocompatibility can enable an efficient and safe implementation of neural interfaces. However, it has been difficult to achieve a robust integration of patterned conducting materials (multichannel electrodes) on flexible SF film substrates due to the absence of some enduring interactions. In this study, a thermo-assisted pattern-transfer technique is demonstrated that can facilely transfer a layer of pre-set poly(3,4-ethylenedioxythiophene) (PEDOT) onto the flexible SF substrate through an interpenetrating network of 2 polymer chains, achieving a desired substrate/conductor intertwined interface with good flexibility (≈33MPa), conductivity (386Scm-1) and stability in liquid state over 4 months simultaneously. Importantly, this technique can be combined with ink-jet printing to prepare a multichannel SF-based neural interface for the electrocorticogram (ECoG) recording and inflammation remission in rat models. The SF-based neural interface with satisfied tissue conformability, biocompatibility, and bioelectric conductivity is a promising ECoG acquisition tool, where the demonstrated approach can also be useful to develop other SF-based flexible bioelectronics.

Silk Fibroin Film Decorated with Ultralow FeCo Content by Sputtering Deposition Results in a Flexible and Robust Biomaterial for Magnetic Actuation.

Magnetically responsive soft biomaterials are at the forefront of bioengineering and biorobotics. We have created a magnetic hybrid material by coupling silk fibroin─i.e., a natural biopolymer with an optimal combination of biocompatibility and mechanical robustness─with the FeCo alloy, the ferromagnetic material with the highest saturation magnetization. The material is in the form of a 6 μm-thick silk fibroin film, coated with a FeCo layer (nominal thickness: 10 nm) grown by magnetron sputtering deposition. The sputtering deposition technique is versatile and eco-friendly and proves effective for growing the magnetic layer on the biopolymer substrate, also allowing one to select the area to be decorated. The hybrid material is biocompatible, lightweight, flexible, robust, and water-resistant. Electrical, structural, mechanical, and magnetic characterization of the material, both as-prepared and after being soaked in water, have provided information on the adhesion between the silk fibroin substrate and the FeCo layer and on the state of internal mechanical stresses. The hybrid film exhibits a high magnetic bending response under a magnetic field gradient, thanks to an ultralow fraction of the FeCo component (less than 0.1 vol %, i.e., well below 1 wt %). This reduces the risk of adverse health effects and makes the material suitable for bioactuation applications.

Silk fibroin-based wearable SERS biosensor for simultaneous sweat monitoring of creatinine and uric acid

Sweat biomarkers have the potential to offer valuable clinical insights into an individual's health and disease condition. Current sensors predominantly utilize enzymes and antibodies as biometric components to measure biomarkers present in sweat quantitatively. However, enzymes and antibodies are susceptible to interference by environmental factors, which may affect the performance of the sensor. Herein, we present a wearable microfluidic surface-enhanced Raman scattering (SERS) biosensor that enables the non-invasive and label-free detection of biomarkers in sweat. Concretely, we developed a bimetallic self-assembled anti-opal array structure with uniform hot spots, enhanced the Raman scattering effect, and integrated it into a silk fibroin-based sensing patch. Utilizing a silk fibroin substrate in the wearable SERS sensor imparts desirable properties such as softness, breathability, and biocompatibility, which enables the sensor to establish close contact with the skin without causing chemical or physical irritation. In addition, introducing microfluidic channels enables the controlled and high temporal resolution management of sweat, facilitating more efficient sweat collection. The proposed label-free SERS sensor can offer chemical ‘fingerprint’ information, enabling the identification of sweat analytes. As an illustration of the feasibility, we have effectively monitored the creatinine and uric acid levels in sweat. This study presents a versatile and highly sensitive approach for the simultaneous detection of biomarkers in human sweat, showcasing significant potential for application in point-of-care monitoring.

