Methods for Silk Property Analyses across Structural Hierarchies and Scales.

Silk from silkworms and spiders is an exceptionally important natural material, inspiring a range of new products and applications due to its high strength, elasticity, and toughness at low density, as well as its unique conductive and optical properties. Transgenic and recombinant technologies offer great promise for the scaled-up production of new silkworm- and spider-silk-inspired fibres. However, despite considerable effort, producing an artificial silk that recaptures the physico-chemical properties of naturally spun silk has thus far proven elusive. The mechanical, biochemical, and other properties of pre-and post-development fibres accordingly should be determined across scales and structural hierarchies whenever feasible. We have herein reviewed and made recommendations on some of those practices for measuring the bulk fibre properties; skin-core structures; and the primary, secondary, and tertiary structures of silk proteins and the properties of dopes and their proteins. We thereupon examine emerging methodologies and make assessments on how they might be utilized to realize the goal of developing high quality bio-inspired fibres.

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.

Spinning Regenerated Silk Fibers with Improved Toughness by Plasticizing with Low Molecular Weight Silk.

Low-molecular weight (LMW) silk was utilized as a LMW silk plasticizer for regenerated silk, generating weak physical crosslinks between high-molecular weight (HMW) silk chains in the amorphous regions of a mixed solution of HMW/LMW silk. The plasticization effect of LMW silk was investigated using mechanical testing, Raman spectroscopy, and wide-angle X-ray scattering (WAXS). Small amounts (10%) of LMW silk resulted in a 19.4% enhancement in fiber extensibility and 37.8% increase in toughness. The addition of the LMW silk facilitated the movement of HMW silk chains during drawing, resulting in an increase in molecular chain orientation when compared with silk spun from 100% HMW silk solution. The best regenerated silk fibers produced in this work had an orientation factor of 0.94 and crystallinity of 47.82%, close to the values of natural degummedBombyx mori silk fiber. The approach and mechanism elucidated here can facilitate artificial silk systems with enhanced properties.

Protein Paper from Exfoliated Eri Silk Nanofibers.

The exfoliation of silk fiber is an attractive method to produce silk micro- and nanofibers that retain the secondary structure of native silk. However, most fibrillation methods used to date require the use of toxic and/or expensive solvents and the use of high energy. This study describes a low cost, scalable method to produce microfibrillated silk nanofibers without the use of toxic chemicals by controlling the application of shear using commercially scalable milling and homogenization equipment. Manipulation of the degumming conditions (alkaline concentration and degumming temperature) and the shear in milling and/or homogenization enabled control over the degree of fibrillation. The microfibrillated silk was then characterized to determine structural change during processing and the stability of the resulting suspensions at different pH. Silk nanofibers obtained from milling degummed silk were characterized using atomic force microscopy. Nanofibers obtained both with and without high-pressure homogenization were then used to produce silk "protein paper" through casting. Silk degumming conditions played a critical role in determining the degree of microfibrillation and the properties of the cast silk papers. The silk papers produced from homogenized nanofibers showed excellent mechanical properties, high water absorption, and wicking properties. The silk papers were excellent for supporting the attachment and growth of human skin keratinocytes, demonstrating application possibilities in healthcare such as wound healing.

Facts and myths of antibacterial properties of silk.

Silk cocoons provide protection to silkworm from biotic and abiotic hazards during the immobile pupal phase of the lifecycle of silkworms. Protection is particularly important for the wild silk cocoons reared in an open and harsh environment. To understand whether some of the cocoon components resist growth of microorganisms, in vitro studies were performed using gram negative bacteria Escherichia coli (E. coli) to investigate antibacterial properties of silk fiber, silk gum, and calcium oxalate crystals embedded inside some cocoons. The results show that the previously reported antibacterial properties of silk cocoons are actually due to residues of chemicals used to isolate/purify cocoon elements, and properly isolated silk fiber, gum, and embedded crystals free from such residues do not have inherent resistance to E. coli. This study removes the uncertainty created by previous studies over the presence of antibacterial properties of silk cocoons, particularly the silk gum and sericin.

Unveiling the potential of cellulose nanofibre based nitrogen fertilizer and its transformative effect on Vigna radiata (Mung Bean): nanofibre for sustainable agriculture.

