Comparative Analysis of Various Spider Silks in Regard to Nerve Regeneration: Material Properties and Schwann Cell Response.

Peripheral nerve reconstruction through the employment of nerve guidance conduits with Trichonephila dragline silk as a luminal filling has emerged as an outstanding preclinical alternative to avoid nerve autografts. Yet, it remains unknown whether the outcome is similar for silk fibers harvested from other spider species. This study compares the regenerative potential of dragline silk from two orb-weaving spiders, Trichonephila inaurata and Nuctenea umbratica, as well as the silk of the jumping spider Phidippus regius. Proliferation, migration, and transcriptomic state of Schwann cells seeded on these silks are investigated. In addition, fiber morphology, primary protein structure, and mechanical properties are studied. The results demonstrate that the increased velocity of Schwann cells on Phidippus regius fibers can be primarily attributed to the interplay between the silk's primary protein structure and its mechanical properties. Furthermore, the capacity of silk fibers to trigger cells toward a gene expression profile of a myelinating Schwann cell phenotype is shown. The findings for the first time allow an in-depth comparison of the specific cellular response to various native spider silks and a correlation with the fibers' material properties. This knowledge is essential to open up possibilities for targeted manufacturing of synthetic nervous tissue replacement.

Natural spider silk nanofibrils produced by assembling molecules or disassembling fibers.

Spider silk is biocompatible, biodegradable, and rivals some of the best synthetic materials in terms of strength and toughness. Despite extensive research, comprehensive experimental evidence of the formation and morphology of its internal structure is still limited and controversially discussed. Here, we report the complete mechanical decomposition of natural silk fibers from the golden silk orb-weaver Trichonephila clavipes into ≈10 nm-diameter nanofibrils, the material's apparent fundamental building blocks. Furthermore, we produced nanofibrils of virtually identical morphology by triggering an intrinsic self-assembly mechanism of the silk proteins. Independent physico-chemical fibrillation triggers were revealed, enabling fiber assembly from stored precursors "at-will". This knowledge furthers the understanding of this exceptional material's fundamentals, and ultimately, leads toward the realization of silk-based high-performance materials. STATEMENT OF SIGNIFICANCE: Spider silk is one of the strongest and toughest biomaterials, rivaling the best man-made materials. The origins of these traits are still under debate but are mostly attributed to the material's intriguing hierarchical structure. Here we fully disassembled spider silk into 10 nm-diameter nanofibrils for the first time and showed that nanofibrils of the same appearance can be produced via molecular self-assembly of spider silk proteins under certain conditions. This shows that nanofibrils are the key structural elements in silk and leads toward the production of high-performance future materials inspired by spider silk.

Multi-Point Nanoindentation Method to Determine Mechanical Anisotropy in Nanofibrillar Thin Films.

Biomaterials with outstanding mechanical properties, including spider silk, wood, and cartilage, often feature an oriented nanofibrillar structure. The orientation of nanofibrils gives rise to a significant mechanical anisotropy, which is extremely challenging to characterize, especially for microscopically small or inhomogeneous samples. Here, a technique utilizing atomic force microscope indentation at multiple points combined with finite element analysis to sample the mechanical anisotropy of a thin film in a microscopically small area is reported. The system studied here is the tape-like silk of the Chilean recluse spider, which entirely consists of strictly oriented nanofibrils giving rise to a large mechanical anisotropy. The most detailed directional nanoscale structure-property characterization of spider silk to date is presented, revealing the tensile and transverse elastic moduli as 9 and 1 GPa, respectively, and the binding strength between silk nanofibrils as 159 ± 13 MPa. Furthermore, based on this binding strength, the nanofibrils' surface energy is derived as 37 mJ m-2 , and concludes that van der Waals forces play a decisive role in interfibrillar binding. Due to its versatility, this technique has many potential applications, including early disease diagnostics, as underlying pathological conditions can alter the local mechanical properties of tissues.

Protein secondary structure in spider silk nanofibrils.

