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Abstract Cribellate silks, produced by ancient spiders, are fascinating because they feature a highly sophisticated, 3D hierarchical structure consisting of filaments with different diameters and shapes. Here, the smallest and thinnest constituents of the cribellate silk are investigated: nanofibrils that form a dense mesh that is supported by larger fibers. Analysis of their structure via atomic force and transmission electron microscopies shows that they are flattened fibrils, only ≈5 nm thick — thinner than any other natural spider silk fibrils previously reported. In this work, the first mechanical tensile testing experiments on these fibrils are carried out, which reveals that the fibrils show an outstanding extensibility of at least 1100%, almost twice as much as the most stretchable spider silk previously reported. Based on these extraordinary findings, this work significantly expands the parameter space of materials properties attainable by spider silks and provides further insights into their nanomechanics. 

Abstract 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 β-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. 

Abstract 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. 

Coconuts are one of nature’s toughest lignocellulosic materials, possessing a fracture toughness on par with dentin and a compressive strength ten times that of bamboo. The coconut’s hierarchical structure has been characterized before, except prior studies left out one key aspect, the smallest length scales, approaching the molecular level. Here we exfoliate the hard shell of Cocos nucifera, revealing the true cellular organization and the dimensions of the crystalline cellulose nanofibrils found in the cell walls. After chemical pretreatments, we found entanglement between elongated sclereid cells that was not visible in the untreated coconut shell. This may contribute to the mechanical performance of the endocarp; it also utilizes elongated, high-aspect ratio structural elements at the cellular level, in addition to the nanofibrillar level previously known. Compared to other wood-like materials, the cellulose nanofibrils were shorter and represented a smaller weight fraction. This reduced length and the lower filler-to-matrix ratio could be the optimal lignocellulosic nanostructure for tough biomaterials. These newly discovered unique features explain how the endocarp of Cocos nucifera mechanically outperforms materials consisting of the same molecular components. 

Spider silk (SPSI) is a promising candidate for use as a filler material in nerve guidance conduits (NGCs), facilitating peripheral nerve regeneration by providing a scaffold for Schwann cells (SCs) and axonal growth. However, the specific properties of SPSI that contribute to its regenerative success remain unclear. In this study, the egg sac silk of Trichonephila (T.) inaurata is investigated, which contains two distinct fiber types: tubuliform (TU) and major ampullate (MA) silk. These fibers serve as models to derive material parameters governing SC migration on natural silk substrates, since they are produced by the same spider, yet exhibiting distinct composition and morphology. In this paper, detailed characterization of the fibers’ material properties and in vitro evaluation of their SC-guiding performance were conducted. Live cell imaging revealed significantly enhanced SC mobility and directionality on TU silk compared to MA silk, which is remarkable, given the lack of studies on TU silk for nerve regeneration. Our results suggest that the distinct morphological and material properties of these fibers are critical to their nerve-guiding potential. These insights contribute to the optimization of NGC filler materials by identifying key parameters essential for effective nerve regeneration. 

While spider silk threads mainly consist of a core of partially crystalline silk proteins, it has been found that they also exhibit a very thin skin layer of distinct structure and a coating rich in lipids and glycoproteins. These outer layers are poorly researched, but can be assumed to be a major player governing the interaction of cells with spider silk threads, as observed in cell culture. Here we propose SAXS/WAXS mapping with ultra-high spatial resolution to examine the surface layer of thin cryo-cut sections of different spider silks that have shown different cell guiding behavior in cell culture. This approach allows studying surface layers from two orientations (along and normal to fiber axis) and the cryo-approach minimizes morphological changes. In a recent nano-SAXS/WAXS beamtime at ID13, we obtained very promising data, however with whole threads and with lower resolution. This follow-up work aims to characterize the surface layer systematically. 

Raw data of optical microscopy (OM), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), and data analysis. The data is organized by the figure numbers used in the manuscript, in the order of appearance. This organization is best seen if viewed in the "Tree" mode. File Formats * AFM raw data is provided in NT-MDT's proprietary format (MDT) as well as Gwyddion format (GWY), which can both be viewed using the Gwyddion AFM viewer, which has been released under the GNU public software license GPLv3 and can be downloaded for free at http://gwyddion.net/. * AFM line profile raw data is provided in plain text ASCII (TXT) format. * XRD raw data is provided in plain text ASCII (TXT) format. * FE-SEM raw data always has the SEM image data, provided in TIF format, along with a parameter file produced by the SEM instrument in plain text ASCII (TXT). * Optical microscopy raw data is provided in PNG format. * Data analysis results of nanofibril dimensions are provided in an Excel sheet (XLSX). Data (Folder Structure) Figure 1 * FE-SEM raw data and PNG file from optical microscopy of the coconut. Figure 2 * FE-SEM raw data of all images of the coconut. Figure 3 * MDT and GWY files of all AFM scans of the exfoliated coconut cellulose. Lengths of crystalline nanofibrils were determined manually by running a line profile longitudinally across each nanofibril and determining its end-to-end length, ignoring any bends or kinks. The results of this procedure are shown in an XLSX file (column A). Other columns of this spread sheet contain the number average (B), its standard deviation (C), the sum of lengths (D), and the length weights (E) used to calculate the length-weighted average (G). Figure 4 * MDT and GWY file of the exfoliated coconut cellulose AFM scans. TXT files from XRD and line profile of AFM image. 

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 naurata 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. 

Raw data of optical microscopy, field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), force spectroscopy, and data analysis. File Formats * AFM and force spectroscopy raw data, is provided in NT-MDT's proprietary format (MDT) as well as Gwyddion format (GWY), which can both be viewed using the Gwyddion AFM viewer, which has been released under the GNU public software license GPLv3 and can be downloaded for free at http://gwyddion.net/ * FE-SEM and TEM raw data is provided in TIF format * Optical microscopy is provided in PNG format * Data analysis is provided in an excel sheet (XLSX) Data (Folder Structure) Figure 1 * All TIF files (with accompanying TXT files) from FE-SEM and PNG files from optical microscopy of the cribellate silk structure from the K. hibernalis. Figure 2 * All TIF files from TEM, TIF files (with accompanying TXT files) from FE-SEM, MDT and GWY files from AFM of the cribellate silk nanofibrils. Figure 3 + S3 * MDT and GWY files from AFM and force spectroscopy of the cribellate silk nanofibrils as well as the hard substrate and holes within the substrate. XLSX file containing data analysis process with descriptive boxes of what each row does. Figure S4 * MDT and GWY files from AFM and force spectroscopy of the cribellate silk nanofibrils. 

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.