Effect of water on Bombyx mori regenerated silk fibers and its application in modifying their mechanical properties

The effect of water on regenerated silkworm silk fibers has been studied and compared with that of water on natural silkworm silk fibers. Regenerated fibers are spun from an N-methylmorpholine-N-oxide (NMMO) fibroin solution through a wet-spinning process, leading to fibers with two distinct tensile behaviors, labeled as brittle and ductile, respectively. Regenerated fibers show a significant contraction when immersed in water. Contraction increases further after drying. In contrast, natural silkworm silk fibers show a negligible contraction when submerged in water. Regenerated fibers tested in water are considerably more compliant than samples tested in air, though their stiffness and tensile strength are significantly reduced. It has been shown that the tensile properties of brittle regenerated fibers can be modified by a wet-stretching process, which consists of deforming the fiber while immersed in water. Regenerated wet-stretched fibers always show a ductile behavior independent from their initial tensile behavior

Stability and activity of lactate dehydrogenase on biofunctional layers deposited by activated vapour silanization (AVS) and immersion silanization (IS).

The interaction between surfaces and biological elements, in particular, proteins is critical for the performance of biomaterials and biosensors. This interaction can be controlled by modifying the surface in a process known as biofunctionalization. In this work, the enzyme lactate dehydrogenase (LDH) is used to study the stability of the interaction between a functional protein and amine-functionalized surfaces. Two different functionalization procedures were compared: Activated Vapour Silanization (AVS) and Immersion Silanization (IS). Adsorption kinetics is shown to follow the Langmuir model for AVS-functionalized samples, while IS-functionalized samples show a certain instability if immersed in an aqueous medium for several hours. In turn, the enzymatic activity of LDH is preserved for longer times by using glutaraldehyde as crosslinker between the AVS biofunctional surface and the enzyme.pre-print966 K

The hidden link between supercontraction and mechanical behavior of spider silks

The remarkable properties of spider silks have stimulated an increasing interest in understanding the roles of their composition and processing, as well as in the mass-production of these fibers. Previously, the variability in the mechanical properties of natural silk fibers was a major drawback in the elucidation of their behavior, but the authors have found that supercontraction of these fibers allows one to characterize and reproduce the whole range of tensile properties in a consistent way. The purpose of this review is to summarize these findings. After a review of the pertinent mechanical properties, the role of supercontraction in recovering and tailoring the tensile properties is explained, together with an alignment parameter to characterize silk fibers. The concept of the existence of a mechanical ground state is also mentioned. These behaviors can be modeled, and two such models–at the molecular and macroscopic levels–are briefly outlined. Finally, the assessment of the existence of supercontraction in bio-inspired fibers is considered, as this property may have significant consequences in the design and production of artificial fibers

Polymeric fibers with tunable properties Lessons from spider silk

Making artificial fibers inspired in spider silks is considered as one of the milestones in the field of biomimetics. The interest is usually justified by the outstanding tensile properties of natural fibers, but it is usually overlooked that spider silk is endowed with a number of related properties – supercontraction, recovery and the existence of a ground state – that impart the material with additional desirable features, such as the possibility of tuning its mechanical behaviour. In this work we present a review on the experimental analysis and significance of these properties, stressing the contributions of our research group to the field. It is also demonstrated how the knowledge gained in the basic study of the natural material has been essential for the improvement of the properties exhibited by artificially processed bio-inspired silk fiber

Efecto de la Longitud de onda de la radiación UV sobre la seda de araña

En el presente trabajo se continúa el análisis de la influencia de la radiación UV sobre las propiedades mecánicas de las fibras de seda de araña. Para ello se ha empleado la seda producida por la glándula ampollácea mayor de la especie Argiope trifasciata y se ha estudiado el comportamiento en tracción simple de fibras de seda sometidas a diferentes tiempos de irradiación con luz UV de longitudes de onda de 254, 312 y 365 nm. Se ha encontrado que la radiación ultravioleta disminuye la tensión de rotura y la deformación de rotura modificando, en algunos casos, el aspecto general de las curvas tensión-deformación. Además se ha hecho uso de la radiación UV de longitud de onda de 254 nm para introducir daño en las fibras con objeto de realizar un análisis fractográfico sistemático. El estudio se complementa con la evaluación del efecto producido por la irradiación en el fenómeno de supercontracción