Nanofibrils play a pivotal role in spider silk and are responsible for many of the impressive properties of this unique natural material. However, little is known about the internal structure of these protein fibrils. We carry out polarized Raman and polarized Fourier-transform infrared spectroscopies on native spider silk nanofibrils and determine the concentrations of six distinct protein secondary structures, including ß-sheets, and two types of helical structures, for which we also determine orientation distributions. Our advancements in peak assignments are in full agreement with the published silk vibrational spectroscopy literature. We further corroborate our findings with X-ray diffraction and magic-angle spinning nuclear magnetic resonance experiments. Based on the latter and on polypeptide Raman spectra, we assess the role of key amino acids in different secondary structures. For the recluse spider we develop a highly detailed structural model, featuring seven levels of structural hierarchy. The approaches we develop are directly applicable to other proteinaceous materials.

Self-strengthening tape junctions inspired by recluse spider webs.

Adhesive tapes are versatile and widely used yet lack adhesion strength due to their tendency to fail via peeling, a weak failure mode. A tape with surprising adhesive properties is the recluse spider's 50 nm-thin silk ribbon with a 1 : 150 aspect ratio. Junctions of these microscopic sticky tapes can withstand the material's tensile failure stress of ≈1 GPa. We modeled these natural tape-tape junctions and revealed a bi-modal failure behavior, critically dependent on the two tapes' intersection angle. One mode leads to regular, low-strength peeling failure, while the other causes the junction to self-strengthen, eliminating the inherent weakness in peeling. This self-strengthening mechanism locks the two tapes together, increasing the junction strength by 550% and allowing some junctions to remain intact after tensile failure. This impressive adhesive strength of tapes has never before been observed or predicted. We found that recluse spiders make tape junctions with pre-stress to force the locked, high-strength failure mode. We used this approach to make junctions with synthetic adhesive tapes that overcame the weak peeling failure.

Silk Protein Paper with In Situ Synthesized Silver Nanoparticles.

Silver nanoparticles (AgNPs) are in situ synthesized for the first time on microfibrillated silk (MFS) exfoliated from domesticated Philosamia cynthia ricini (eri) and Bombyx mori (mulberry) silkworm silk fibers. The process is rapid (hours time), does not rely on harmful chemicals, and produces robust and flexible AgNPs coated MFS (MFS-AgNPs) protein papers with excellent handling properties. None of these can be achieved by approaches used in the past to fabricate AgNPs silk systems. MFS bonds the AgNPs strongly, providing good support and stabilization for the NPs, leading to strong wash fastness. The mechanical properties of the MFS-AgNPs papers largely do not change compared to the MFS papers without nanoparticles, except for some higher concentration of AgNPs in the case of mulberry silk. The improved tensile properties of eri silk papers with or without AgNPs compared to mulberry silk papers can be attributed to the higher degree of fibrillation achieved in eri silk and its inherent higher ductility. MFS-AgNPs from eri silk also exhibit strong antibacterial activity. This study provides the basis for the development of smart protein papers based on silk fiber and functional nanomaterials.

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.

High-throughput optical thickness and size characterization of 2D materials.

We describe a method using simple optical microscopy and image processing that simultaneously characterizes thousands of nanosheets in a sample area on the order of 1 mm2. Including data acquisition and processing, both the number of atomic layers and the lateral sizes of all sheets can be obtained within a few hours-approximately 100 times faster than with previous methods, such as atomic force microscopy. This is achieved by normalizing the optical image based on substrate brightness, which eliminates inhomogeneities usually limiting optical techniques. Ultimately, the method enables robust statistical analysis of populations of nanosheet materials. We demonstrate the utility of this method by examining fractions made from a sample of graphene oxide (GO) made using an emulsion-based method. Beyond providing the morphological composition of the samples, the reported method is sensitive enough to provide information about the oxidation level of a population of GO sheets and, correspondingly, optical constants of the material.

Strength of Recluse Spider's Silk Originates from Nanofibrils.

