Biomimetic Silk Fibre Assembly: Mimicking Nature's Pultrusion Process

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Moreno-Tortolero, Juliusz Michalski, Eleanor Wells, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4130861/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Among the best natural structural materials, silks have remarkable properties due to their hierarchical structure. The silk proteins from spiders or caterpillars, despite being distinct Classes, are produced by similar mechanisms with conserved features. They are stored as aqueous liquid solutions that undergo irreversible liquid-to-solid transformations driven by different stimuli, primarily pH and shear strain. This transformation has attracted the attention of many researchers aiming to replicate this apparently facile process. However, most biomimetic assembly processes that have been developed rely on extrusion-based technologies or flow-focusing microfluidic devices, typically using coagulating baths with unnatural solvent conditions. These synthetic processing strategies differ substantially from natural, all-aqueous, pultrusion-based fibre production and increase the overall energy input required to drive the transformation. In contrast, we observe that native-like silk fibroin (NLSF) rapidly forms a highly viscoelastic film at the air–water interface. This phenomenon is then exploited by applying an extensional strain field to produce multimeter silk-like fibres with observable coaligned nanofibrillar bundles. Our studies showed that the proteins undergo stress-induced denaturation, consistent with a model of hexagonal packing of β-solenoid units, at low pulling speeds, at which point the proteins switch to a β-sheet-rich structure as the speed increases. Moreover, the produced fibres showed optimal mechanical properties when the pulling speeds were near the maximum physiologically relevant speeds (ca. 30 mm/s). s pulled at 26.3 mm/s had an elastic modulus of 8 ± 1 GPa and a toughness of 8 ± 5 MJ/m2, which is commensurate with the mechanical performance of natural fibres. Moreover, the method demonstrated here is readily compatible with complex material fabrication under ambient conditions, opening up the possibility of facile incorporation of cells and biomolecules. Overall, the developed method replicates the natural pultrusion process entirely water-based and offers great potential for the future development of novel fibre-based composite materials. Biomaterials biomimetic silk fibres hierarchical biomaterials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Silk fibroin has captivated researchers for generations owing to its remarkable mechanical properties and unique self-assembly behavior. 1 Unlike synthetic materials, silk fibroin undergoes a programmed transition from a liquid aqueous solution to a solid-state with minimal energy input, making it a fascinating subject of study in biomaterials research. 2 , 3 Recent investigations have provided further insight into the molecular self-assembly mechanism of Lepidopteran silk fibroin, revealing its nanofibrillar structure in the Silk-I configuration and the intricate interactions driving its solidification. 4 At the macroscopic level, natural silk fibres are not extruded but rather pulled into shape through a process akin to pultrusion. 5 , 6 This pultrusive mechanism, observed in silkworms and spiders, underscores the biomechanical sophistication of silk production in nature. 7 Reconstituted silk fibroin (RSF), although often used as a precursor in biomimetic silk material research, has several inherent limitations due to its reduced molecular weight and the lack of the N-terminal domain (NTD) responsible for pH-controlled supramolecular assembly. 4 As a result, RSF requires nonnative conditions, such as organic solvents, 8 coagulation baths, 9 or prepolymerized aggregates, 10 for fibre formation. However, these methods impose environmental stress on the protein and deviate from the natural spinning process, where only minimal energy intake is required to fabricate the silk fibre. 5 , 10 , 11 Interestingly, both silk fibroin and spidroins exhibit surface-active properties, rapidly forming elastic films at the water‒air interface. 12 , 13 Although the surface activity of native or native-like fibroin (SF) materials has been underexplored, recent insights into the structural dynamics of proteins suggest that the water–air interface plays a crucial role in directing assembly in vitro. 14 By leveraging this behavior, our aim was to fabricate silk-like fibres with enhanced control and efficiency. In this study, we propose a novel approach to silk fibre fabrication that harnesses the interfacial self-assembly of silk fibroin and its sensitivity to stresses. The method was developed from observations made while working with dilute native-like silk fibroin (NLSF) solution (see ST 1 and Figures S1 and S2). In brief, fibroin molecules adsorb at the water‒air interface, and by applying perpendicular extension, we induce a strain field. Pulling the film in this way promotes the more ordered aggregation of solenoid units via lateral interactions, and subsequent denaturation leads to fibre formation. Our proposed molecular-level mechanism is illustrated in Fig. 1 (Video 1). We believe this method offers a promising alternative to conventional methods, allowing for the production of silk-like fibres under more physiologically relevant conditions. Similar methods have also been reported for recombinant spidroin systems 15 , 16 but without demonstrating the critical role of the water-air interface in driving assembly or scalability. Overall, our study seeks to deepen our understanding of silk fibroin assembly and pave the way for the development of biomimetic materials with tailored properties and applications. By bridging the gap between fundamental research and practical application, we aim to unlock the full potential of silk fibroin as a versatile biomaterial. Results and discussion Interfacial assembly of the protein at the water‒air interface With respect to the model, the surface assembly properties of the proteins were studied. Although the concentration is known to play a role in the interfacial properties of proteins, given the high complexity of the system and for simplicity, this work used only a single concentration, ca. 6 mg/mL, and studied the effect of pH on protein assembly at the water‒air interface. Therefore, the first experiment was to determine the adsorption behaviour of the proteins at the water‒air interface and the effect of pH. To do this, interfacial surface tension (IFT) analysis was performed using a Wilhelmy plate. In this experiment, the force that a platinum plate is subjected to when wetted with a liquid is related to surface tension Eq. 1: \({\gamma }=\frac{F}{L\text{cos}\theta }\) Equation 1 where \({\gamma }\) is the interfacial surface tension, L is the length of the plate, and \(\theta\) is the wetting angle. The advantage of this system is that it allows for the measurement of adsorption kinetics on long timescales without suffering as much from evaporative effects. Inspired by the natural pH gradient within the insect and our own observations, only pH values of 8, 7 and 6 were used. Figure S3 shows the results from these experiments. In all the cases, very rapid adsorption was observed, with all the experiments showing a reduction in IFT even at the beginning of the experiment; the IFT of pure water was 72 mN/m. Despite the similar behaviors of the samples at pH 6 and 7, the protein absorbs to the interface much more quickly at high pH, with an apparent lower IFT value. Notably, the observed values agree with the RSF values observed at similar concentrations. 