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

The Spider Silk Standardization Initiative (S3I): A powerful tool to harness biological variability and to systematize the characterization of major ampullate silk fibers spun by spiders from suburban Sydney, Australia

The true stress-true strain curves of 11 Australian spider species from the Entelegynae lineage were tensile tested and classified based on the values of the alignment parameter, α*, in the framework of the Spider Silk Standardization Initiative (S3I). The application of the S3I methodology allowed the determination of the alignment parameter in all cases, and were found to range between α* = 0.03 and α* = 0.65. These data, in combination with previous results on other species included in the Initiative, were exploited to illustrate the potential of this approach by testing two simple hypotheses on the distribution of the alignment parameter throughout the lineage: (1) whether a uniform distribution may be compatible with the values obtained from the studied species, and (2) whether any trend may be established between the distribution of the α* parameter and phylogeny. In this regard, the lowest values of the α* parameter are found in some representatives of the Araneidae group, and larger values seem to be found as the evolutionary distance from this group increases. However, a significant number of outliers to this apparent general trend in terms of the values of the α* parameter are described

Unexpected high toughness of Samia cynthia ricini silk gut

Silk gut fibers were produced from the silkworm Samia cynthia ricini silk glands by the usual procedure of immersion in a mildly acidic solution and subsequent stretching. The morphology of the silk guts was assessed by scanning electron microscopy, and their microstructure was assessed by infrared spectroscopy and X-ray diffraction. It was found that both naturally spun and Samia silk guts share a common semicrystalline microstructure. The mechanical characterization of the silk guts revealed that these fibers show an elastomeric behavior when tested in water, and exhibit a genuine ground state to which the fiber may revert independently of its previous loading history. In spite of its large cross-sectional area compared with naturally spun silk fibers, Samia silk guts show values of work to fracture up to 160 MJ m, much larger than those of most of their natural counterparts, and establish a new record value for this parameter in silk guts

Axonal Guidance Using Biofunctionalized Straining Flow Spinning Regenerated Silk Fibroin Fibers as Scaffold

After an injury, the limited regenerative capacity of the central nervous system makes the reconnection and functional recovery of the affected nervous tissue almost impossible. To address this problem, biomaterials appear as a promising option for the design of scaffolds that promote and guide this regenerative process. Based on previous seminal works on the ability of regenerated silk fibroin fibers spun through the straining flow spinning (SFS) technique, this study is intended to show that the usage of functionalized SFS fibers allows an enhancement of the guidance ability of the material when compared with the control (nonfunctionalized) fibers. It is shown that the axons of the neurons not only tend to follow the path marked by the fibers, in contrast to the isotropic growth observed on conventional culture plates, but also that this guidance can be further modulated through the biofunctionalization of the material with adhesion peptides. Establishing the guidance ability of these fibers opens the possibility of their use as implants for spinal cord injuries, so that they may represent the core of a therapy that would allow the reconnection of the injured ends of the spinal cord.Unidad Docente de Biodiversidad, Ecología y EvoluciónFac. de Óptica y OptometríaTRUEMinisterio de Ciencia e Innovación de EspañaComunidad de Madrid (España)Banco Santander (España)Universidad Complutense de Madrid (España)pu

Design of a shape-adaptable silk fibroin biohybrid to overcome drug delivery barriers in the treatment of cerebral injuries