Spider silk exhibits a combination of outstanding tensile strength and extensibility unique among all synthetic and biogenic polymer fibers. It has thus generated great interest to understand protein-based high-toughness materials and inspired the design of similar synthetic materials. The unrivaled properties of silk fibers have been recognized to be intimately related to their hierarchical structure. However, in the absence of unambiguous experimental evidence, competing and incompatible structural models of natural silk fibers have been proposed, some of them including various types of fibrillar components. Here we show that the fibers of the recluse (Loxosceles) spider exhibit the typical tensile properties of a very good spider silk and are entirely composed of 20 nm diameter protein fibrils that are more than 1 µm long. Based on these findings, we developed the most detailed structural model for any silk directly supported by experimental evidence. Our work suggests that all the key properties of a spider silk are implemented within a single nanofibril, and we have isolated and imaged such a nanofibril from a native spider silk fiber. The nanofibril breaking force was estimated to be ≈120 nN. Our work underlines the importance of nanofibrils and furthers the understanding of the structure-property relationships of silk, with wide-ranging implications for silk research and the design of silk-inspired high-performance materials.

Graphene Oxide's Reduction of Hydrolytic Degradation in Polyamide-11.

Graphene oxide (GO) was incorporated into polyamide-11 (PA11) via in-situ polymerization. The GO-PA11 nano-composite had elevated resistance to hydrolytic degradation. At a loading of 1 mg/g, GO to PA11, the accelerated aging equilibrium molecular weight of GO-PA11 was higher (33 and 34 kDa at 100 and 120 °C, respectively) compared to neat PA11 (23 and 24 kDa at 100 and 120 °C, respectively). Neat PA11 had hydrolysis rate constants (kH) of 2.8 and 12 (×10-2 day-1) when aged at 100 and 120 °C, respectively, and re-polymerization rate constants (kP) of 5.0 and 23 (×10-5 day-1), respectively. The higher equilibrium molecular weight for GO-PA11 loaded at 1 mg/g was the result of a decreased kH, 1.8 and 4.5 (×10-2 day-1), and an increased kP, 10 and 17 (×10-5 day-1) compared with neat PA11 at 100 and 120 °C, respectively. The decreased rate of degradation and resulting 40 % increased equilibrium molecular weight of GO-PA11 was attributed to the highly asymmetric planar GO nano-sheets that inhibited the molecular mobility of water and the polymer chain. The crystallinity of the polymer matrix was similarly affected by a reduction in chain mobility during annealing due to the GO nanoparticles' chemistry and highly asymmetric nano-planar sheet structure.

AFM-based mechanical characterization of single nanofibres.

Nanofibres are found in a broad variety of hierarchical biological systems as fundamental structural units, and nanofibrillar components are playing an increasing role in the development of advanced functional materials. Accurate determination of the mechanical properties of single nanofibres is thus of great interest, yet measurement of these properties is challenging due to the intricate specimen handling and the exceptional force and deformation resolution that is required. The atomic force microscope (AFM) has emerged as an effective, reliable tool in the investigation of nanofibrillar mechanics, with the three most popular approaches-AFM-based tensile testing, three-point deformation testing, and nanoindentation-proving preferable to conventional tensile testing in many (but not all) cases. Here, we review the capabilities and limitations of each of these methods and give a comprehensive overview of the recent advances in this field.

Silk Reconstitution Disrupts Fibroin Self-Assembly.

Using atomic force microscopy, we present the first molecular-scale comparison of two of the most important silk dopes, native (NSF) and reconstituted (RSF) silkworm fibroin. We found that both systems depended on shear to show self-assembly. Significant differences in the nature of self-assembly between NSF and RSF were shown. In the highest studied concentration of 1000 mg/L, NSF exhibited assembly into 20-30 nm-wide nanofibrils closely resembling the surface structures found in natural silk fibers. RSF, in contrast, showed no self-assembly whatsoever at the same concentration, which suggests that the reconstitution process significantly disrupts silk's inherent self-assembly capability. At lower concentrations, both RSF and NSF formed fibrils under shear, apparently denatured by the substrate. Using image analysis, we quantified the properties of these self-assembled fibrils as a function of concentration and found low-concentration fibrils of NSF to form larger continuous structures than those of RSF, further supporting NSF's superior self-assembly capabilities.

Brown recluse spider's nanometer scale ribbons of stiff extensible silk.

The silk of the recluse spider features a ribbon-like morphology unlike any other spider silk or synthetically spun polymer fiber. These protein ribbons represent free-standing polymer films with a thickness of about 50 nm. Stress-strain characterization of individual fibers via atomic force microscopy reveals that these ribbons, only a few molecular layers of protein thin, rival the mechanical performance of the best silks.