Silk fibroin wetting stability film induced by polyamide-amine-epichlorohydrin (PAE) for intelligent sensing system

Protein materials gain new functions and applicability through redesigns in protein structure and engineering confer. However, the application and development of proteins for use in flexible devices that fit in flexible devices that fit the surface of human skin is hindered by their poor wet stability. Here, we described the design of wet-stable materials based on the reconstruction of silk fibroin (SF). The combination of polyamide-amine-epichlorohydrin (PAE) was used as a traction rope to bring SF molecular chains closer to each other, to facilitate the self-assembly of SF through branching and lengthening of molecular chains, and change its crystalline structure. SF/PAE composite films that exhibited huge improvement in ductility and wet stability were combined with flexible SF substrates via patterning and ion sputtering to prepare flexible sensors. In addition, the SF/PAE sensing system equipped with a microprocessor and Bluetooth module enabled the real-time remote acquisition of human health signals such as vocal cords, joints, pulse and meridians. This reconfiguration of the SF structure will advance the systematic exploration of protein structures and the development of protein materials for intelligent device applications.

Environmental-friendly, flexible silk fibroin-based film as dual-responsive shape memory material

Bio-based shape memory materials have attracted wide attention due to their biocompatibility, degradability and safety. However, designing and manufacturing wearable bio-based shape memory films with excellent flexibility and toughness is still a challenge. In this work, silk fibroin substrate with a β-sheet structure was combined with a tri-block shape memory copolymer to prepare a transparent composited shape memory film. The silk fibroin-based film showed a dual-responsive shape memory function, which can respond to both temperature and water stimuli. This film has a sensitive water-responsive shape memory, which starts deforming after exposure to water for 3 s and fully recovers in 30 s. In addition, the composite film shows highly stretchable (>300 %) and could maintain its high tensile properties after 5 cycles of regeneration. The films also exhibited rapid degradation ability. This study provides new insights for the design of dual-responsive shape memory materials by combining biocompatible matrix and multi-block SMP to simultaneously enhance the mechanical properties, which can be used for intelligent packaging, medical supplies, soft actuators and wearable devices.

Mechanical Transfer of Black Phosphorus on a Silk Fibroin Substrate: A Viable Method for Photoresponsive and Printable Biomaterials.

Despite the technological importance of semiconductor black phosphorus (BP) in materials science, maintaining the stability of BP crystals in organic media and protecting them from environmental oxidation remains challenging. In this study, we present the synthesis of bulk BP and the exploitation of the viscoelastic properties of a regenerated silk fibroin (SF) film as a biocompatible substrate to transfer BP flakes, thereby preventing oxidation. A model based on the flow of polymers revealed that the applied flow-induced stresses exceed the yield stress of the BP aggregate. Raman spectroscopy was used to investigate the exfoliation efficiency as well as the environmental stability of BP transferred on the SF substrate. Notably, BP flakes transferred to the SF substrate demonstrated improved stability when SF was dissolved in a phosphate-buffered saline medium, and in vitro cancer cell viability experiments demonstrate the tumor ablation efficiency under visible to near-infrared (Vis-nIR) radiation. Moreover, the SF and BP-enriched SF (SF/BP) solution was shown to be processable via extrusion-based three-dimensional (3D) printing. Therefore, this work paves the way for a general method for the transferring of BP on natural biodegradable polymers and processing them via 3D printing toward novel functionalities and complex shapes for biomedical purposes.

Selective Encapsulation of the Polyphenols on Silk Fibroin Nanoparticles: Optimization Approaches.

The present study proposes a method for designing small bioactive nanoparticles using silk fibroin as a carrier to deliver hydrophobic polyphenols. Quercetin and trans-resveratrol, widely distributed in vegetables and plants, are used here as model compounds with hydrophobic properties. Silk fibroin nanoparticles were prepared by desolvation method and using various concentrations of ethanol solutions. The optimization of the nanoparticle formation was achieved by applying Central Composite Design (CCD) and the response surface methodology (RSM). The effects of silk fibroin and ethanol solution concentrations together with the pH on the selective encapsulation of phenolic compounds from a mixture were reported. The obtained results showed that nanoparticles with an average particle size of 40 to 105 nm can be prepared. The optimized system for the selective encapsulation of the polyphenols on the silk fibroin substrate was determined to be 60% ethanol solution and 1 mg/mL silk fibroin concentration at neutral pH. The selective encapsulation of the polyphenols was achieved, with the best results being obtained in the case of resveratrol and quercetin and encapsulation of gallic and vanillic acids being rather poor. Thin-layer chromatography confirmed the selective encapsulation and the loaded silk fibroin nanoparticles exhibited antioxidant activity.