Introduction: Fertilizer management is crucial to maintaining a balance between environmental health, plant health, and total crop yield. Farmers are overutilizing fertilizers with a mind set to enhance the productive capacity of the field, which adversely impacts soil fertility and causes serious environmental hazards. To mitigate the issues of over-utilization of fertilizers, controlled-release fertilizers were developed using nitrogen fertilizer (ammonium chloride) loaded on cellulose nanofibres (named CNF*N). Methodology: In this study, the effects of CNF*N were compared with commercial nitrogen fertilizer (ammonium chloride) on Vigna radiata (Mung) under greenhouse conditions. The pot experiment was conducted using six treatments: first treatment was control, where the plant was cultivated (T1); second treatment was T2, where the plant was cultivated with CNF to determine the impact of CNF on the plant; third was T3 where commercial ammonium chloride (24 mg/ 2 kg soil) was added to the plant; fourth was T4, where the plant was loaded with CNF, viz. CNF*N contains 4.8 mg of nitrogen; fifth was T5 CNF*N pellet contains 12 mg of nitrogen, and the last sixth treatment (T6) where CNF*N pellet containing 24 mg of nitrogen. Results: It indicated that the growth parameters were best achieved in T6 treatment. Plant height was at its maximum in the T6 treatment (44.4 ±0.1cm) after the second harvest, whereas the minimum plant height was observed in T1, which was 39.1 ±0.1 cm. Root-to-shoot weight ratio was also maximum in T6 (0.183± 0.02) and minimum in T1 (0.07± 0.01) after second harvesting. The significant difference among the treatments was determined with Tukey's honestly significant difference (HSD). The nitrogen content (available and total) was significantly higher in the T4, T5, and T6 treatments (0.22, 0.25, and 0.28%) as compared to the control treatments (T1 (0.12%), T2 (0.13%), and T3 (0.14%) during the second harvesting stage (90 days), as nitrogen plays a crucial role in the development of vegetative growth in Vigna radiata. The rate of controlled-release nitrogen-fertilizer was found to be optimal in terms of plant growth and soil nutrients; hence, it could potentially play a crucial role in improving soil health and the yield of the crop.

Silk Industry Waste Protein-Derived Sericin Hybrid Nanoflowers for Antibiotics Remediation via Circular Economy.

Hybrid protein-copper nanoflowers have emerged as promising materials with diverse applications in biocatalysis, biosensing, and bioremediation. Sericin, a waste biopolymer from the textile industry, has shown potential for fabricating such nanoflowers. However, the influence of the molecular weight of sericin on nanoflower morphology and peroxidase-like activity remains unexplored. This work focused on the self-assembly of nanoflowers using high- and low-molecular-weight (HMW and LMW) silk sericin combined with copper(II) as an inorganic moiety. The peroxidase-like activity of the resulting nanoflowers was evaluated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H2O2). The findings revealed that high-molecular-weight sericin hybrid nanoflowers (HMW-ShNFs) exhibited significantly higher peroxidase-like activity than low-molecular-weight sericin hybrid nanoflowers (LMW-ShNFs). Furthermore, HMW-ShNFs demonstrated superior reusability and storage stability, thereby enhancing their potential for practical use. This study also explored the application of HMW-ShNF for ciprofloxacin degradation to address the environmental and health hazards posed by this antibiotic in water. The results indicated that HMW-ShNFs facilitated the degradation of ciprofloxacin, achieving a maximum degradation of 33.2 ± 1% at pH 8 and 35 °C after 72 h. Overall, the enhanced peroxidase-like activity and successful application in ciprofloxacin degradation underscore the potential of HMW-ShNFs for a sustainable and ecofriendly remediation process. These findings open avenues for the further exploration and utilization of hybrid nanoflowers in various environmental applications.

Banana fibre-chitosan-guar gum composite as an alternative wound healing material.

Bio-composites, which can be obtained from the renewable natural resources, are fascinating material for use as sustainable biomaterials with essential properties like biodegradable, bio-compatibility as well cyto-compatibility etc. These properties are useful for bio-medical including wound healing applications. In this study, fibre obtained banana pseudo stem of banana plant, which is otherwise wasted, was used as a material along with chitosan and guar gum to fabricate a banana fibre-biopolymer composite patch. The physiochemical properties of the patches were examined using Fourier Transformed Infra-red spectrophotometer (FT-IR), tensile tester, Scanning Electron Microscope (SEM), contact angle tester, swelling and degradation studies. We further demonstrated that a herbal drug, Nirgundi could be loaded to the patch showed controlled its release at different pHs. The patch had good antibacterial property and supported proliferation of mouse fibroblast cells. The study thus indicates that banana fibre-chitosan-guar gum composite can be developed into an alternative wound healing material.

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms.

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk-based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials.

Photoprotection by silk cocoons.