Effects of coating a titanium alloy with fibronectin on the expression of osteoblast gene markers in the MC3T3 osteoprogenitor cell line.

PURPOSE: A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material in combination with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation. MATERIALS AND METHODS: Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat- sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes. RESULTS: Fibronectin increased the peak expression of all analyzed osteoblast gene markers. "Early" genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, "late" genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture. CONCLUSIONS: Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis.

Stabilization of graphene sheets by a structured benzene/hexafluorobenzene mixed solvent.

Applications requiring pristine graphene derived from graphite demand a solution stabilization method that utilizes an easily removable media. Using a combination of molecular dynamics simulations and experimental techniques, we investigate the solublization/suspension of pristine graphene sheets by an equimolar mixture of benzene and hexafluorobenzene (C(6)H(6)/C(6)F(6)) that is known to form an ordered structure solidifying at 23.7 °C. Our simulations show that the graphene surface templates the self-assembly of the mixture into periodic layers extending up to 30 Å from both sides of the graphene sheet. The solvent structuring is driven by quadrupolar interactions and consists of stacks of alternating C(6)H(6)/C(6)F(6) molecules rising from the surface of the graphene. These stacks result in density oscillations with a period of about 3.4 Å. The high affinity of the 1:1 C(6)H(6)/C(6)F(6) mixture with graphene is consistent with observed hysteresis in Wilhelmy plate measurements using highly ordered pyrolytic graphite (HOPG). AFM, SEM, and TEM techniques verify the state of the suspended material after sonication. As an example of the utility of this mixture, graphene suspensions are freeze-dried at room temperature to produce a sponge-like morphology that reflects the structure of the graphene sheets in solution.

Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy.

Noncontact mode atomic force microscopy was used to investigate native silk proteins prepared in different ways. Low protein concentrations revealed that single protein molecules exhibit a simple, round shape with apparent diameters of 20-25 nm. Shearing the native protein solutions after extraction from the gland and prior to drying led to a beads-on-a-string assembly at the nanometer scale. Protein concentration had a significant effect on the morphology of the protein assemblies. At higher protein concentrations, shear-induced alignment into nanofibrils was observed, while lower concentrations lead to the formation of much thinner fibrils with a width of about 8 nm.

Surface oxide net charge of a titanium alloy: comparison between effects of treatment with heat or radiofrequency plasma glow discharge.

In the current study, we have compared the effects of heat and radiofrequency plasma glow discharge (RFGD) treatment of a Ti6Al4V alloy on the physico-chemical properties of the alloy's surface oxide. Titanium alloy (Ti6Al4V) disks were passivated alone, heated to 600 °C, or RFGD plasma treated in pure oxygen. RFGD treatment did not alter the roughness, topography, elemental composition or thickness of the alloy's surface oxide layer. In contrast, heat treatment altered oxide topography by creating a pattern of oxide elevations approximately 50-100 nm in diameter. These nanostructures exhibited a three-fold increase in roughness compared to untreated surfaces when RMS roughness was calculated after applying a spatial high-pass filter with a 200 nm-cutoff wavelength. Heat treatment also produced a surface enrichment in aluminum and vanadium oxides. Both RFGD and heat treatment produced similar increases in oxide wettability. Atomic force microscopy (AFM) measurements of metal surface oxide net charge signified by a long-range force of attraction to or repulsion from a (negatively charged) silicon nitride AFM probe were also obtained for all three experimental groups. Force measurements showed that the RFGD-treated Ti6Al4V samples demonstrated a higher net positive surface charge at pH values below 6 and a higher net negative surface charge at physiological pH (pH values between 7 and 8) compared to control and heat-treated samples. These findings suggest that RFGD treatment of metallic implant materials can be used to study the role of negatively charged surface oxide functional groups in protein bioactivity, osteogenic cell behavior and osseointegration independently of oxide topography.

Tuning of structural color using a dielectric actuator and multifunctional compliant electrodes.

We have developed electrically conducting silicone elastomer nanocomposites that serve both as compliant electrodes in an electrostatic actuator and, at the same time, as optically active elements creating structural color. We demonstrate the capabilities of our setup by actuating an elastomeric diffraction grating and colloidal-crystal-based photonic structures.