13 To some extent, these experiments verify the observations of DLS, 4 where the proteins had smaller diameters; hence, faster diffusion at higher pH values occurred. This indicates that the protein at pH 8 has a faster adsorption to the interface due to faster diffusion speeds under these conditions. Nevertheless, the difference in the IFT itself is more nuanced. It is possible to argue that there might be changes in the total accessible surface area of the protein, whereby at pH 8, the protein exists as a monomeric unit, with a greater likelihood of forming a different type of oligomeric unit as the pH decreases. Oligomerization or aggregation would then reduce the total accessible surface area of the protein. Because the concentration was constant, such a reduction in the IFT can be described by Eq. 2 in the SI. 17 Further discussion and observations can be found in ST 2, describing more in-depth observations of a solid-to-solid transformation driven by stress; see Video 2 and Figures S4 and S5. To characterize the morphology of the protein film at the water‒air interface, we used confocal laser fluorescence microscopy (CLFM). Here, intrinsic fluorescence was used with no labeling to avoid changes in assembly behaviour by incorporating fluorescent labels, which commonly react with free primary amines or carboxylate groups (i.e., Lys or Asp/Glu residues), which are present only at terminal domains or linker units. This finding is particularly relevant given the important role of the NTD in driving assembly. In these experiments, approximately 10 µL of solution equilibrated at either pH 8 or 6 was placed in individual wells and left to age for approximately 5 h before observing the air/water interface using a long working distance objective (x10). The results are shown in Fig. 2 A and B. Despite the films being of similar thickness (10–20 µm, as shown in Fig. 2 C), the relative fluorescence intensity of the protein at pH 6 was greater than that at other pH values, which could indicate an aggregation-induced emission (AIE) process. 18 Freely rotating Tyr (mainly) might be locked in a single rotameric state, enhancing the fluorescence of these multimers/aggregates against the protein in solution. These observations support our hypothesis of a higher degree of order/oligomerization at lower pH. However, a high degree of order is also imparted by the geometrical restriction of the interface itself and the elongated nature of the protein, which also produces a significant signal at higher pH. To further characterize the mechanics of the films, they were subjected to dynamic microindentation measurements. A small indenter was gently placed in contact with the film and later oscillated, with small oscillation amplitudes, at a range of frequencies (0.1–10 Hz). Using this method, we determined the viscoelastic response in terms of the separate elastic and viscous components of the same material. The results are shown in Fig. 2 D-F, with the sample pH shown in the right lower corner. In these experiments, the measurements are reported as stiffness (K) against frequency, with stiffness calculated as the measured force divided by the indentation depth, or half the oscillation amplitude. K’ and K” refer to the elastic and viscous components, respectively. As expected, at pH 6, the material exhibited a relatively higher K’ (160 ± 1 mN/m) than did its counterparts at pH 7 or 8 (60 ± 2 and 60 ± 3 mN/m, respectively); these two pH values exhibited little difference. Once again, we propose that the switch-like behavior occurs at pH values just below 7. 4 At lower pH, the nature of the protein in solution is likely oligomeric, driven by NTD interactions; hence, the effective MW of the system increases, increasing the total cohesiveness, which translates to a higher modulus. Biomimetic silk-like fibre fabrication and characterization Taken together, these observations indicate that an elastic yet dynamic film forms very rapidly at the water/air interface, and upon extension of the strain/stress applied by pulling, it is possible to assemble insoluble silk-like fibres. For simplicity and because the film was more resilient at pH 6, this condition was used and exploited to produce multimeter long single fibres by continuously pulling the formed fibre using the method depicted in Video 3 and simplified in Fig. 3 A. Simply by depositing small droplets onto standard plastic petri dishes, it was possible to pull fibres at different reeling speeds. The achieved reeling speeds ranged from 1.8 mm/s to approximately 53 mm/s. However, the fibre formation was less stable at higher speeds. We hypothesize that there is competition between film formation and protein depletion through the formation of fibres. However, the coverage range contains the natural silkworm spinning speeds estimated to be between 10 and 30 mm/s. 19 Using this method, from droplets between 50 and 100 µL (Fig. 3 B), it was possible to collect several meters of single fibres (Fig. 3 C). All the fibres produced presented a highly hierarchical morphology, with the main fibre formed by bundles of submicron fibres composed of smaller nanofibrils, as shown in Figure S2. The results obtained by SEM corroborate these observations and are summarized in Fig. 4 A-G. The fibrillar network extends far from the location where the mature fibre emerges, as outlined in Fig. 4 A, with the experimental observations shown in Fig. 4 B. We were able to observe individual nanofibrils without much evidence of branching. In other words, the smaller observable fibrils seem to grow in one dimension and only interact by relatively weaker lateral interactions to form larger bundles. These observations are not only in line with the current understanding of natural silk fibre, 20 but also predicted from our proposed fibre assembly model; Tyr residues limit the lateral docking of strands, much like the lateral docking of β-solenoids, facilitating the nanofibrillar interface but also facilitating other types of interactions, such as π-π or methyl-π interactions. It has been observed that native silk fibroin fibres can be exfoliated into increasingly thinner fibrils, ranging from 20–100 nm bundles down to 3.1 ± 0.8 nm and ultimately into an extended chain with a diameter of approximately 3.7 ± 0.9 Å. 20 In the interest of understanding the biological relevance of our method in the context of in vivo fibre production, we measured the forces that were required to pull these fibres. At steady-state. We measured a force of approximately 0.45 ± 0.04 mN at a reeling speed of 15.5 mm/s, well within the forces that the insect can exert. 21 , 22 No significant differences in the forces were observed at the different speeds (Figure S6 A), indicating that by varying the reeling speeds, we were more likely to change the strain rate rather than the overall stress. Further chemical analysis of the fibres, beyond the nanoscopic similarity with natural silk fibres, showed a remarkable resemblance of the molecular structure, with the amide-I peak showing similar nominal β-sheet, β-turn and statistical coil compositions (Fig. 4 D). Thus, soluble silk fibroin (silk-I) was transformed to the well-known insoluble conformation (silk-II) by just using pH control and mechanical stimulation. Notably, no significant differences were observed in the FTIR spectra when the reeling speed was varied. To further understand the effect of reeling speed at the molecular level, we conducted fibre X-ray diffraction (fXRD) experiments on the different fibres immediately after they were produced at different reeling speeds. In silk fibres, the protein adopts an extended chain conformation, wherein the hydrogen bond network is perpendicular to the long axis of the fibre. However, this conformation is drastically different from that of our Silk-I model, which has a β-solenoid structure. In the proposed fibre formation process, the solenoids would be aligned parallel to the water/air interface plane, and upon drawing, these would align with the long axis of the created filament. The solenoids are denatured/stretched upon application of a critical strain rate and stress, and the known Silk-II configuration emerges. Hence, we would expect to observe a transition from a dominating Silk-I structure to an increasingly more dominating and aligned Silk-II structure upon increasing the reeling speed. Indeed, this was observed going from a reeling speed of approximately 1.5 mm/s to approximately the maximum possible speed of approximately 52.7 mm/s. As shown in Fig. 5 A, there is a reflection corresponding to approximately 17 Å at the lowest reeling speeds, which disappears as the reeling speed increases. We interpret this reflection as coming from the hexagonal packing of hydrated solenoid units, which upon reaching a critical strain rate are disrupted, prompting extension of the backbone and collapse of the chains in β-sheets, consistent with the prediction in our previous work. 4 Following these experiments, we conducted single-fibre tensile testing, and the results are presented in Fig. 5 B-F. Figure 5 B shows the average stress‒strain curves obtained for the fibres produced at different reeling speeds; these curves already show noticeable differences that are more detailed in Fig. 5 C-F. As one would expect, we observed optimal mechanical properties at a reeling speed that corresponded to the maximum natural spinning speeds (ca. 30 mm/s), with the mechanical properties decreasing thereafter. Briefly, the elastic modulus showed no significant