A silk cocoon protects a silkworm during its pupal stage from various threats. We systematically investigated the role of fiber, sericin, and embedded crystals in the UV protection of a silk cocoon. Diffuse reflectance and UV absorbance were measured and free radicals generated during exposure to UV radiation were quantified using photoinduced chemiluminescence (PICL). We identified the response to both UV-A and UV-B radiations by silk materials and found that sericin was primarily responsible for UV-A absorption. When sericin was removed, the photoinduced chemiluminescence intensity increased significantly, indicating higher UV-A-induced reactions of cocoons in the absence of sericin. There is progressively higher sericin content toward the outer part of the cocoon shell that allows an effective shield to pupae from UV radiation and resists photodegradation of silk fibers. The study will inspire development of advanced organic photoprotective materials and designing silk-based, free-radical-scavenging antioxidants.

Controlled release fertilizer delivery system derived from rice straw cellulose nanofibres: a circular economy based solution for sustainable development.

Recently, the development of sustainable and environmentally friendly biomaterials has gained the attention of researchers as potential alternatives to petroleum-based materials. Biomaterials are a promising candidate to mitigate sustainability issues due to their renewability, biodegradability, and cost-effectiveness. Thus, the purpose of this study is to explore a cost-effective biomaterial-based delivery system for delivering fertilizers to plants. To achieve this, rice straw (agro-waste) was selected as a raw material for the extraction of cellulose. The cellulose was extracted through alkali treatment (12% NaOH), followed by TEMPO-based oxidation. The cellulose nanofibers were characterized using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy, and transmission electron microscopy. In scanning electron microscopy, a loosening of the fibrillar structure in cellulose nanofibers (CNFs) was observed with a diameter of 17 ± 4 nm. The CNFs were loaded with nitrogen-based fertilizer (ammonium chloride) in 1:1, 1:2, and 2:1 (w/w) proportions. The loading was estimated through surface charge variation; in the case of the 1:1 sample, maximum reductions in surface charge were seen from -42.0 mV to -12.8 mV due to the binding of positive ammonium ions. In the release kinetics study, a controlled release pattern was observed at 1:1, which showed a 58% cumulative release of ammonium ions within 8 days. Thus, the study paves the way for value-added uses of rice straw as an alternative to the current environmentally harmful practices.

Bio-based cellulose nanofibers (CNFs) from rice straw via circular economy approach.Controlled release fertilizers for sustainable agriculture.Nanotechnology for precision agriculture and decarbonization via agricultural waste management.

Rice straw-derived cellulose: a comparative study of various pre-treatment technologies and its conversion to nanofibres.

Rice straw is a waste product generated after the harvesting of rice crops and is commonly disposed of by burning it off in open fields. This study explored the potential for the extraction and conversion of cellulose to cellulose nanofibres (CNFs) to be used as smart delivery systems for fertilizers applications. In this study, alkali, steam explosion, and organosolv treatments were investigated for cellulose extraction efficiency. The morphological characterization of cellulose showed smooth fibrillar structures. Fourier transform infrared spectroscopy represented significant removal of non-cellulosic components in treatments. The crystallinity increased from 52.2 to 65% in CNFs after fibrillation. Cellulose nanofibres (CNFs) had an average diameter of 37.4 nm and - 25.2 mV surface charges as determined by SEM and zeta potential, respectively, which have desired properties for holding fertilizers. Therefore, this study paves the way for value-added uses of rice straw as alternatives to current environmentally harmful practices.

Tuning the microstructure and mechanical properties of lyophilized silk scaffolds by pre-freezing treatment of silk hydrogel and silk solution.

This work aims to understand how pre-freezing treatments (-20 °C, -80 °C or -196 °C (liquid nitrogen)) affect the microstructure, mechanical properties and secondary structure of silk scaffolds prepared from lyophilization of silk hydrogels and silk solutions. It is found that in comparison with silk solutions, silk hydrogels at the same silk fibroin concentrations produce scaffolds with more nanofibrous structures when they are pre-frozen at the different temperatures. Although pre-freezing with liquid nitrogen can produce nanofibrous scaffolds from either a silk solution (low concentration of 2%) or silk hydrogel (produced from 2 to 6% silk fibroin solutions), aligned macro-channels can be produced only from silk hydrogels. In addition, scaffolds obtained from silk hydrogels are dominated by ß-sheets due to the crystallization process for gel network formation, while scaffolds prepared from silk solutions are largely amorphous. The findings of this work are important to tune the microstructure and mechanical properties of silk scaffolds.

Novel hybrid biocomposites for tendon grafts: The addition of silk to polydioxanone and poly(lactide-co-caprolactone) enhances material properties, in vitro and in vivo biocompatibility.