Material properties of evolutionary diverse spider silks described by variation in a single structural parameter

Spider major ampullate gland silks (MAS) vary greatly in material properties among species but, this variation is shown here to be confined to evolutionary shifts along a single universal performance trajectory. This reveals an underlying design principle that is maintained across large changes in both spider ecology and silk chemistry. Persistence of this design principle becomes apparent after the material properties are defined relative to the true alignment parameter, which describes the orientation and stretching of the protein chains in the silk fiber. Our results show that the mechanical behavior of all Entelegynae major ampullate silk fibers, under any conditions, are described by this single parameter that connects the sequential action of three deformation micromechanisms during stretching: stressing of protein-protein hydrogen bonds, rotation of the ?-nanocrystals and growth of the ordered fraction. Conservation of these traits for over 230 million years is an indication of the optimal design of the material and gives valuable clues for the production of biomimetic counterparts based on major ampullate spider silk

The variability and interdependence of spider viscid line tensile properties

True stress-true strain curves of naturally spun viscid line fibers retrieved directly from the spiral of orb-webs built by Argiope trifasciata spiders were measured using a novel methodology. This new procedure combines a method for removing the aqueous coating of the fibers and a technique that allows the accurate measurement of their cross sectional area. Comparison of the tensile behaviour of different samples indicates that naturally spun viscid lines show a large variability, comparable to that of other silks, such as major ampullate gland silk and silkworm silk. Nevertheless, application of a statistical analysis allowed identifying two independent parameters that underlie the variability and characterize the observed range of true stress-true strain curves. Combination of this result with previous mechanical and microstructural data suggested the assignment of these two independent effects to the degree of alignment of the protein chains and to the local relative humidity which, in turn, depends on the composition of the viscous coating and on the external environmental conditions

Conduits based on the combination of hyaluronic acid and silk fibroin: Characterization, in vitro studies and in vivo biocompatibility