differences from the lowest reeling speed of 1.8 mm/s up to 15.9 mm/s, with values ranging between 4 and 5 GPa; however, from 21 to 32.2 mm/s, we observed an increased modulus, with three increasing between 8 and 10 GPa, decreasing back to 6 ± 1 GPa for the highest reeling speed (52.7 mm/s); see Fig. 5 C. The values of tensile strength followed a similar trend, with strength increasing for reeling speeds between 21 and 26.3 mm/s to almost 200 MPa for these speeds and decreasing monotonically as the reeling speed increased thereafter; see Fig. 5 D. Interestingly, a slightly different trend was followed by the maximum strain and toughness values, where these values showed an obvious second maxima at the minimal reeling speed (1.8 mm/s). The maximum extensibility was approximately 6% for both 1.8 and 5.9 mm/s, decreasing to approximately 3% for reeling speeds from 10.7 to 21 mm/s, after which the extensibility increased again to 6 ± 4% for the fibres produced at 26 mm/s, decreasing to approximately 3% for any of the faster reeling speeds (see Fig. 5 E). Similarly, the same trend was observed for toughness (see Fig. 5 F). Interestingly, the observed trends are different from the fibre diameter trends, which only showed a monotonic decrease from 6 ± 1 µm to 3.7 ± 0.2 µm for fibres produced at 1.8 and 52.7 mm/s (see Figure S6 B). Notably, the best performance of our fibres replicates the properties shown by degummed cocoon fibres to a great extent, as reported in the literature. 23 Moreover, the overall results are readily explained by our fXRD data and indeed support our model. At the lowest reeling speeds, the relatively higher content of the Silk-I conformation would enable greater extensibility (and toughness) without necessarily enhancing other properties, such as the elastic modulus or tensile strength, as lower strain rates would also imply a lower orientation of the formed β-sheet crystallites. On the other hand, the observation of the overall properties showing a maximum at the peak of natural spinning speeds might indicate the formation of an optimal protein network architecture, where the balance of crystallite size, distribution and orientation maximizes the properties of the material, particularly its toughness. Although our system might not fully recapitulate the in vivo system, mainly due to concentration differences, it is notable that we observed an optimum at natural spinning speeds. Our proposed fibre formation mechanism involves two separate steps: first, the oligomerization of NTD driven by pH reduction; second, the reconfiguration of the network and further denaturation of the protein fold driven by stress. In this sense, the oligomerization of NTD effectively reduces the degrees of freedom of the protein while also breaking the symmetry of the system. This first step promotes network formation and primes the protein for stress-driven assembly. The system we propose here recapitulates the two-step system, first by breaking the symmetry of the system by exposing it to an interface, becoming inherently asymmetric, and further driving the assembly by stress. Here, in purely aqueous solutions without any precipitants (salts or solvents) and by using high-quality protein feedstock, we were able to replicate the Silk-I to Silk-II transformation using relative speeds and forces that are accessible to the animal. In addition to the fundamental implications of our observations, the presented method represents a facile, biomimetic process that allows for the easy and efficient fabrication of silk-like fibres. Its simplicity is amenable to the fabrication of several composite materials. For example, multiple fibres can be pulled simultaneously (Fig. 6 A) while moving the platform along the collector/mandrel to fabricate nonwoven mats (see Video 4 and Figure S7 for further explanation of the principle). Using standard ideas for the fabrication of orthotopically modified materials, multiple fibrous mats approximately 2 × 4 cm in length were manufactured using approximately 500 µL of the protein solution (Fig. 6 B). Moreover, the process allows for the facile bottom-up incorporation of functionality into the produced fibres. In the past, functional silk fibres were formed by either modifying the surface chemistry of natural fibres 24 or directly feeding insects functional particles. 25 Here, by simply introducing functional particles into a low-viscosity protein solution, we were able to fabricate magnetic silk-like fibres with incorporated magnetite nanoparticles (Fig. 6 C and Figure S8), as well as living composites with Escherichia coli ( E. coli ) transformed with green fluorescence protein (GFP) for use as fluorescent stimuli-responsive fibres. Despite the relatively large size of E. coli , these bacteria were readily homogeneously incorporated (Fig. 6 D and E), with strong evidence of preferential alignment of the elongated bacteria in the same direction as the fibrils (Video 5), as shown by the directional analysis of the images in Figure S9. We believe that this orientation is induced mainly by the confinement offered by the hierarchical nanofibrillar morphology of the fibres and not necessarily by the flow, as the forces used here are low. We are now attempting to further optimize and fabricate devices. Conclusions In this work, we present a novel methodology for the production of silk-like fibres that recapitulates many of the natural steps and requires minimal energy. This process exploits the rapid formation of a protein film at the water‒air interface, which upon stretching, undergoes crystallization. Although we only present results with NLSF, the process can likely be generalized to other surface-active, sticky proteins and polymers. On the other hand, these results reinforce our proposed self-assembly pathway where pH and shear promote fibre formation from a protein that belongs to the β-solenoid family. The applied stress/strain not only aligns the fibrillar protein but also, upon a critical stress/strain rate, promotes unfolding and extension of the protein backbone, allowing for extensive β-sheet formation. Using this simple process, multimeter long silk-like fibres were produced that exhibited native-like mechanical properties without further need for posttreatment. Moreover, the simplicity and versatility of this method allows for the facile bottom-up fabrication of a myriad of fibrous composites, demonstrated here by incorporating both magnetic nanoparticles and GFP-transformed E. coli . Although the method is limited in throughput compared to electrospinning, it offers a cost-effective, minimal energy and soft method for the fabrication of microfibres. Overall, in this work, we present a novel material fabrication method that can likely be expanded beyond silk and offers unprecedented insight into the assembly mechanism of silkworm fibroin into macroscopic fibres. Declarations Acknowledgments The authors would like to express their gratitude to the following individuals and facilities for their contributions and assistance: J.C. Eloi from the Chemistry Imaging Facility. Katy Jepson from the Wolfson Bioimaging facility and Anna Slatanova for their assistance with interfacial analysis. Funding : The EPSRC National Productivity Investment Fund grant EP/R51245/XF (R.O.M.T. ). EPSRC Doctoral Prize Fellowship at the University of Bristol grant EP/W524414/1 (R.O.M.T. ). Wellcome Trust grants 086906/Z/08/Z and 100917/Z/13/Z (N.S. and R.W.). The EIC Accelerator grant 947454 (N.S. and R.W.). The NIHR i4i Invention for Innovation award II-LB-0417-20005 (N.S. and R.W.). † EPSRC, grants EP/K035746/1 and EP/M028216/1 (TEM). † The views expressed in this work are those of the author(s) and do not necessarily reflect those of the National Institute of Health and Social Care (NIHR), the Department of Health and Social Care or any of their funding bodies. Author Contributions: Conceptualization: ROMT, SAD, LS, CH, NS, RW Methodology: ROMT, SAD, CH, LS Investigation: ROMT, LS, JM, EW, FG. Visualization: ROMT Funding acquisition: SAD, NS, RW, ROMT Project administration: ROMT, SAD Supervision: SAD, NS, RW Resources: NS, RW, ROMT Writing – original draft: ROMT, SAD Writing – review & editing: ROMT, NS, RW, LS, CH, SAD Competing interests: The authors declare no competing interests. Data and materials availability: All the data are available in the main text or the supplementary materials. The raw data and relevant materials are available upon reasonable request. References Omenetto FG, Kaplan DL (2010) New opportunities for an ancient material. Sci (80-) 329:528–531 Walker AA, Holland C, Sutherland TD (2015) More than one way to spin a crystallite: multiple trajectories through liquid crystallinity to solid silk. Proc. R. Soc. B Biol. Sci. 282 Sparkes J, Holland C (2019) The Energy Requirements for Flow-Induced Solidification of Silk. 