Mesoscale Confinement in Bagworm Silk: A Hidden Structural Organization.

While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline ß-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider's dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Secretion-Catalyzed Assembly of Protein Biomaterials on a Bacterial Membrane Surface.

Protein-based biomaterials have played a key role in tissue engineering, and additional exciting applications as self-healing materials and sustainable polymers are emerging. Over the past few decades, recombinant expression and production of various fibrous proteins from microbes have been demonstrated; however, the resulting proteins typically must then be purified and processed by humans to form usable fibers and materials. Here, we show that the Gram-positive bacterium Bacillus subtilis can be programmed to secrete silk through its translocon via an orthogonal signal peptide/peptidase pair. Surprisingly, we discover that this translocation mechanism drives the silk proteins to assemble into fibers spontaneously on the cell surface, in a process we call secretion-catalyzed assembly (SCA). Secreted silk fibers form self-healing hydrogels with minimal processing. Alternatively, the fibers retained on the membrane provide a facile route to create engineered living materials from Bacillus cells. This work provides a blueprint to achieve autonomous assembly of protein biomaterials in useful morphologies directly from microbial factories.

Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks.

Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers' mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in ß-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high ß-strand propensity and can mediate tight inter-ß-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger ß-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L-1 which is in line with requirements for economically feasible bulk scale production.

Toughening Wet-Spun Silk Fibers by Silk Nanofiber Templating.

Regenerated silk fibers typically fall short of silkworm cocoon fibers in mechanical properties due to reduced fiber crystal structure and alignment. One approach to address this has been to employ inorganic materials as reinforcing agents. The present study avoids the need for synthetic additives, demonstrating the first use of exfoliated silk nanofibers to control silk solution crystallization, resulting in all-silk pseudocomposite fibers with remarkable mechanical properties. Incorporating only 0.06 wt% silk nanofibers led to a ≈44% increase in tensile strength (over 600 MPa) and ≈33% increase in toughness (over 200 kJ kg-1 ) compared with fibers without silk nanofibers. These remarkable properties can be attributed to nanofiber crystal seeding in conjunction with fiber draw. The crystallinity nearly doubled from ≈17% for fiber spun from pure silk solution to ≈30% for the silk nanofiber reinforced sample. The latter fiber also shows a high degree of crystal orientation with a Herman's orientation factor of 0.93, a value which approaches that of natural degummed B. mori silk cocoon fiber (0.96). This study provides a strong foundation to guide the development of simple, eco-friendly methods to spin regenerated silk with excellent properties and a hierarchical structure that mimics natural silk.

Silk Composite Electronic Textile Sensor for High Space Precision 2D Combo Temperature-Pressure Sensing.

Wearable electronic textiles based on natural biocompatible/biodegradable materials have attracted great attention due to applications in health care and smart clothes. Silkworm fibers are durable, good heat conductors, insulating, and biocompatible, and are therefore regarded as excellent mediating materials for flexible electronics. In this paper, a strategy on the design and fabrication of highly flexible multimode electronic textiles (E-textile) based on functionalized silkworm fiber coiled yarns and weaving technology is presented. To achieve enhanced temperature sensing performance, a mixture of carbon nanotubes and an ionic liquid ([EMIM]Tf2 N) is embedded, which displays top sensitivity of 1.23% °C-1 and stability compared with others. Furthermore, fibrous pressure sensing based on the capacitance change of each cross-point of two yarns gives rise to highly position dependent and sensitivity sensing of 0.136 kPa-1 . Based on weaving technologies, a unique combo textile sensor, which can sense temperature and pressure independently with a position precision of 1 mm2 , is obtained. The application to intelligent gloves endows the position dependent sensing of the weight, and temperature distribution sensing of the temperature.

Carbonized silk fibers for in-tube solid-phase microextraction to detect polycyclic aromatic hydrocarbons in water samples.