[EN] We address the production of structures intended as conduits made from natural biopolymers, capable of promoting the regeneration of axonal tracts. We combine hyaluronic acid (HA) and silk fibroin (SF) with the aim of improving mechanical and biological properties of HA. The results show that SF can be efficiently incorporated into the production process, obtaining conduits with tubular structure with a matrix of HA-SF blend. HA-SF has better mechanical properties than sole HA, which is a very soft hydrogel, facilitating manipulation. Culture of rat Schwann cells shows that cell adhesion and proliferation are higher than in pure HA, maybe due to the binding motifs contributed by the SF protein. This increased proliferation accelerates the formation of a tight cell layer, which covers the inner channel surface of the HA-SF tubes. Biocompatibility of the scaffolds was studied in immunocompetent mice. Both HA and HA-SF scaffolds were accepted by the host with no residual immune response at 8 weeks. New collagen extracellular matrix and new blood vessels were visible and they were present earlier when SF was present. The results show that incorporation of SF enhances the mechanical properties of the materials and results in promising biocompatible conduits for tubulization strategies.The authors acknowledge financing from the Spanish Ministry of Economy and Competitiveness through grants RTI2018-095872-B-C22/ERDF, DPI2015-72863-EXP, MAT2016-79832-R, MAT2016-76847-R and Community of Madrid through grant Neurocentro-B2017/BMD-3760. FGR acknowledges scholarship FPU16/01833 of the Spanish Ministry of Education, Culture and Sports. We thank the Electron Microscopy Service at the UPV, where the FESEM images were obtainedGisbert-Roca, F.; Lozano Picazo, P.; Pérez-Rigueiro, J.; Guinea Tortuero, GV.; Monleón Pradas, M.; Martínez-Ramos, C. (2020). Conduits based on the combination of hyaluronic acid and silk fibroin: Characterization, in vitro studies and in vivo biocompatibility. International Journal of Biological Macromolecules. 148:378-390. https://doi.org/10.1016/j.ijbiomac.2020.01.149S378390148Fawcett, J. W., & Asher, R. . (1999). The glial scar and central nervous system repair. Brain Research Bulletin, 49(6), 377-391. doi:10.1016/s0361-9230(99)00072-6Koeppen, A. H. (2004). Wallerian degeneration: history and clinical significance. Journal of the Neurological Sciences, 220(1-2), 115-117. doi:10.1016/j.jns.2004.03.008Hall, S. (2005). The response to injury in the peripheral nervous system. The Journal of Bone and Joint Surgery. British volume, 87-B(10), 1309-1319. doi:10.1302/0301-620x.87b10.16700Dubový, P., Klusáková, I., & Hradilová Svíženská, I. (2014). Inflammatory Profiling of Schwann Cells in Contact with Growing Axons Distal to Nerve Injury. BioMed Research International, 2014, 1-7. doi:10.1155/2014/691041Houschyar, K. S., Momeni, A., Pyles, M. N., Cha, J. Y., Maan, Z. N., Duscher, D., … Schoonhoven, J. van. (2016). The Role of Current Techniques and Concepts in Peripheral Nerve Repair. Plastic Surgery International, 2016, 1-8. doi:10.1155/2016/4175293Tian, L., Prabhakaran, M. P., & Ramakrishna, S. (2015). Strategies for regeneration of components of nervous system: scaffolds, cells and biomolecules. Regenerative Biomaterials, 2(1), 31-45. doi:10.1093/rb/rbu017Kehoe, S., Zhang, X. F., & Boyd, D. (2012). FDA approved guidance conduits and wraps for peripheral nerve injury: A review of materials and efficacy. Injury, 43(5), 553-572. doi:10.1016/j.injury.2010.12.030Collins, M. N., & Birkinshaw, C. (2013). Hyaluronic acid based scaffolds for tissue engineering—A review. Carbohydrate Polymers, 92(2), 1262-1279. doi:10.1016/j.carbpol.2012.10.028Cowman, M. K., & Matsuoka, S. (2005). Experimental approaches to hyaluronan structure. Carbohydrate Research, 340(5), 791-809. doi:10.1016/j.carres.2005.01.022Liang, Y., Walczak, P., & Bulte, J. W. M. (2013). The survival of engrafted neural stem cells within hyaluronic acid hydrogels. Biomaterials, 34(22), 5521-5529. doi:10.1016/j.biomaterials.2013.03.095Wang, T.