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Sci Rep 9:1–10 Chai S et al (2024) Progress in Research and Application of Modified Silk Fibroin Fibres. Adv Mater Technol 9:2301659 Wang JT et al (2014) Directly obtaining pristine magnetic silk fibres from silkworm. Int J Biol Macromol 63:205–209 Additional Declarations The authors declare no competing interests. Supplementary Files BiomimeticSilkFibreAssemblyMimickingNaturesPultrusionProcessSI.docx ESI Video1.gif Video 1 Video2.mov Video 2 Video3.mov Video 3 Video4.mp4 Video 4 Video5.avi Video 5 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4130861","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281404697,"identity":"157bed17-5a5b-40c5-81c1-af24ab24aaef","order_by":0,"name":"Rafael O. Moreno-Tortolero","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie3RsWrDMBCAYQmDsiiEbmdcnFdQyJAU8jDWUk/ZPSoILkvIWJq38NZks/HQ5R6gpYtLXsCla6Gx2zEge8ygf5IOPjghxny+m4xvav2zihXjpr0t/2fGSQKrvszj/I8UDIaQEd4dTKXz7jyILJ5KDMbHIH2JKvvdZMAm20KEzw5y/6HxPCaxPu01QkHAgBIR5g4Ckd7OpZDrnDiyEtvF3pgIazfBSApIFXHbdGQ6hIQHVElLDHREdaRnMasaSmanHUcgAjkjbR9cz4co/ayT7He6kKNzk2WrOH6tyvedg1wlez/S5/P5fP1dAJD9Tl374HV7AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1413-3344","institution":"University of Bristol","correspondingAuthor":true,"prefix":"","firstName":"Rafael","middleName":"O.","lastName":"Moreno-Tortolero","suffix":""},{"id":281404699,"identity":"9ec1e1f3-e154-432a-9ffb-b9344eaae0d1","order_by":1,"name":"Juliusz Michalski","email":"","orcid":"","institution":"University of Bristol","correspondingAuthor":false,"prefix":"","firstName":"Juliusz","middleName":"","lastName":"Michalski","suffix":""},{"id":281404700,"identity":"213b64be-9d97-4812-ad48-9e8c2a34e6c9","order_by":2,"name":"Eleanor Wells","email":"","orcid":"","institution":"University of Bristol","correspondingAuthor":false,"prefix":"","firstName":"Eleanor","middleName":"","lastName":"Wells","suffix":""},{"id":281404701,"identity":"208c68a9-1a29-4064-8e55-1025b64747d6","order_by":3,"name":"Flora Gibb","email":"","orcid":"","institution":"University of Bristol","correspondingAuthor":false,"prefix":"","firstName":"Flora","middleName":"","lastName":"Gibb","suffix":""},{"id":281404702,"identity":"58a1096e-94d2-424d-a2a1-140a63d924d8","order_by":4,"name":"Nick Skaer","email":"","orcid":"","institution":"Orthox LTD","correspondingAuthor":false,"prefix":"","firstName":"Nick","middleName":"","lastName":"Skaer","suffix":""},{"id":281404703,"identity":"d9a526fb-a5e5-4da1-afbc-dd48c2555717","order_by":5,"name":"Robert Walker","email":"","orcid":"","institution":"Orthox LTD","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Walker","suffix":""},{"id":281404704,"identity":"a4c483a6-4727-47ae-94f8-eea7a45f684c","order_by":6,"name":"Louise Serpell","email":"","orcid":"","institution":"University of Sussex","correspondingAuthor":false,"prefix":"","firstName":"Louise","middleName":"","lastName":"Serpell","suffix":""},{"id":281404705,"identity":"5abfc17c-2e51-45c3-bbbd-3bdab7107b62","order_by":7,"name":"Chris Holland","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Chris","middleName":"","lastName":"Holland","suffix":""},{"id":281405163,"identity":"be94894e-b0a6-415d-8071-12243954c9a2","order_by":8,"name":"Sean Davis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie2RsUrEQBCG/2Vh04zaBpTsKwwsWB3cqyQEUoVDEMTC4qpNI9YeeA9xb3ASuCpaCzaxP2GrwyKIWSwluZQW+zEs7LAf+w8DBAL/FfIV3QNpfzn97aUTFGr8O4aaqgBx6c8Jil7KFvu7+kKvPneu7bpEQX44YYtBhbeKxdOuJn5f5I+ZZaOgTCxsOaz0JUkVxOelQbbkzAKXEPZ2JFjkJH0XpFeNQdp5JTqMKtgSyxM7I7xRryivkP9lJFhNV8/rhxlxs8iRWWOUpOs4fR0eX1fVpt0f4rmuXmrx1SXJWVRtnLvJh4PJPtufzrFFBgKBQOAIPw5tRR+GEMJDAAAAAElFTkSuQmCC","orcid":"","institution":"University of Bristol","correspondingAuthor":true,"prefix":"","firstName":"Sean","middleName":"","lastName":"Davis","suffix":""}],"badges":[],"createdAt":"2024-03-19 13:50:01","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4130861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4130861/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53104998,"identity":"e5676a93-b816-4330-90d5-604f7c4d8b72","added_by":"auto","created_at":"2024-03-20 15:58:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":301014,"visible":true,"origin":"","legend":"\u003cp\u003eProposed assembly and fabrication of the biomimetic pulled silk-like fibre. Briefly, a protein film is formed at the water‒air interface, where proteins form a cohesive material driven by different interactions. As the film is pulled, for example, with a pipette tip, the film is extended, forcing the elongated protein to extend and promote further widespread lateral interactions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/6ae51c0cfd3feedf2c5981ff.png"},{"id":53105003,"identity":"7eab15d9-64c0-4c6b-8582-f99861dc653b","added_by":"auto","created_at":"2024-03-20 15:58:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":359382,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy (CLSM) analysis of the self-assembled silk fibroin film at the water/air interface. X-Z projection of the film formed from solution buffered at pH 8 (A) and 6 (B). The fluorescence intensity 1D profile across the interface shows the thickness of the estimated films at pH 8 and 6 (C). Dynamic microindentation of the protein film formed at pH 6, 7 and 8 showing elastic (blue) and viscous (red) components of stiffness plotted as stiffness against frequency (D, E, F, respectively). The scale bars (A) and (B) are 20 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/9a39488664d7c21ed7a30784.png"},{"id":53104996,"identity":"2003fb70-ae2d-4a09-ae0a-c24c21e8025e","added_by":"auto","created_at":"2024-03-20 15:58:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":395913,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the general setup for fibre fabrication, with a rotating mandrel collecting the fibre as it is being pulled from the interfacial film (A), a photograph of the droplet as it is being pulled (B), an example of a 5 m long fibre collected onto a stainless steel mandrel (C) and FTIR analysis of natural degummed fibroin fibre and biomimetic fibre produced at 15.6 mm/s of the protein fingerprint region (1800–100 cm-1) (D).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/b515d1290692fc2b81a29f44.png"},{"id":53104999,"identity":"7f9502d1-670e-43b3-b68f-fc4c8835e3e4","added_by":"auto","created_at":"2024-03-20 15:58:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":836728,"visible":true,"origin":"","legend":"\u003cp\u003eStructural characterization of the hierarchical silk-like fibres produced by pulling protein films from the water–air interface. Drawing the hierarchical organization of the fibres, from the time of fabrication at the water‒air interface of a droplet. (A) SEM micrograph of a fibre pulled and dried on mica. (B) SEM micrograph of a different fibre at higher magnification showing evidence of a striated texture indicative of a microfibrillar internal structure. (C) SEM micrograph of the same fibre showing detail in another area of nanofibrils (D), nanofibril bundles joining (E), and additional details of the bundle (F) and detail of the individual nanofibrils (G). The scale bars in (B) are 100 µm. 5 µm in image (C, E), 1 µm in (F, G) and 500 nm in image (D).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/ffb8b2517c07ba8c95a26a92.png"},{"id":53105004,"identity":"c1f814e4-4196-46c2-8f8a-02f865cdaee1","added_by":"auto","created_at":"2024-03-20 15:58:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308897,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis Fibre XRD patterns were obtained for different fibres formed under various reeling speeds. The white arrow indicates the reflection assigned to the hexagonal packing of the solenoid units (ca. 16 Å), and the black arrows indicate typical Silk-II reflections (ca. 9.5 and 4.5 Å) (A). Average single-fibre tensile test results for fibres produced at different reeling speeds (B) and the elastic modulus, E (C), tensile strength (D), strain at break (E) and toughness (F) of the fibres. The error bars in B-F represent 1.5 standard errors.