Silk fibroin based wearable electrochemical sensors with biomimetic enzyme-like activity constructed for durable and on-site health monitoring

Flexible biomimetic sensors have encountered a bottleneck of sensitivity and durability, as the sensors must directly work within complex body fluid with ultra-trace biomarkers. In this work, a wearable electrochemical sensor on a modified silk fibroin substrate is developed using gold nanoparticles hosted into N-doped porous carbonizated silk fibroin (AuNPs@CSF) as active materials. Taking advantage of the inherent biocompatibility and flexibility of CSF, and the high stability and enzyme-like catalytic activity of AuNPs, AuNPs@CSF-based sensor exhibits durable stability and superior sensitivity to monitor H2O2 released from cancer cell (4T1) and glucose in sweat. The detection limits for H2O2 and glucose are low to be 1.88 μM and 23 μM respectively, and the sensor can be applied in succession within 30 days at room temperature. Further, physical cross-linking of polyurethane (PU) with SF well matches with the skin tissue mechanically and provides a flexible, robust and stable electrode-tissue interface. AuNPs@CSF is applied successfully for wearable electrochemical monitoring of glucose in human sweat.The present AuNPs@CSF will possess a potential application in clinical diagnosing of H2O2- or glucose-related diseases in future.

All-Solution-Processed Polythiophene/Carbon Nanotube Nanocomposites Integrated on Biocompatible Silk Fibroin Substrates for Wearable Thermoelectric Generators

This paper presents proof-of-concept all-solution-processed wearable thermoelectric generators made of single-walled carbon nanotube/poly(3-hexylthiophene) (SWCNT/P3HT) nanocomposites integrated with biocompatible silk fibroin substrates and Ag nanowires (NWs)/poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT/PSS) layers. Since well-dispersed SWCNT/P3HT nanocomposites are obtained through π–π interactions between conjugated backbones, continuous and repeated spraying/vacuum processes enabled the silk fibroin-substrated SWCNT/P3HT films to exhibit an optimized power factor of 204 ± 4.6 μW m–1 K–2 at 50 °C, an electrical conductivity of 1170 ± 52.8 S cm–1, and a Seebeck coefficient of 41.8 ± 0.9 μV K–1, which are ∼1.5 times those of the glass-substrated device. This may be attributed to the significantly enhanced conductivity caused by the increased junction density of the SWCNT bundles within the compact and dense SWCNT/P3HT nanocomposites. When a spray-patterned Ag NWs/PEDOT/PSS electrode was assembled, the constructed in-plane SWCNT/P3HT thermoelectric generators with 14 legs produced an open-circuit voltage (Voc) of 22.6 mV and a peak power of 25.1 nW at a temperature difference (ΔT) of 28.8 °C while maintaining good flexibility during bending tests. This demonstrates that these silk fibroin-substrated thermoelectric generator prototypes, which are worn on the forearm, can be used to harvest human body heat, thereby generating a Voc of ∼6.1 mV with a ΔT as low as 8.3 °C between the skin and surrounding air. This study offers a promising strategy for exploiting simple solution-processed thermoelectric nanocomposites assembled using biomass-based substrates and patterned electrodes for low-grade waste heat-harvesting wearable devices.

Influence of spiral topographies on human limbal-derived immortalized corneal epithelial cells.

Cells migrate from the limbus to the corneal epithelium following a centripetal pathway. Corneal epithelial cells tend to orientate in spiral or vortex patterns. However, when cultured in-vitro, limbal derived corneal epithelia do not tend to align or migrate in a spiral pattern. Here, we used soft lithography to create silk fibroin substrates with spiral topographies that direct the human limbal-derived immortalized corneal epithelial cells (hTCEpi) to form a spiral orientation. The impact of this topography on the cells was then characterized. The spiral patterns affected cytoskeletal organization, cell spreading, and nuclei shapes. Spiral width and numbers had a significant impact on proliferation of cells, their focal adhesion, their chromatin condensation, and number of actin filaments. Immunocytochemical staining showed that the spiral pattern enhanced the expression of markers associated with limbal stem cells. The current work illustrates micro spiral patterns can serve to control the nature of limbal derived epithelial cells by providing relevant biophysical cues.