-W., & Spector, M. (2009). Development of hyaluronic acid-based scaffolds for brain tissue engineering. Acta Biomaterialia, 5(7), 2371-2384. doi:10.1016/j.actbio.2009.03.033Ma, J., Tian, W.-M., Hou, S.-P., Xu, Q.-Y., Spector, M., & Cui, F.-Z. (2007). An experimental test of stroke recovery by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model. Biomedical Materials, 2(4), 233-240. doi:10.1088/1748-6041/2/4/005Tian, W. M., Hou, S. P., Ma, J., Zhang, C. L., Xu, Q. Y., Lee, I. S., … Cui, F. Z. (2005). Hyaluronic Acid–Poly-D-Lysine-Based Three-Dimensional Hydrogel for Traumatic Brain Injury. Tissue Engineering, 11(3-4), 513-525. doi:10.1089/ten.2005.11.513Vilariño-Feltrer, G., Martínez-Ramos, C., Monleón-de-la-Fuente, A., Vallés-Lluch, A., Moratal, D., Barcia Albacar, J. A., & Monleón Pradas, M. (2016). Schwann-cell cylinders grown inside hyaluronic-acid tubular scaffolds with gradient porosity. Acta Biomaterialia, 30, 199-211. doi:10.1016/j.actbio.2015.10.040Ortuño-Lizarán, I., Vilariño-Feltrer, G., Martínez-Ramos, C., Pradas, M. M., & Vallés-Lluch, A. (2016). Influence of synthesis parameters on hyaluronic acid hydrogels intended as nerve conduits. Biofabrication, 8(4), 045011. doi:10.1088/1758-5090/8/4/045011Vepari, C., & Kaplan, D. L. (2007). Silk as a biomaterial. Progress in Polymer Science, 32(8-9), 991-1007. doi:10.1016/j.progpolymsci.2007.05.013Murphy, A. R., & Kaplan, D. L. (2009). Biomedical applications of chemically-modified silk fibroin. Journal of Materials Chemistry, 19(36), 6443. doi:10.1039/b905802hSofia, S., McCarthy, M. B., Gronowicz, G., & Kaplan, D. L. (2000). Functionalized silk-based biomaterials for bone formation. Journal of Biomedical Materials Research, 54(1), 139-148. doi:10.1002/1097-4636(200101)54:13.0.co;2-7Altman, G. H., Diaz, F., Jakuba, C., Calabro, T., Horan, R. L., Chen, J., … Kaplan, D. L. (2003). Silk-based biomaterials. Biomaterials, 24(3), 401-416. doi:10.1016/s0142-9612(02)00353-8Horan, R. L., Antle, K., Collette, A. L., Wang, Y., Huang, J., Moreau, J. E., … Altman, G. H. (2005). In vitro degradation of silk fibroin. Biomaterials, 26(17), 3385-3393. doi:10.1016/j.biomaterials.2004.09.020Chi, N.-H., Yang, M.-C., Chung, T.-W., Chou, N.-K., & Wang, S.-S. (2013). Cardiac repair using chitosan-hyaluronan/silk fibroin patches in a rat heart model with myocardial infarction. Carbohydrate Polymers, 92(1), 591-597. doi:10.1016/j.carbpol.2012.09.012Chi, N.-H., Yang, M.-C., Chung, T.-W., Chen, J.-Y., Chou, N.-K., & Wang, S.-S. (2012). Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model. Biomaterials, 33(22), 5541-5551. doi:10.1016/j.biomaterials.2012.04.030Yang, M.-C., Chi, N.-H., Chou, N.-K., Huang, Y.-Y., Chung, T.-W., Chang, Y.-L., … Wang, S.-S. (2010). The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin – Hyaluronic acid cardiac patches. Biomaterials, 31(5), 854-862. doi:10.1016/j.biomaterials.2009.09.096Zhou, J., Zhang, B., Liu, X., Shi, L., Zhu, J., Wei, D., … He, D. (2016). Facile method to prepare silk fibroin/hyaluronic acid films for vascular endothelial growth factor release. Carbohydrate Polymers, 143, 301-309. doi:10.1016/j.carbpol.2016.01.023Yan, S., Li, M., Zhang, Q., & Wang, J. (2013). Blend films based on silk fibroin/hyaluronic acid. Fibers and Polymers, 14(2), 188-194. doi:10.1007/s12221-013-0188-2Foss, C., Merzari, E., Migliaresi, C., & Motta, A. (2012). Silk Fibroin/Hyaluronic Acid 3D Matrices for Cartilage Tissue Engineering. Biomacromolecules, 14(1), 38-47. doi:10.1021/bm301174xJaipaew, J., Wangkulangkul, P., Meesane, J., Raungrut, P., & Puttawibul, P. (2016). Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues. Materials Science and Engineering: C, 64, 173-182. doi:10.1016/j.msec.2016.03.063Fan, Z., Zhang, F., Liu, T., & Zuo, B. Q. (2014). Effect of hyaluronan molecular weight on structure and biocompatibility of silk fibroin/hyaluronan scaffolds. International Journal of Biological Macromolecules, 65, 516-523. doi:10.1016/j.ijbiomac.2014.01.058Chung, T.