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/c7164374764586d71f5d76c7.png"},{"id":53105002,"identity":"7a997588-889a-4251-a9ce-48804dd551e5","added_by":"auto","created_at":"2024-03-20 15:58:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1487866,"visible":true,"origin":"","legend":"\u003cp\u003ePhotograph of several droplets of NLSF being simultaneously pulled (A). Photograph of a fabricated nonwoven multiply mat (B). Photograph of a silk-like fibre composite with magnetite nanoparticles interacting with a tungsten magnet (C). Maximum intensity projection along Z-stacks obtained via confocal fluorescence of an \u003cem\u003eE. coli\u003c/em\u003e/silk-like fibre composite with bacteria producing GFP in green and silk protein intrinsic fluorescence in red (D). Color-enhanced SEM image of the \u003cem\u003eE. coli\u003c/em\u003e-silk-like fibre composite showing integrated \u003cem\u003eE. coli\u003c/em\u003ecells (green) within the nanofibrillar bundles (red) (E); scale bars are 1 mm (B), 50 µm (D) and 4 µm (E).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/02aa0e33aad4b1aee85e7453.png"},{"id":53105540,"identity":"2cc304b4-ac9c-4219-8585-57d26517a548","added_by":"auto","created_at":"2024-03-20 16:06:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3562228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/91290337-f22f-4ae8-ba32-03bf93657e51.pdf"},{"id":53104997,"identity":"1679c511-0bb4-40cd-913f-74c2c827c354","added_by":"auto","created_at":"2024-03-20 15:58:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15876877,"visible":true,"origin":"","legend":"\u003cp\u003eESI\u003c/p\u003e","description":"","filename":"BiomimeticSilkFibreAssemblyMimickingNaturesPultrusionProcessSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/e4a6477e42ed2f9cb6be2292.docx"},{"id":53105007,"identity":"f5cdb59b-9831-4f9f-8154-f22db45b48e9","added_by":"auto","created_at":"2024-03-20 15:58:13","extension":"gif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":120142056,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 1\u003c/p\u003e","description":"","filename":"Video1.gif","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/b9d39e9b1a8dff1deffe0544.gif"},{"id":53105010,"identity":"502ddcd4-f62f-48d9-a775-7fb9af1a60ed","added_by":"auto","created_at":"2024-03-20 15:58:16","extension":"mov","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":292439317,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 2\u003c/p\u003e","description":"","filename":"Video2.mov","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/4bb295630b027db458cac334.mov"},{"id":53105008,"identity":"37a0af98-d011-44a0-a15e-b653764bcc92","added_by":"auto","created_at":"2024-03-20 15:58:13","extension":"mov","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":156001528,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 3\u003c/p\u003e","description":"","filename":"Video3.mov","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/a8048ce1ff946173bad4c973.mov"},{"id":53105001,"identity":"aa0daf6a-d188-4886-85d0-8bd2702a43a1","added_by":"auto","created_at":"2024-03-20 15:58:11","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14778267,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 4\u003c/p\u003e","description":"","filename":"Video4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/28cdda48851b32145ed28a59.mp4"},{"id":53105005,"identity":"0ae721af-7824-4f94-83d4-3be087fcb0cd","added_by":"auto","created_at":"2024-03-20 15:58:13","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1067908,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 5\u003c/p\u003e","description":"","filename":"Video5.avi","url":"https://assets-eu.researchsquare.com/files/rs-4130861/v1/39522f0ee041e1ee5315175a.avi"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBiomimetic Silk Fibre Assembly: Mimicking Nature's Pultrusion Process\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSilk fibroin has captivated researchers for generations owing to its remarkable mechanical properties and unique self-assembly behavior.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Unlike synthetic materials, silk fibroin undergoes a programmed transition from a liquid aqueous solution to a solid-state with minimal energy input, making it a fascinating subject of study in biomaterials research.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Recent investigations have provided further insight into the molecular self-assembly mechanism of \u003cem\u003eLepidopteran\u003c/em\u003e silk fibroin, revealing its nanofibrillar structure in the Silk-I configuration and the intricate interactions driving its solidification.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e At the macroscopic level, natural silk fibres are not extruded but rather pulled into shape through a process akin to pultrusion.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e This pultrusive mechanism, observed in silkworms and spiders, underscores the biomechanical sophistication of silk production in nature.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eReconstituted silk fibroin (RSF), although often used as a precursor in biomimetic silk material research, has several inherent limitations due to its reduced molecular weight and the lack of the N-terminal domain (NTD) responsible for pH-controlled supramolecular assembly.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e As a result, RSF requires nonnative conditions, such as organic solvents,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e coagulation baths,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e or prepolymerized aggregates,\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e for fibre formation. However, these methods impose environmental stress on the protein and deviate from the natural spinning process, where only minimal energy intake is required to fabricate the silk fibre.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eInterestingly, both silk fibroin and spidroins exhibit surface-active properties, rapidly forming elastic films at the water‒air interface.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Although the surface activity of native or native-like fibroin (SF) materials has been underexplored, recent insights into the structural dynamics of proteins suggest that the water\u0026ndash;air interface plays a crucial role in directing assembly in vitro.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e By leveraging this behavior, our aim was to fabricate silk-like fibres with enhanced control and efficiency.\u003c/p\u003e \u003cp\u003eIn this study, we propose a novel approach to silk fibre fabrication that harnesses the interfacial self-assembly of silk fibroin and its sensitivity to stresses. The method was developed from observations made while working with dilute native-like silk fibroin (NLSF) solution (see ST 1 and Figures S1 and S2). In brief, fibroin molecules adsorb at the water‒air interface, and by applying perpendicular extension, we induce a strain field. Pulling the film in this way promotes the more ordered aggregation of solenoid units via lateral interactions, and subsequent denaturation leads to fibre formation. Our proposed molecular-level mechanism is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Video 1). We believe this method offers a promising alternative to conventional methods, allowing for the production of silk-like fibres under more physiologically relevant conditions. Similar methods have also been reported for recombinant spidroin systems\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e but without demonstrating the critical role of the water-air interface in driving assembly or scalability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, our study seeks to deepen our understanding of silk fibroin assembly and pave the way for the development of biomimetic materials with tailored properties and applications. By bridging the gap between fundamental research and practical application, we aim to unlock the full potential of silk fibroin as a versatile biomaterial.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInterfacial assembly of the protein at the water‒air interface\u003c/h2\u003e \u003cp\u003eWith respect to the model, the surface assembly properties of the proteins were studied. Although the concentration is known to play a role in the interfacial properties of proteins, given the high complexity of the system and for simplicity, this work used only a single concentration, ca. 6 mg/mL, and studied the effect of pH on protein assembly at the water‒air interface. Therefore, the first experiment was to determine the adsorption behaviour of the proteins at the water‒air interface and the effect of pH. To do this, interfacial surface tension (IFT) analysis was performed using a Wilhelmy plate. In this experiment, the force that a platinum plate is subjected to when wetted with a liquid is related to surface tension Eq.