Silk fibers were carbonized to develop a biomass carbon material as an adsorbent for solid-phase microextraction. The surface structure of the carbonized silk fibers was characterized by scanning electron microscopy, and the graphitization degree was determined by Raman spectrometry. After carbonization under high temperature, the orderliness and structural regularity of carbon atoms on silk fibers were promoted. Extraction tube packed with carbonized silk fibers was prepared for in-tube solid-phase microextraction. Coupled with high performance liquid chromatography, it exhibited good extraction performance for hydrophobic polycyclic aromatic hydrocarbons. Main parameters including sampling volume, sampling rate, methanol content in sample, and desorption time were systematically investigated. Under the optimum conditions, the analysis method was established and it exhibited wide linear range (0.016-20 µg/L) with good linearity (correlation coefficient ≥ 0.9947), low limits of detection (0.005-0.050 µg/L), and high enrichment factors (1189-2775). Relative standard deviations (n = 3) for intraday (≤3.3%) and interday (≤9.6%) tests indicated that the extraction material had satisfactory repeatability. Finally, the analytical method was successfully applied to detect trace polycyclic aromatic hydrocarbons in real water samples, demonstrating its satisfactory practicability.

Properties of Engineered and Fabricated Silks.

Silk is a protein-based material which is predominantly produced by insects and spiders. Hundreds of millions of years of evolution have enabled these animals to utilize different, highly adapted silk types in a broad variety of applications. Silk occurs in several morphologies, such as sticky glue or in the shape of fibers and can, depending on the application by the respective animal, dissipate a high mechanical energy, resist heat and radiation, maintain functionality when submerged in water and withstand microbial settling. Hence, it's unsurprising that silk piqued human interest a long time ago, which catalyzed the domestication of silkworms for the production of silk to be used in textiles. Recently, scientific progress has enabled the development of analytic tools to gain profound insights into the characteristics of silk proteins. Based on these investigations, the biotechnological production of artificial and engineered silk has been accomplished, which allows the production of a sufficient amount of silk materials for several industrial applications. This chapter provides a review on the biotechnological production of various silk proteins from different species, as well as on the processing techniques to fabricate application-oriented material morphologies.

Major Ampullate Spider Silk with Indistinguishable Spidroin Dope Conformations Leads to Different Fiber Molecular Structures.

To plentifully benefit from its properties (mechanical, optical, biological) and its potential to manufacture green materials, the structure of spider silk has to be known accurately. To this aim, the major ampullate (MA) silk of Araneus diadematus (AD) and Nephila clavipes (NC) has been compared quantitatively in the liquid and fiber states using Raman spectromicroscopy. The data show that the spidroin conformations of the two dopes are indistinguishable despite their specific amino acid composition. This result suggests that GlyGlyX and GlyProGlyXX amino acid motifs (X = Leu, Glu, Tyr, Ser, etc.) are conformationally equivalent due to the chain flexibility in the aqueous environment. Species-related sequence specificity is expressed more extensively in the fiber: the ß-sheet content is lower and width of the orientation distribution of the carbonyl groups is broader for AD (29% and 58°, respectively) as compared to NC (37% and 51°, respectively). ß-Sheet content values are close to the proportion of polyalanine segments, suggesting that ß-sheet formation is mainly dictated by the spidroin sequence. The extent of molecular alignment seems to be related to the presence of proline (Pro) that may decrease conformational flexibility and inhibit chain extension and alignment upon drawing. It appears that besides the presence of Pro, secondary structure and molecular orientation contribute to the different mechanical properties of MA threads.

Regenerated Fiber's Ideal Target: Comparable to Natural Fiber.

The toughness of silk naturally obtained from spiders and silkworms exceeds that of all other natural and man-made fibers. These insects transform aqueous protein feedstocks into mechanically specialized materials, which represents an engineering phenomenon that has developed over millions of years of natural evolution. Silkworms have become a new research hotspot due to the difficulties in collecting spider silk and other challenges. According to continuous research on the natural spinning process of the silkworm, it is possible to divide the main aspects of bionic spinning into two main segments: the solvent and behavior. This work focuses on the various methods currently used for the spinning of artificial silk fibers to replicate natural silk fibers, providing new insights based on changes in the fiber properties and production processes over time.

Identification, extraction, and application of orange peel color extracts for silk fabric coloration.