-W., & Chang, Y.-L. (2010). Silk fibroin/chitosan–hyaluronic acid versus silk fibroin scaffolds for tissue engineering: promoting cell proliferations in vitro. Journal of Materials Science: Materials in Medicine, 21(4), 1343-1351. doi:10.1007/s10856-009-3876-0Garcia-Fuentes, M., Meinel, A. J., Hilbe, M., Meinel, L., & Merkle, H. P. (2009). Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering. Biomaterials, 30(28), 5068-5076. doi:10.1016/j.biomaterials.2009.06.008Raia, N. R., Partlow, B. P., McGill, M., Kimmerling, E. P., Ghezzi, C. E., & Kaplan, D. L. (2017). Enzymatically crosslinked silk-hyaluronic acid hydrogels. Biomaterials, 131, 58-67. doi:10.1016/j.biomaterials.2017.03.046Yan, S., Zhang, Q., Wang, J., Liu, Y., Lu, S., Li, M., & Kaplan, D. L. (2013). Silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue reconstruction. Acta Biomaterialia, 9(6), 6771-6782. doi:10.1016/j.actbio.2013.02.016Garcia-Fuentes, M., Giger, E., Meinel, L., & Merkle, H. P. (2008). The effect of hyaluronic acid on silk fibroin conformation. Biomaterials, 29(6), 633-642. doi:10.1016/j.biomaterials.2007.10.024Hu, X., Lu, Q., Sun, L., Cebe, P., Wang, X., Zhang, X., & Kaplan, D. L. (2010). Biomaterials from Ultrasonication-Induced Silk Fibroin−Hyaluronic Acid Hydrogels. Biomacromolecules, 11(11), 3178-3188. doi:10.1021/bm1010504Ren, Y.-J., Zhou, Z.-Y., Liu, B.-F., Xu, Q.-Y., & Cui, F.-Z. (2009). Preparation and characterization of fibroin/hyaluronic acid composite scaffold. International Journal of Biological Macromolecules, 44(4), 372-378. doi:10.1016/j.ijbiomac.2009.02.004Cazzaniga, A., Ballin, A., & Brandt, F. (2008). Hyaluronic acid gel fillers in the management of facial aging. Clinical Interventions in Aging, Volume 3, 153-159. doi:10.2147/cia.s2135Sun, S.-F., Chou, Y.-J., Hsu, C.-W., & Chen, W.-L. (2009). Hyaluronic acid as a treatment for ankle osteoarthritis. Current Reviews in Musculoskeletal Medicine, 2(2), 78-82. doi:10.1007/s12178-009-9048-5Yucel, T., Lovett, M. L., & Kaplan, D. L. (2014). Silk-based biomaterials for sustained drug delivery. Journal of Controlled Release, 190, 381-397. doi:10.1016/j.jconrel.2014.05.059Bettinger, C. J., Cyr, K. M., Matsumoto, A., Langer, R., Borenstein, J. T., & Kaplan, D. L. (2007). Silk Fibroin Microfluidic Devices. Advanced Materials, 19(19), 2847-2850. doi:10.1002/adma.200602487Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676-682. doi:10.1038/nmeth.2019Taddei, P., Pavoni, E., & Tsukada, M. (2016). Stability toward alkaline hydrolysis ofB.morisilk fibroin grafted with methacrylamide. Journal of Raman Spectroscopy, 47(6), 731-739. doi:10.1002/jrs.4892Perea, G. B., Solanas, C., Marí-Buyé, N., Madurga, R., Agulló-Rueda, F., Muinelo, A., … Pérez-Rigueiro, J. (2016). The apparent variability of silkworm ( Bombyx mori ) silk and its relationship with degumming. European Polymer Journal, 78, 129-140. doi:10.1016/j.eurpolymj.2016.03.012Hu, M., Sabelman, E. E., Tsai, C., Tan, J., & Hentz, V. R. (2000). Improvement of Schwann Cell Attachment and Proliferation on Modified Hyaluronic Acid Strands by Polylysine. Tissue Engineering, 6(6), 585-593. doi:10.1089/10763270050199532Monteiro, G. A., Fernandes, A. V., Sundararaghavan, H. G., & Shreiber, D. I. (2011). Positively and Negatively Modulating Cell Adhesion to Type I Collagen Via Peptide Grafting. Tissue Engineering Part A, 17(13-14), 1663-1673. doi:10.1089/ten.tea.2008.0346Ude, A. U., Eshkoor, R. A., Zulkifili, R., Ariffin, A. K., Dzuraidah, A. W., & Azhari, C. H. (2014). Bombyx mori silk fibre and its composite: A review of contemporary developments. Materials & Design, 57, 298-305. doi:10.1016/j.matdes.2013.12.052Atkins, E. D. T., Phelps, C. F., & Sheehan, J. K. (1972). The conformation of the mucopolysaccharides. Hyaluronates. Biochemical Journal, 128(5), 1255-1263. doi:10.1042/bj128125