\u0026nbsp;1:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }=\\frac{F}{L\\text{cos}\\theta }\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEquation 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }\\)\u003c/span\u003e\u003c/span\u003eis the interfacial surface tension, L is the length of the plate, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\theta\\)\u003c/span\u003e\u003c/span\u003e is the wetting angle. The advantage of this system is that it allows for the measurement of adsorption kinetics on long timescales without suffering as much from evaporative effects. Inspired by the natural pH gradient within the insect and our own observations, only pH values of 8, 7 and 6 were used. Figure S3 shows the results from these experiments. In all the cases, very rapid adsorption was observed, with all the experiments showing a reduction in IFT even at the beginning of the experiment; the IFT of pure water was 72 mN/m. Despite the similar behaviors of the samples at pH 6 and 7, the protein absorbs to the interface much more quickly at high pH, with an apparent lower IFT value. Notably, the observed values agree with the RSF values observed at similar concentrations.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo some extent, these experiments verify the observations of DLS,\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e where the proteins had smaller diameters; hence, faster diffusion at higher pH values occurred. This indicates that the protein at pH 8 has a faster adsorption to the interface due to faster diffusion speeds under these conditions. Nevertheless, the difference in the IFT itself is more nuanced. It is possible to argue that there might be changes in the total accessible surface area of the protein, whereby at pH 8, the protein exists as a monomeric unit, with a greater likelihood of forming a different type of oligomeric unit as the pH decreases. Oligomerization or aggregation would then reduce the total accessible surface area of the protein. Because the concentration was constant, such a reduction in the IFT can be described by Eq.\u0026nbsp;2 in the SI.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Further discussion and observations can be found in ST 2, describing more in-depth observations of a solid-to-solid transformation driven by stress; see Video 2 and Figures S4 and S5.\u003c/p\u003e \u003cp\u003eTo characterize the morphology of the protein film at the water‒air interface, we used confocal laser fluorescence microscopy (CLFM). Here, intrinsic fluorescence was used with no labeling to avoid changes in assembly behaviour by incorporating fluorescent labels, which commonly react with free primary amines or carboxylate groups (i.e., Lys or Asp/Glu residues), which are present only at terminal domains or linker units. This finding is particularly relevant given the important role of the NTD in driving assembly. In these experiments, approximately 10 \u0026micro;L of solution equilibrated at either pH 8 or 6 was placed in individual wells and left to age for approximately 5 h before observing the air/water interface using a long working distance objective (x10). The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B. Despite the films being of similar thickness (10\u0026ndash;20 \u0026micro;m, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), the relative fluorescence intensity of the protein at pH 6 was greater than that at other pH values, which could indicate an aggregation-induced emission (AIE) process.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Freely rotating Tyr (mainly) might be locked in a single rotameric state, enhancing the fluorescence of these multimers/aggregates against the protein in solution. These observations support our hypothesis of a higher degree of order/oligomerization at lower pH. However, a high degree of order is also imparted by the geometrical restriction of the interface itself and the elongated nature of the protein, which also produces a significant signal at higher pH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the mechanics of the films, they were subjected to dynamic microindentation measurements. A small indenter was gently placed in contact with the film and later oscillated, with small oscillation amplitudes, at a range of frequencies (0.1\u0026ndash;10 Hz). Using this method, we determined the viscoelastic response in terms of the separate elastic and viscous components of the same material. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F, with the sample pH shown in the right lower corner. In these experiments, the measurements are reported as stiffness (K) against frequency, with stiffness calculated as the measured force divided by the indentation depth, or half the oscillation amplitude. K\u0026rsquo; and K\u0026rdquo; refer to the elastic and viscous components, respectively. As expected, at pH 6, the material exhibited a relatively higher K\u0026rsquo; (160\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mN/m) than did its counterparts at pH 7 or 8 (60\u0026thinsp;\u0026plusmn;\u0026thinsp;2 and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mN/m, respectively); these two pH values exhibited little difference. Once again, we propose that the switch-like behavior occurs at pH values just below 7.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e At lower pH, the nature of the protein in solution is likely oligomeric, driven by NTD interactions; hence, the effective MW of the system increases, increasing the total cohesiveness, which translates to a higher modulus.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiomimetic silk-like fibre fabrication and characterization\u003c/h3\u003e\n\u003cp\u003eTaken together, these observations indicate that an elastic yet dynamic film forms very rapidly at the water/air interface, and upon extension of the strain/stress applied by pulling, it is possible to assemble insoluble silk-like fibres. For simplicity and because the film was more resilient at pH 6, this condition was used and exploited to produce multimeter long single fibres by continuously pulling the formed fibre using the method depicted in Video 3 and simplified in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Simply by depositing small droplets onto standard plastic petri dishes, it was possible to pull fibres at different reeling speeds. The achieved reeling speeds ranged from 1.8 mm/s to approximately 53 mm/s. However, the fibre formation was less stable at higher speeds. We hypothesize that there is competition between film formation and protein depletion through the formation of fibres. However, the coverage range contains the natural silkworm spinning speeds estimated to be between 10 and 30 mm/s.\u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing this method, from droplets between 50 and 100 \u0026micro;L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), it was possible to collect several meters of single fibres (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). All the fibres produced presented a highly hierarchical morphology, with the main fibre formed by bundles of submicron fibres composed of smaller nanofibrils, as shown in Figure S2. The results obtained by SEM corroborate these observations and are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-G. The fibrillar network extends far from the location where the mature fibre emerges, as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, with the experimental observations shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. We were able to observe individual nanofibrils without much evidence of branching. In other words, the smaller observable fibrils seem to grow in one dimension and only interact by relatively weaker lateral interactions to form larger bundles. These observations are not only in line with the current understanding of natural silk fibre,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e but also predicted from our proposed fibre assembly model; Tyr residues limit the lateral docking of strands, much like the lateral docking of β-solenoids, facilitating the nanofibrillar interface but also facilitating other types of interactions, such as π-π or methyl-π interactions. It has been observed that native silk fibroin fibres can be exfoliated into increasingly thinner fibrils, ranging from 20\u0026ndash;100 nm bundles down to 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 nm and ultimately into an extended chain with a diameter of approximately 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 \u0026Aring;.