Under-utilized orange peel waste contains natural colorants that might be used for textile dyeing. Research into orange peel waste as natural colorants provides benefits for both the agricultural and fashion industry with a creative and sustainable solution. This research performed the extraction of colorants from the orange peel as plant dyes and investigated their potential dyeing capability of silk fabrics. With full factorial analysis, we determined the optimal extraction conditions by comparing 100 % ethanol, 70 % ethanol, and water, aiming to achieve the highest absorbance for the extracted solution. Conditions obtained with the best performance include an extraction temperature of 60 °C, an extraction time of 120 min, and a material-to-liquor ratio of 1:20 (wt/vol) for both 100 % and 0 % ethanol. To attain the highest K/S values on textiles with orthogonal experimental design, the optimal dyeing profiles of silk fabrics with water solution were found to be 100 °C, 60 min, pH 3, and Liquid Ratio of 1:15. Colorfastness results of crocking, washing, and sunlight are in favor of the usage of orange peel color extracts for textile application.

Fine Structural Analysis of Degummed Fibroin Fibers Reveals Its Superior Mechanical Capabilities.

Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of functionalization and reprocessing into various material formats. The properties of these fibers are typically affected by the intense thermal treatments needed to remove the sericin gum coating layer. Additionally, their mechanical characteristics are often misinterpreted by assuming the asymmetrical cross-sectional area (CSA) as a perfect circle. The thermal treatments impact not only the mechanics of the degummed fibroin fibers, but also the structural configuration of the resolubilized protein, thereby limiting the performance of the resulting silk-based materials. To mitigate these limitations, we explored varying alkali conditions at low temperatures for surface treatment, effectively removing the sericin gum layer while preserving the molecular structure of the fibroin protein, thus, maintaining the hierarchical integrity of the exposed fibroin microfiber core. The precise determination of the initial CSA of the asymmetrical silk fibers led to a comprehensive analysis of their mechanical properties. Our findings indicate that the alkali surface treatment raised the Young's modulus and tensile strength, by increasing the extent of the fibers' crystallinity, by approximately 40 % and 50 %, respectively, without compromising their strain. Furthermore, we have shown that this treatment facilitated further production of high-purity soluble silk protein with rheological and self-assembly characteristics comparable to those of native silk feedstock, initially stored in the animal's silk gland. The developed approaches benefits both the development of silk-based materials with tailored properties and the proper mechanical characterization of asymmetrical fibrous biological materials made of natural building blocks.

Bio-Inspired Multiscale Design for Strong and Tough Biological Ionogels.

Structure design provides an effective solution to develop advanced soft materials with desirable mechanical properties. However, creating multiscale structures in ionogels to obtain strong mechanical properties is challenging. Here, an in situ integration strategy for producing a multiscale-structured ionogel (M-gel) via ionothermal-stimulated silk fiber splitting and moderate molecularization in the cellulose-ions matrix is reported. The produced M-gel shows a multiscale structural superiority comprised of microfibers, nanofibrils, and supramolecular networks. When this strategy is used to construct a hexactinellid inspired M-gel, the resultant biomimetic M-gel shows excellent mechanical properties including elastic modulus of 31.5 MPa, fracture strength of 6.52 MPa, toughness reaching 1540 kJ m-3 , and instantaneous impact resistance of 3.07 kJ m-1 , which are comparable to those of most previously reported polymeric gels and even hardwood. This strategy is generalizable to other biopolymers, offering a promising in situ design method for biological ionogels that can be expanded to more demanding load-bearing materials requiring greater impact resistance.

Biomimetic polymer fibers-function by design.

Biomimicry applies the fundamental principles of natural materials, processes, and structures to technological applications. This review presents the two strategies of biomimicry-bottom-up and top-down approaches, using biomimetic polymer fibers and suitable spinning techniques as examples. The bottom-up biomimicry approach helps to acquire fundamental knowledge on biological systems, which can then be leveraged for technological advancements. Within this context, we discuss the spinning of silk and collagen fibers due to their unique natural mechanical properties. To achieve successful biomimicry, it is imperative to carefully adjust the spinning solution and processing parameters. On the other hand, top-down biomimicry aims to solve technological problems by seeking solutions from natural role models. This approach will be illustrated using examples such as spider webs, animal hair, and tissue structures. To contextualize biomimicking approaches in practical applications, this review will give an overview of biomimetic filter technologies, textiles, and tissue engineering.

Sustainable Antibacterial Surgical Suture Based on Recycled Silk Resource by an Internal Combination of Inorganic Nanomaterials.