Combination of Porous Silk Fibroin Substrate and Gold Nanocracks as a Novel SERS Platform for a High-Sensitivity Biosensor.

Novel concepts for developing a surface-enhanced Raman scattering (SERS) sensor based on biocompatible materials offer great potential in versatile applications, including wearable and in vivo monitoring of target analytes. Here, we report a highly sensitive SERS sensor consisting of a biocompatible silk fibroin substrate with a high porosity and gold nanocracks. Our silk-based SERS detection takes advantage of strong local field enhancement in the nanoscale crack regions induced by gold nanostructures evaporated on a porous silk substrate. The SERS performance of the proposed sensor is evaluated in terms of detection limit, sensitivity, and linearity. Compared to the performance of a counterpart SERS sensor with a thin gold film, SERS results using 4-ABT analytes present that a significant improvement in the detection limit and sensitivity by more than 4 times, and a good linearity and a wide dynamic range is achieved. More interestingly, overlap is integral, and a quantitative measure of the local field enhancement is highly consistent with the experimental SERS enhancement.

Novel TiO2 nanotubes films with silk fibroin substrate

The poor adhesion of anodic TiO2 nanotubes (TiO2 NTbs) on titanium substrates obviously affects applications in many fields. Herein, novel films of silk fibroin (SF) substrate based TiO2 nanotubes were successfully prepared. The work of this thesis include the followings: 1. The composite films with only hundreds of nanometers tube length can be obtained. 2. The film is fiexible and has enough mechanical strength after peel off from Ti substrate, which is beneficial to further application. 3. The nanotube structure remains unchanged. 4. The barrier layer of TiO2 nanotubes can be removed by HF etching and the opened TiO2 nanotube arrays lied on SF substrate were successfully prepared.

Morphology and cellular-traction of fibroblasts on 2D silk-fibroin hydrogel substrates

ABSTRACT Development of clinically amenable bio-implants with silk-fibroin (SF) necessitates characterization of cellular-traction generated between cells and the substrate. However, studies on the biomechanical response of cells on SF substrates are limited. In this study, we prepared SF hydrogels of varying compliance (SF30 and SF50) and varying surface-ligands (derivatized with poly-L-lysine (PLL) or Arg-Gly-Asp (RGD) peptide). Subsequently, NIH-3T3 fibroblast cells were grown on these substrates, and the morphological changes was examined. It was observed that the increase in SF stiffness from 0.7 kPa to 3.1 kPa decreased nucleus-to-cytoplasm area-ratio and increased asymmetricity along the major-axis of cells. Moreover, while functionalization of SF with RGD induced increase in cell-area and circularity, functionalization with PLL did not cause any change. Next, using traction-force-microscopy (TFM), we quantified 2D cell-traction for NIH-3T3 cells cultured on SF hydrogels. Cells plated on SF50 hydrogel exhibited significantly high traction stress as compared to SF30; change of functionalization did not show significant change. Also, protrusion traction stress was found to be greater than nuclear traction stress. Put together, our results suggest that surface-stiffness of SF-hydrogel, rather than nature of surface-ligand, regulates both cellular morphology and cellular traction stresses.

The molecular conformation of silk fibroin regulates osteogenic cell behavior by modulating the stability of the adsorbed protein-material interface

Silk fibroin (SF) can be used to construct various stiff material interfaces to support bone formation. An essential preparatory step is to partially transform SF molecules from random coils to β-sheets to render the material water insoluble. However, the influence of the SF conformation on osteogenic cell behavior at the material interface remains unknown. Herein, three stiff SF substrates were prepared by varying the β-sheet content (high, medium, and low). The substrates had a comparable chemical composition, surface topography, and wettability. When adsorbed fibronectin was used as a model cellular adhesive protein, the stability of the adsorbed protein-material interface, in terms of the surface stability of the SF substrates and the accompanying fibronectin detachment resistance, increased with the increasing β-sheet content of the SF substrates. Furthermore, (i) larger areas of cytoskeleton-associated focal adhesions, (ii) higher orders of cytoskeletal organization and (iii) more elongated cell spreading were observed for bone marrow-derived mesenchymal stromal cells (BMSCs) cultured on SF substrates with high vs. low β-sheet contents, along with enhanced nuclear translocation and activation of YAP/TAZ and RUNX2. Consequently, osteogenic differentiation of BMSCs was stimulated on high β-sheet substrates. These results indicated that the β-sheet content influences osteogenic differentiation of BMSCs on SF materials in vitro by modulating the stability of the adsorbed protein-material interface, which proceeds via protein-focal adhesion-cytoskeleton links and subsequent intracellular mechanotransduction. Our findings emphasize the role of the stability of the adsorbed protein-material interface in cellular mechanotransduction and the perception of stiff SF substrates with different β-sheet contents, which should not be overlooked when engineering stiff biomaterials.