Biopolymers play a critical role as scaffolds used in tendon and ligament (TL) regeneration. Although advanced biopolymer materials have been proposed with optimised mechanical properties, biocompatibility, degradation, and processability, it is still challenging to find the right balance between these properties. Here, we aim to develop novel hybrid biocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL) and silk to produce high-performance grafts suitable for TL tissue repair. Biocomposites containing 1-15% of silk were studied through a range of characterisation techniques. We then explored biocompatibility through in vitro and in vivo studies using a mouse model. We found that adding up to 5% silk increases the tensile properties, degradation rate and miscibility between PDO and LCL phases without agglomeration of silk inside the composites. Furthermore, addition of silk increases surface roughness and hydrophilicity. In vitro experiments show that the silk improved attachment of tendon-derived stem cells and proliferation over 72 h, while in vivo studies indicate that the silk can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. Finally, we selected a promising biocomposite and created a prototype TL graft based on extruded fibres. We found that the tensile properties of both individual fibres and braided grafts could be suitable for anterior cruciate ligament (ACL) repair applications.

Structure and biodegradation mechanism of milled Bombyx mori silk particles.

The aim of this study was to understand the structure and biodegradation relationships of silk particles intended for targeted biomedical applications. Such a study is also useful in understanding structural remodelling of silk debris that may be generated from silk-based implants. Ultrafine silk particles were prepared using a combination of efficient wet-milling and spray-drying processes with no addition of chemicals other than those used in degumming. Milling reduced the intermolecular stacking forces within the ß-sheet crystallites without changing the intramolecular binding energy. Because of the rough morphology and the ultrafine size of the particles, degradation of silk particles by protease XIV was increased by about 3-fold compared to silk fibers. Upon biodegradation, the thermal degradation temperature of silk increased, which was attributed to the formation of tight aggregates by the hydrolyzed residual macromolecules. A model of the biodegradation mechanism of silk particles was developed based on the experimental data. The model explains the process of disintegration of ß-sheets, supported by quantitative secondary structural analysis and microscopic images.

A Substantial Role of Agro-Textiles in Agricultural Applications.

Agro-textiles have been used in the agriculture sector for thousands of years and are an attractive tool for the protection of crops during their entire lifecycle. Currently, the agro-textile market is dominated by polyolefins or petrochemical-based agro-textiles. However, climate change and an increase in greenhouse gas emissions have raised concern about the future oil-based economy, and petroleum-based agro-textiles have become expensive and less desirable in the modern world. Other products include agro-textiles based on natural fibers which degrade so fast in the environment that their recovery from the field becomes difficult and unattractive even by efficient recycling or combustion, and their lifetime is usually limited to 1 or a maximum of 2 years. Hence, the development of bio-based agro-textiles with a reduced impact on the environment and with extended durability is foreseen to initiate the growth in the bio-based economy. The world is gradually preparing the shift toward a bio-based economy, and research for sustainable bio-based alternatives has already been initiated. This review provides insight into the various agro-textiles used currently in agriculture and the research going on in the area of agro-textiles to offer alternative solutions to the current agro-textile market.

Enhanced formation of bioactive and strong silk-bioglass hybrid materials through organic-inorganic mutual molecular nucleation induction and templating.

Materials based on silk fibroin (SF) are important for many biomedical applications due to their excellent biocompatibility and tunable biodegradability. However, the insufficient mechanical strength and low bioactivity of these materials have limited their applications. For silk hydrogels, slow gelation is also a crucial problem. In this work, a simple approach is developed to address these challenging problems all at once. By mixing SF solution with bioglass (BG) sol, instant gelation of silk is induced, the storage modulus of the hydrogel and the compressive modulus of the aerogel are significantly enhanced. The formation of a complex of SF and tetraethyl orthosilicate (TEOS), either through hydrogen bonding or TEOS condensation on SF, facilitated the aggregation of SF and, on the other hand, created active sites for the condensation of TEOS and BG formation on the surface of silk nanofibrils. The resultant hybrid gels have much higher capacity for biomineralization, indicating their higher bioactivity, compared with the pristine silk gels. This organic (SF)-inorganic (BG) mutual nucleation induction and templating can be used for a general approach to produce bioactive silk materials of various formats not limited to gels and may also inspire the formation of other functional protein-BG hybrid materials.

Nanovoid formation induces property variation within and across individual silkworm silk threads.

Silk is a unique fiber, having a strength and toughness that exceeds other natural fibers. While inroads have been made in our understanding of silkworm silk structure and function, few studies have measured structure and function at nanoscales. As a consequence, the sources of variation in mechanical properties along single silk fibers remain unresolved at multiple scales. Here we utilized state of the art spectroscopic and microscopic methodologies to show that the silks of species of wild and domesticated silkworms vary in mechanical properties along a single fiber and, what is more, this variation correlates with nanoscale void formations. These results can also explain the strain hardening behaviours observed in the silks where structural features of the proteins could not. We thereupon devised a predictive thermal model and showed that the voids contribute to temperature regulation within the silkworm cocoons.

1000 spider silkomes: Linking sequences to silk physical properties.

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.