Cortical Reshaping and Functional Recovery Induced by Silk Fibroin Hydrogels-Encapsulated Stem Cells Implanted in Stroke Animals

The restitution of damaged circuitry and functional remodeling of peri-injured areas constitute two main mechanisms for sustaining recovery of the brain after stroke. In this study, a silk fibroin-based biomaterial efficiently supports the survival of intracerebrally implanted mesenchymal stem cells (mSCs) and increases functional outcomes over time in a model of cortical stroke that affects the forepaw sensory and motor representations. We show that the functional mechanisms underlying recovery are related to a substantial preservation of cortical tissue in the first days after mSCs-polymer implantation, followed by delayed cortical plasticity that involved a progressive functional disconnection between the forepaw sensory (FLs1) and caudal motor (cFLm1) representations and an emergent sensory activity in peri-lesional areas belonging to cFLm1. Our results provide evidence that mSCs integrated into silk fibroin hydrogels attenuate the cerebral damage after brain infarction inducing a delayed cortical plasticity in the peri-lesional tissue, this later a functional change described during spontaneous or training rehabilitation-induced recovery. This study shows that brain remapping and sustained recovery were experimentally favored using a stem cell-biomaterial-based approach

Influencia de la radiación UV en las propiedades mecánicas y en el comportamiento en fractura de un polimero artificial bioinspirado en la seda de araña

En el presente trabajo se estudia la influencia de la radiación UV sobre las propiedades mecánicas y las superficies de fractura de un polímero artificial bioinspirado en la seda de araña. Las fibras de seda de araña constituyen un material enormemente atractivo ya que su elevada resistencia y deformabilidad lo convierten en el material con mayor trabajo hasta rotura de los conocidos hasta el momento. Además se ha encontrado que posee una elevada biocompatibilidad y un comportamiento biodegradable. Debido a estas excelentes propiedades se han dedicado importantes esfuerzos a intentar producir fibras inspiradas en la seda de araña. Fruto de estos esfuerzos es el polímero artificial estudiado en este trabajo. Dicho polímero presenta una secuencia de aminoácidos inspirada en la spidroína 1, que es una de las dos proteínas que conforman la seda de araña natural. Uno de los factores más perjudiciales para los polímeros es la radiación ultravioleta (UV), de presencia ubicua en aplicaciones al aire libre, ya que puede provocar la modificación de sus enlaces covalentes y, como consecuencia, modificar sus propiedades mecánicas. Para evaluar el efecto de la radiación UV sobre el material bioinspirado se ha estudiado el comportamiento a tracción simple de fibras sometidas a diferentes tiempos de irradiación con luz UV de longitud de onda de 254 nm. Se ha observado que la radiación UV de 254 nm modifica considerablemente las propiedades mecánicas de este material a tiempos de exposición elevados (a partir de 3 días de irradiación). Además se ha estudiado el comportamiento a fractura de este material cuando es irradiado con luz UV. Se ha observado que a medida que aumenta el tiempo de irradiación las superficies de fractura comienzan a ser cada vez más planas, obteniéndose un aspecto extremadamente especular para muestras irradiadas durante 16 día