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the interest of understanding the biological relevance of our method in the context of in vivo fibre production, we measured the forces that were required to pull these fibres. At steady-state. We measured a force of approximately 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mN at a reeling speed of 15.5 mm/s, well within the forces that the insect can exert.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e No significant differences in the forces were observed at the different speeds (Figure S6 A), indicating that by varying the reeling speeds, we were more likely to change the strain rate rather than the overall stress. Further chemical analysis of the fibres, beyond the nanoscopic similarity with natural silk fibres, showed a remarkable resemblance of the molecular structure, with the amide-I peak showing similar nominal β-sheet, β-turn and statistical coil compositions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Thus, soluble silk fibroin (silk-I) was transformed to the well-known insoluble conformation (silk-II) by just using pH control and mechanical stimulation. Notably, no significant differences were observed in the FTIR spectra when the reeling speed was varied.\u003c/p\u003e \u003cp\u003eTo further understand the effect of reeling speed at the molecular level, we conducted fibre X-ray diffraction (fXRD) experiments on the different fibres immediately after they were produced at different reeling speeds. In silk fibres, the protein adopts an extended chain conformation, wherein the hydrogen bond network is perpendicular to the long axis of the fibre. However, this conformation is drastically different from that of our Silk-I model, which has a β-solenoid structure. In the proposed fibre formation process, the solenoids would be aligned parallel to the water/air interface plane, and upon drawing, these would align with the long axis of the created filament. The solenoids are denatured/stretched upon application of a critical strain rate and stress, and the known Silk-II configuration emerges. Hence, we would expect to observe a transition from a dominating Silk-I structure to an increasingly more dominating and aligned Silk-II structure upon increasing the reeling speed. Indeed, this was observed going from a reeling speed of approximately 1.5 mm/s to approximately the maximum possible speed of approximately 52.7 mm/s. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, there is a reflection corresponding to approximately 17 \u0026Aring; at the lowest reeling speeds, which disappears as the reeling speed increases. We interpret this reflection as coming from the hexagonal packing of hydrated solenoid units, which upon reaching a critical strain rate are disrupted, prompting extension of the backbone and collapse of the chains in β-sheets, consistent with the prediction in our previous work.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFollowing these experiments, we conducted single-fibre tensile testing, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-F. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows the average stress‒strain curves obtained for the fibres produced at different reeling speeds; these curves already show noticeable differences that are more detailed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F. As one would expect, we observed optimal mechanical properties at a reeling speed that corresponded to the maximum natural spinning speeds (ca. 30 mm/s), with the mechanical properties decreasing thereafter. Briefly, the elastic modulus showed no significant differences from the lowest reeling speed of 1.8 mm/s up to 15.9 mm/s, with values ranging between 4 and 5 GPa; however, from 21 to 32.2 mm/s, we observed an increased modulus, with three increasing between 8 and 10 GPa, decreasing back to 6\u0026thinsp;\u0026plusmn;\u0026thinsp;1 GPa for the highest reeling speed (52.7 mm/s); see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC. The values of tensile strength followed a similar trend, with strength increasing for reeling speeds between 21 and 26.3 mm/s to almost 200 MPa for these speeds and decreasing monotonically as the reeling speed increased thereafter; see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. Interestingly, a slightly different trend was followed by the maximum strain and toughness values, where these values showed an obvious second maxima at the minimal reeling speed (1.8 mm/s). The maximum extensibility was approximately 6% for both 1.8 and 5.9 mm/s, decreasing to approximately 3% for reeling speeds from 10.7 to 21 mm/s, after which the extensibility increased again to 6\u0026thinsp;\u0026plusmn;\u0026thinsp;4% for the fibres produced at 26 mm/s, decreasing to approximately 3% for any of the faster reeling speeds (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Similarly, the same trend was observed for toughness (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Interestingly, the observed trends are different from the fibre diameter trends, which only showed a monotonic decrease from 6\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026micro;m to 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;m for fibres produced at 1.8 and 52.7 mm/s (see Figure S6 B). Notably, the best performance of our fibres replicates the properties shown by degummed cocoon fibres to a great extent, as reported in the literature.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Moreover, the overall results are readily explained by our fXRD data and indeed support our model. At the lowest reeling speeds, the relatively higher content of the Silk-I conformation would enable greater extensibility (and toughness) without necessarily enhancing other properties, such as the elastic modulus or tensile strength, as lower strain rates would also imply a lower orientation of the formed β-sheet crystallites. On the other hand, the observation of the overall properties showing a maximum at the peak of natural spinning speeds might indicate the formation of an optimal protein network architecture, where the balance of crystallite size, distribution and orientation maximizes the properties of the material, particularly its toughness. Although our system might not fully recapitulate the in vivo system, mainly due to concentration differences, it is notable that we observed an optimum at natural spinning speeds.\u003c/p\u003e \u003cp\u003eOur proposed fibre formation mechanism involves two separate steps: first, the oligomerization of NTD driven by pH reduction; second, the reconfiguration of the network and further denaturation of the protein fold driven by stress. In this sense, the oligomerization of NTD effectively reduces the degrees of freedom of the protein while also breaking the symmetry of the system. This first step promotes network formation and primes the protein for stress-driven assembly. The system we propose here recapitulates the two-step system, first by breaking the symmetry of the system by exposing it to an interface, becoming inherently asymmetric, and further driving the assembly by stress. Here, in purely aqueous solutions without any precipitants (salts or solvents) and by using high-quality protein feedstock, we were able to replicate the Silk-I to Silk-II transformation using relative speeds and forces that are accessible to the animal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to the fundamental implications of our observations, the presented method represents a facile, biomimetic process that allows for the easy and efficient fabrication of silk-like fibres. Its simplicity is amenable to the fabrication of several composite materials. For example, multiple fibres can be pulled simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) while moving the platform along the collector/mandrel to fabricate nonwoven mats (see Video 4 and Figure S7 for further explanation of the principle). Using standard ideas for the fabrication of orthotopically modified materials, multiple fibrous mats approximately 2 \u0026times; 4 cm in length were manufactured using approximately 500 \u0026micro;L of the protein solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMoreover, the process allows for the facile bottom-up incorporation of functionality into the produced fibres. In the past, functional silk fibres were formed by either modifying the surface chemistry of natural fibres\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e or directly feeding insects functional particles.