The current antibacterial treatment methods of silk sutures can only be finished by surface modification, leading to problems of short antibacterial effects, easy slow-release consumption, prominent toxicity, and susceptibility to drug resistance. Speculatively, surgical sutures combining antibacterial material internally will possess a more promising efficacy. Hence, we extracted recycled regenerated silk fibroin (RRSF) from waste silk resources to make RRSF solutions. Internally combining with inorganic titanium dioxide (TiO2) nanoparticles, we fabricated antibacterial RRSF-based surgical sutures. The morphologies, mechanical and antibacterial properties, biocompatibility tests, and in vivo experiments were carried out. The results showed that the surgical sutures with 1.25 wt % TiO2 acquired 2.40 N knot strength (143 µm diameter) and achieved a sustainable antibacterial effect of 93.58%. Surprisingly, the sutures significantly reduced inflammatory reactions and promoted wound healing. Surgical sutures in this paper realize high-value recovery of waste silk fibers and provide a novel approach to preparing multifunctional sutures.

Robust and flexible smart silk/PEDOT conductive fibers as wearable sensor for personal health management and information transmission.

Flexible highly conductive fibers have attracted much attention due to their great potential in the field of wearable electronic devices. In this work, silk/PEDOT conductive fibers with a resistivity of 1.73 Ω·cm were obtained by oxidizing Ce3+ with H2O2 under alkaline conditions to produce CeO2 and further promote the in-situ polymerization of 3,4-ethylenedioxythiophene (EDOT) on the surface of silk fibers. The morphology and chemical composition of the silk/PEDOT conductive fibers were characterized and the results confirmed that a large amount of polythiophene was synthesized and deposited on the surface of silk fibers. The conductivity and electrochemical property stability of the silk/PEDOT conductive fibers were evaluated by soaping and organic solvent immersion, and the conductive silk fibers exhibited excellent environmental stability and durability. The silk/PEDOT conductive fibers show good pressure sensing and strain sensing performance, which exhibits high sensitivity, fast response and cyclability, and have excellent applications in personal health monitoring, human-machine information transmission, etc.

Changes in Number and Antibacterial Activity of Silver Nanoparticles on the Surface of Suture Materials during Cyclic Freezing.

This article presents the results of the 10-fold cyclic freezing (-37.0 °C) and thawing (0.0 °C) effect on the number and size range of silver nanoparticles (AgNPs). AgNPs were obtained by the cavitation-diffusion photochemical reduction method and their sorption on the fiber surface of various suture materials, perlon, silk, and catgut, was studied. The distribution of nanoparticles of different diameters before and after the application of the cyclic freezing/thawing processes for each type of fibers studied was determined using electron microscopy. In general, the present study demonstrates the effectiveness of using the technique of 10-fold cyclic freezing. It is applicable to increase the absolute amount of AgNPs on the surface of the suture material with a simultaneous decrease in the size dispersion. It was also found that the application of the developed technique leads to the overwhelming predominance of nanoparticles with 1 to 15 nm diameter on all the investigated fibers. In addition, it was shown that after the application of the freeze/thaw method, the antibacterial activity of silk and catgut suture materials with AgNPs was significantly higher than before their treatment by cyclic freezing.

Silk fibers assisted long-term 3D culture of human primary urinary stem cells via inhibition of senescence-associated genes: Potential use in the assessment of chronic mitochondrial toxicity.