Patterning the neuronal cells via inkjet printing of self-assembled peptides on silk scaffolds

The patterning of neuronal cells and guiding neurite growth are important for neuron tissue engineering and cell-based biosensors. In this paper, inkjet printing has been employed to pattern self-assembled I3QGK peptide nanofibers on silk substrates for guiding the growth of neuron-like PC12 cells. Atomic force microscopy (AFM) confirmed the dynamic self-assembly of I3QGK into nanofiber structures. The printed self-assembled peptide strongly adheres to regenerated silk fibroin (RSF) substrates through charge-charge interactions. It was observed that in the absence of I3QGK, PC12 cells exhibited poor attachment to RSF films, while for RSF surfaces coated or printed with peptide nanofibers, cellular attachment was significantly improved in terms of both cell density and morphology. AFM results revealed that peptide nanofibers can promote the generation of axons and terminal buttons of PC12 cells, indicating that I3QGK nanofibers not only promote cellular attachment but also facilitate differentiation into neuronal phenotypes. Inkjet printing allows complex patterning of peptide nanofibers onto RSF substrates, which enabled us to engineer cell alignment and provide an opportunity to direct axonal development in vitro. The live/dead assay showed that printed I3QGK patterns exhibit no cytotoxicity to PC12 cells demonstrating potential for future nerve tissue engineering applications.

Click Decoration of Bombyx mori Silk Fibroin for Cell Adhesion Control.

Silk fibroin produced by the domesticated silkworm, Bombyx mori, has been studied widely as a substrate for tissue engineering applications because of its mechanical robustness and biocompatibility. However, it is often difficult to precisely tune silk fibroin’s biological properties due to the lack of easy, reliable, and versatile methodologies for decorating it with functional molecules such as those of drugs, polymers, peptides, and enzymes necessary for specific applications. In this study we applied an azido-functionalized silk fibroin, AzidoSilk, produced by a state-of-the-art biotechnology, genetic code expansion, to produce silk fibroin decorated with cell-repellent polyethylene glycol (PEG) chains for controlling the cell adhesion property of silk fibroin film. Azido groups can act as selective handles for chemical reactions such as a strain-promoted azido-alkyne cycloaddition (SPAAC), known as a click chemistry reaction. We found that azido groups in AzidoSilk film were selectively decorated with PEG chains using SPAAC. The PEG-decorated film demonstrated decreased cell adhesion depending on the lengths of the PEG chains. Azido groups in AzidoSilk can be decomposed by UV irradiation. By partially decomposing azido groups in AzidoSilk film in a spatially controlled manner using photomasks, cells could be spatially arranged on the film. These results indicated that SPAAC could be an easy, reliable, and versatile methodology to produce silk fibroin substrates having adequate biological properties.

Aligned graphene/silk fibroin conductive fibrous scaffolds for guiding neurite outgrowth in rat spinal cord neurons.

Graphene, as a highly conducting material, incorporated into silk fibroin (SF) substrates is promising to fabricate an electroactive flexible scaffold toward neural tissue engineering. It is well known that aligned morphology could promote cell adhesion and directional growth. The purpose of this study was to develop aligned conductive scaffolds made of graphene and SF (G/SF) by electrospinning technique for neural tissue engineering applications. The physicochemical characterization of scaffolds revealed that the mechanical and electrochemical property of aligned G/SF scaffolds continually raised with the increasing contents of graphene (A0% G/SF, A1% G/SF, A2% G/SF, and A3% G/SF), but the mechanical property descended when the graphene concentration reached to 4% (the A4% G/SF group). The results of the cell experiment in vitro indicated that all the aligned G/SF scaffolds were no neurotoxic to primary cultured spinal cord neurons. In addition, the neurite elongation in all aligned groups was significantly enhanced by the upregulation of Netrin-1 expression compared to them in the control group. Thus, A3% G/SF scaffolds not only possessed the optimal property based on the mechanical and electrochemical performances but also displayed the beneficial capability to neurite outgrowth, which might perform a suitable candidate to successfully scaffold electrically active tissues during neural regeneration or engineering.