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Here, by simply introducing functional particles into a low-viscosity protein solution, we were able to fabricate magnetic silk-like fibres with incorporated magnetite nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and Figure S8), as well as living composites with \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) transformed with green fluorescence protein (GFP) for use as fluorescent stimuli-responsive fibres. Despite the relatively large size of \u003cem\u003eE. coli\u003c/em\u003e, these bacteria were readily homogeneously incorporated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E), with strong evidence of preferential alignment of the elongated bacteria in the same direction as the fibrils (Video 5), as shown by the directional analysis of the images in Figure S9. We believe that this orientation is induced mainly by the confinement offered by the hierarchical nanofibrillar morphology of the fibres and not necessarily by the flow, as the forces used here are low. We are now attempting to further optimize and fabricate devices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this work, we present a novel methodology for the production of silk-like fibres that recapitulates many of the natural steps and requires minimal energy. This process exploits the rapid formation of a protein film at the water‒air interface, which upon stretching, undergoes crystallization. Although we only present results with NLSF, the process can likely be generalized to other surface-active, sticky proteins and polymers. On the other hand, these results reinforce our proposed self-assembly pathway where pH and shear promote fibre formation from a protein that belongs to the β-solenoid family. The applied stress/strain not only aligns the fibrillar protein but also, upon a critical stress/strain rate, promotes unfolding and extension of the protein backbone, allowing for extensive β-sheet formation. Using this simple process, multimeter long silk-like fibres were produced that exhibited native-like mechanical properties without further need for posttreatment. Moreover, the simplicity and versatility of this method allows for the facile bottom-up fabrication of a myriad of fibrous composites, demonstrated here by incorporating both magnetic nanoparticles and GFP-transformed \u003cem\u003eE. coli\u003c/em\u003e. Although the method is limited in throughput compared to electrospinning, it offers a cost-effective, minimal energy and soft method for the fabrication of microfibres. Overall, in this work, we present a novel material fabrication method that can likely be expanded beyond silk and offers unprecedented insight into the assembly mechanism of silkworm fibroin into macroscopic fibres.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to the following individuals and facilities for their contributions and assistance: J.C. Eloi from the Chemistry Imaging Facility. Katy Jepson from the Wolfson Bioimaging facility and Anna Slatanova for their assistance with interfacial analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;EPSRC National Productivity Investment Fund grant EP/R51245/XF (R.O.M.T. ).\u003c/p\u003e\n\u003cp\u003eEPSRC Doctoral Prize Fellowship at the University of Bristol grant EP/W524414/1 (R.O.M.T. ).\u003c/p\u003e\n\u003cp\u003eWellcome Trust grants 086906/Z/08/Z and 100917/Z/13/Z (N.S. and R.W.).\u003c/p\u003e\n\u003cp\u003eThe EIC Accelerator grant 947454 (N.S. and R.W.).\u003c/p\u003e\n\u003cp\u003eThe NIHR i4i Invention for Innovation award II-LB-0417-20005 (N.S. and R.W.).\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eEPSRC, grants EP/K035746/1 and EP/M028216/1 (TEM).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003eThe views expressed in this work are those of the author(s) and do not necessarily reflect those of the\u0026nbsp;National Institute of Health and Social Care (NIHR), the Department of Health and Social Care or any of their funding bodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: ROMT, SAD, LS, CH, NS, RW\u003c/p\u003e\n\u003cp\u003eMethodology: ROMT, SAD, CH, LS\u003c/p\u003e\n\u003cp\u003eInvestigation: ROMT, LS, JM, EW, FG.\u003c/p\u003e\n\u003cp\u003eVisualization: ROMT\u003c/p\u003e\n\u003cp\u003eFunding acquisition: SAD, NS, RW, ROMT\u003c/p\u003e\n\u003cp\u003eProject administration: ROMT, SAD\u003c/p\u003e\n\u003cp\u003eSupervision: SAD, NS, RW\u003c/p\u003e\n\u003cp\u003eResources: NS, RW, ROMT\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: ROMT, SAD\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: ROMT, NS, RW, LS, CH, SAD\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll the data are available in the main text or the supplementary materials. The raw data and relevant materials are available upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOmenetto FG, Kaplan DL (2010) New opportunities for an ancient material. Sci (80-) 329:528\u0026ndash;531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker AA, Holland C, Sutherland TD (2015) More than one way to spin a crystallite: multiple trajectories through liquid crystallinity to solid silk. \u003cem\u003eProc. R. Soc. B Biol. Sci.\u003c/em\u003e 282\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparkes J, Holland C (2019) The Energy Requirements for Flow-Induced Solidification of Silk. 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Int J Biol Macromol 63:205\u0026ndash;209\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Bristol","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biomimetic, silk, fibres, hierarchical biomaterials","lastPublishedDoi":"10.21203/rs.3.rs-4130861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4130861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmong the best natural structural materials, silks have remarkable properties due to their hierarchical structure. The silk proteins from spiders or caterpillars, despite being distinct Classes, are produced by similar mechanisms with conserved features. They are stored as aqueous liquid solutions that undergo irreversible liquid-to-solid transformations driven by different stimuli, primarily pH and shear strain. This transformation has attracted the attention of many researchers aiming to replicate this apparently facile process. However, most biomimetic assembly processes that have been developed rely on extrusion-based technologies or flow-focusing microfluidic devices, typically using coagulating baths with unnatural solvent conditions. These synthetic processing strategies differ substantially from natural, all-aqueous, pultrusion-based fibre production and increase the overall energy input required to drive the transformation. In contrast, we observe that native-like silk fibroin (NLSF) rapidly forms a highly viscoelastic film at the air\u0026ndash;water interface. This phenomenon is then exploited by applying an extensional strain field to produce multimeter silk-like fibres with observable coaligned nanofibrillar bundles. Our studies showed that the proteins undergo stress-induced denaturation, consistent with a model of hexagonal packing of β-solenoid units, at low pulling speeds, at which point the proteins switch to a β-sheet-rich structure as the speed increases. Moreover, the produced fibres showed optimal mechanical properties when the pulling speeds were near the maximum physiologically relevant speeds (ca. 30 mm/s). s pulled at 26.3 mm/s had an elastic modulus of 8\u0026thinsp;\u0026plusmn;\u0026thinsp;1 GPa and a toughness of 8\u0026thinsp;\u0026plusmn;\u0026thinsp;5 MJ/m2, which is commensurate with the mechanical performance of natural fibres. Moreover, the method demonstrated here is readily compatible with complex material fabrication under ambient conditions, opening up the possibility of facile incorporation of cells and biomolecules. Overall, the developed method replicates the natural pultrusion process entirely water-based and offers great potential for the future development of novel fibre-based composite materials.\u003c/p\u003e","manuscriptTitle":"Biomimetic Silk Fibre Assembly: Mimicking Nature's Pultrusion Process","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 15:58:05","doi":"10.21203/rs.3.rs-4130861/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3755196f-92df-49a5-a7bf-a4ba5d282931","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29631958,"name":"Biomaterials"}],"tags":[],"updatedAt":"2024-03-20T15:58:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-20 15:58:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4130861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4130861","identity":"rs-4130861","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}