Despite being widely applied in drug development, existing in vitro 2D cell-based models are not suitable to assess chronic mitochondrial toxicity. A novel in vitro assay system mimicking in vivo microenvironment for this purpose is urgently needed. The goal of this study is to establish a 3D cell platform as a reliable, sensitive, cost-efficient, and high-throughput assay to predict drug-induced mitochondrial toxicity. We evaluated a long-term culture of human primary urine-derived stem cells (USC) seeded in 3D silk fiber matrix (3D USC-SFM) and further tested chronic mitochondrial toxicity induced by Zalcitabine (ddC, a nucleoside reverse transcriptase inhibitor) as a test drug, compared to USC grown in spheroids. The numbers of USC remain steady in 3D spheroids for 4 weeks and 3D SFM for 6 weeks. However, the majority (95%) of USC survived in 3D SFM, while cell numbers significantly declined in 3D spheroids at 6 weeks. Highly porous SFM provides large-scale numbers of cells by increasing the yield of USC 125-fold/well, which enables the carrying of sufficient cells for multiple experiments with less labor and lower cost, compared to 3D spheroids. The levels of mtDNA content and mitochondrial superoxide dismutase2 [SOD2] as an oxidative stress biomarker and cell senescence genes (RB and P16, p21) of USC were all stably retained in 3D USC-SFM, while those were significantly increased in spheroids. mtDNA content and mitochondrial mass in both 3D culture models significantly decreased six weeks after treatment of ddC (0.2, 2, and 10 µM), compared to 0.1% DMSO control. Levels of complexes I, II, and III significantly decreased in 3D SFM-USC treated with ddC, compared to only complex I level which declined in spheroids. A dose- and time-dependent chronic MtT displayed in the 3D USC-SFM model, but not in spheroids. Thus, a long-term 3D culture model of human primary USC provides a cost-effective and sensitive approach potential for the assessment of drug-induced chronic mitochondrial toxicity.

Non-mulberry silk fiber-based scaffolds reinforced by PLLA porous microspheres for auricular cartilage: An in vitro study.

Designing clinical applicable polymeric composite scaffolds for auricular cartilage tissue engineering requires appropriate mechanical strength and biological characteristics. In this study, silk fiber-based scaffolds co-reinforced with poly-L-lactic acid porous microspheres (PLLA PMs) combined with either Bombyx mori (Bm) or Antheraea pernyi (Ap) silk fibers were fabricated as inspired by the "steel bars reinforced concrete" structure in architecture and their chondrogenic functions were also investigated. We found that the Ap silk fiber-based scaffolds reinforced by PLLA PMs (MAF) exhibited superior physical properties (the mechanical properties in particular) as compared to the Bm silk fiber-based scaffolds reinforced by PLLA PMs (MBF). Furthermore, in vitro evaluation of chondrogenic potential showed that the MAF provided better cell adhesion, viability, proliferation and GAG secretion than the MBF. Therefore, the MAF are promising in auricular cartilage tissue engineering and relevant plastic surgery-related applications.

Ultrafine and High-Strength Silk Fibers Secreted by Bimolter Silkworms.

Ultrafine fibers are widely employed because of their lightness, softness, and warmth retention. Although silkworm silk is one of the most applied natural silks, it is coarse and difficult to transform into ultrafine fibers. Thus, to obtain ultrafine high-performance silk fibers, we employed anti-juvenile hormones in this study to induce bimolter silkworms. We found that the bimolter cocoons were composed of densely packed thin fibers and small apertures, wherein the silk diameter was 54.9% less than that of trimolter silk. Further analysis revealed that the bimolter silk was cleaner and lighter than the control silk. In addition, it was stronger (739 MPa versus 497 MPa) and more stiffness (i.e., a higher Young's modulus) than the trimolter silk. FTIR and X-ray diffraction results revealed that the excellent mechanical properties of bimolter silk can be attributed to the higher ß-sheet content and crystallinity. Chitin staining of the anterior silk gland suggested that the lumen is narrower in bimolters, which may lead to the formation of greater numbers of ß-sheet structures in the silk. Therefore, this study reveals the relationship between the structures and mechanical properties of bimolter silk and provides a valuable reference for producing high-strength and ultrafine silk fibers.

Ultrastrong and Highly Sensitive Fiber Microactuators Constructed by Force-Reeled Silks.

Fiber microactuators are interesting in wide variety of emerging fields, including artificial muscles, biosensors, and wearable devices. In the present study, a robust, fast-responsive, and humidity-induced silk fiber microactuator is developed by integrating force-reeling and yarn-spinning techniques. The shape gradient, together with hierarchical rough surface, allows these silk fiber microactuators to respond rapidly to humidity. The silk fiber microactuator can reach maximum rotation speed of 6179.3° s-1 in 4.8 s. Such a response speed (1030 rotations per minute) is comparable with the most advanced microactuators. Moreover, this microactuator generates 2.1 W kg-1 of average actuation power, which is twice higher than fiber actuators constructed by cocoon silks. The actuating powers of silk fiber microactuators can be precisely programmed by controlling the number of fibers used. Lastly, theory predicts the observed performance merits of silk fiber microactuators toward inspiring the rational design of water-induced microactuators.