Delivery of Salvianolic Acid B for Efficient Osteogenesis and Angiogenesis from Silk Fibroin Combined with Graphene Oxide.

The efficiency of drugs often hinges on drug carriers. To effectively transport therapeutic plant molecules, drug delivery carriers should be able to carry large doses of therapeutic drugs, enable their sustained release, and maintain their biological activity. Here, graphene oxide (GO) is demonstrated to be a valid carrier for delivering therapeutic plant molecules. Salvianolic acid B (SB), which contains a large number of hydroxyl groups, bound to the carboxyl groups of GO by self-assembly. Silk fibroin (SF) substrates were combined with functionalized GO through the freeze-drying method. SF/GO scaffolds could be loaded with large doses of SB, maintain the biological activity of SB while continuously releasing SB, and significantly promote the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). SF/GO/SB also dramatically enhanced endothelial cell (EA-hy9.26) migration and tubulogenesis in vitro. Eight weeks after implantation of SF/GO/SB scaffolds in a rat cranial defect model, the defect area showed more new bone and angiogenesis than that following SF and SF/GO scaffold implantation. Therefore, GO is an effective sustained-release carrier for therapeutic plant molecules, such as SB, which can repair bone defects by promoting osteogenic differentiation and angiogenesis.

Micro- and Nanopatterned Silk Substrates for Antifouling Applications.

A major problem of current biomedical implants is the bacterial colonization and subsequent biofilm formation, which seriously affects their functioning and can lead to serious post-surgical complications. Intensive efforts have been directed toward the development of novel technologies that can prevent bacterial colonization while requiring minimal antibiotics doses. To this end, biocompatible materials with intrinsic antifouling capabilities are in high demand. Silk fibroin, widely employed in biotechnology, represents an interesting candidate. Here, we employ a soft-lithography approach to realize micro- and nanostructured silk fibroin substrates, with different geometries. We show that patterned silk film substrates support mammal cells (HEK-293) adhesion and proliferation, and at the same time, they intrinsically display remarkable antifouling properties. We employ Escherichia coli as representative Gram-negative bacteria, and we observe an up to 66% decrease in the number of bacteria that adhere to patterned silk surfaces as compared to control, flat silk samples. The mechanism leading to the inhibition of biofilm formation critically depends on the microstructure geometry, involving both a steric and a hydrophobic effect. We also couple silk fibroin patterned films to a biocompatible, optically responsive organic semiconductor, and we verify that the antifouling properties are very well preserved. The technology described here is of interest for the next generation of biomedical implants, involving the use of materials with enhanced antibacterial capability, easy processability, high biocompatibility, and prompt availability for coupling with photoimaging and photodetection techniques.

Highly transparent and flexible Ag nanowire-embedded silk fibroin electrodes for biocompatible flexible and transparent heater.

We investigated the electrical, optical and mechanical properties of silver (Ag) nanowire (NW) embedded into a silk fibroin (SF) substrate to create high performance, flexible, transparent, biocompatible, and biodegradable heaters for use in wearable electronics. The Ag NW-embedded SF showed a low sheet resistance of 15 Ω sq−1, high optical transmittance of 85.1%, and a small inner/outer critical bending radius of 1 mm. In addition, the Ag NW-embedded SF showed a constant resistance change during repeated bending, folding, and rolling because the connectivity of the Ag NW embedded into the SF substrate was well maintained. Furthermore, the biocompatible and biodegradable Ag NW-embedded SF substrate served as a flexible interconnector for wearable electronics. The high performance of the transparent and flexible heater demonstrated that an Ag NW-embedded SF-based heater can act as a biocompatible and biodegradable substrate for wearable heaters for the human body.