Glioblastoma (GBM) is the most common aggressive brain tumour in adults with a mean survival of 12-15 months. The standard treatment of GBM consists of the resection of the bulk tumour, followed by the implantation in the edge of non-resected brain area of a controlled release drug delivery system based on carmustine loaded wafers, and before the administration of radiotherapy and chemotherapy with temozolomide. This system overcomes the main barriers associated with conventional administration of drugs, such as the quick drug metabolization or clearance, the side effects in no target tissues, and especially for cerebral injuries, the presence of the blood-brain barrier (BBB) that hinders the uptake of the drug at therapeutic concentrations. However, these wafers have a modest benefit due to fast delivery kinetics, implant dislodgments for their lack of adaptability, and not overcome the chemoresistance observed in some patients. Here, we designed a shape-adaptable silk fibroin (SF) biohybrid for optimal control of drug delivery. This biohybrid was composed by an inner core of hydrogel contained into an external cover made of mats and fibres. The hydrogel would provide mechanical features like the brain and would adapt to the resection cavity; the mats would constitute a barrier against the leakage of the gelling SF solution, and the fibres would maintain the structure as a whole and would deform by the injection of the gelling solution, adapting to the resection cavity. Moreover, we tested the ability of this biohybrid to deliver doxorubicin (DOX), a chemotherapeutic molecule which use for central nervous system cancers has been limited by its poor penetration through the BBB. Our results demonstrated that the SF hydrogels had mechanical properties that matched with the brain, high porosity, stability against degradation in the short term, and they were sterilizable. It was possible to obtain fibres with high deformations in water and high performances in air, which are important for the easy handling of the posterior biohybrid material. The mats also showed high capability of deformation in water and a porous structure that would not hamper the drug release. Finally, the assembly of the three formats offered mechanical behaviour that was compatible with the brain tissue. Although fibres and mats resulted to be harmful for cells, the capability of sustained delivery from the hydrogels was demonstrated as well as the preservation of the stability and cytotoxic effect of DOX. New optimized fabrication methods for mats and fibres are needed to enhance the biocompatibility of the whole biohybrid, especially for biomedical applications. RESUMEN El glioblastoma (GBM) es el tumor cerebral agresivo más común en adultos con una supervivencia media de los pacientes de 12 a 15 meses. El tratamiento estándar del GBM consiste en la resección de la mayor parte del tumor, seguida de la implantación en el borde de la región cerebral no extraída de un sistema de administración de fármacos de liberación controlada basado en obleas cargadas con carmustina, y antes de la administración de radioterapia y quimioterapia con temozolomida. Este sistema supera las principales barreras asociadas a la administración convencional de fármacos, como son la rápida metabolización o eliminación del fármaco, los efectos secundarios en tejidos no diana, y especialmente para las lesiones cerebrales, la presencia de la barrera hematoencefálica (BBB, por su nombre en inglés) que dificulta la captación del fármaco a concentraciones terapéuticas. Sin embargo, estas obleas tienen un beneficio moderado debido a la rápida cinética de liberación del fármaco, el desprendimiento de los implantes por su falta de adaptabilidad, y por no superar los problemas de quimiorresistencia experimentada por algunos pacientes. En este trabajo, diseñamos un biohíbrido de fibroína de seda (SF, por su nombre en inglés) adaptable al entorno, compuesto por un núcleo interno de hidrogel contenido en una cubierta hecha de mallas y fibras. El hidrogel proporcionaría características mecánicas similares al cerebro y se adaptaría a la cavidad de resección; las mallas constituirían una barrera contra la fuga de la solución de SF en proceso de gelificación, y las fibras mantendrían la estructura del conjunto y se deformarían por la inyección de la solución gelificante, adaptándose a la cavidad de resección. Además, estudiamos la capacidad del biohíbrido para liberar doxorrubicina (DOX), una molécula quimioterapéutica cuyo uso para los cánceres del sistema nervioso central se ha visto limitado por su escasa penetración a través de la BBB. Nuestros resultados demostraron que los hidrogeles de SF tenían propiedades mecánicas similares a las del cerebro, alta porosidad, estabilidad contra la degradación a corto plazo y que eran esterilizables. Se logró obtener fibras con altas deformaciones en agua y altas prestaciones en aire, las cuales son importantes para facilitar el manejo del posterior material biohíbrido. Las mallas también mostraron una alta capacidad de deformación en agua y una estructura porosa que no dificultaría la liberación del fármaco. Finalmente, el ensamblaje de los tres formatos ofreció un comportamiento mecánico compatible con el tejido cerebral. Aunque las fibras y las mallas resultaron ser dañinas para las células, se pudo demostrar la capacidad de liberación sostenida a partir de los hidrogeles, así como la preservación de la estabilidad y el efecto citotóxico de la DOX. Es necesario implementar nuevos métodos de fabricación optimizados tanto para las mallas como para las fibras, con el fin de mejorar la biocompatibilidad de todo el biohíbrido, especialmente para aplicaciones biomédicas

New Semi-Biodegradable Materials from Semi-Interpenetrated Networks of Poly(epsilon-caprolactone) and Poly(ethyl acrylate)

[EN] Semi-degradable materials may have many applications. Here poly(ethyl acrylate) and poly(ϵ-caprolactone) were combined as semi-interpenetrated networks, and thoroughly characterized in terms of final composition, interactions between components, wettability, and mechanical properties. PCL modulates the mechanical properties of the PEA elastomeric network. Cultures of fibroblasts and adipose-tissue derived stem cells showed excellent biological performance of the materials. The results are relevant for applications seeking materials leaving a permanent supporting skeleton after the partial degradation, as in patches for cardiac regeneration or in abdominal wall meshes.The authors acknowledge funding through the European Commission FP7 project RECATABI (NMP3-SL-2009-229239), and from the Spanish Ministerio de Ciencia e Innovacion through projects MAT2011-28791-C03-02 and -03. Dr. J. C. Chachques (Hopital Europeen Georges Pompidou, Paris, France) and Drs. A. Bayes-Genis and C. Soler-Botija (Hospital Germans Trias i Pujol, Badalona, Spain) are thanked for kindly providing and expanding the ASCs employed in this study.Lozano Picazo, P.; Perez Garnes, M.; Martínez Ramos, C.; Vallés Lluch, A.; Monleón Pradas, M. (2015). New Semi-Biodegradable Materials from Semi-Interpenetrated Networks of Poly(epsilon-caprolactone) and Poly(ethyl acrylate). Macromolecular Bioscience. 15(2):229-240. https://doi.org/10.1002/mabi.201400331229240152Jawad, H., Ali, N. N., Lyon, A. R., Chen, Q. Z., Harding, S. E., & Boccaccini, A. R. (2007). Myocardial tissue engineering: a review. Journal of Tissue Engineering and Regenerative Medicine, 1(5), 327-342. doi:10.1002/term.46Caspi, O., Lesman, A., Basevitch, Y., Gepstein, A., Arbel, G., Habib, I. H. M., … Levenberg, S. (2007). Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells. Circulation Research, 100(2), 263-272. doi:10.1161/01.res.0000257776.05673.ffSteinhauser, M. L., & Lee, R. T. (2011). Regeneration of the heart. EMBO Molecular Medicine, 3(12), 701-712. doi:10.1002/emmm.201100175Atzet, S., Curtin, S., Trinh, P., Bryant, S., & Ratner, B. (2008). Degradable Poly(2-hydroxyethyl methacrylate)-co-polycaprolactone Hydrogels for Tissue Engineering Scaffolds. Biomacromolecules, 9(12), 3370-3377. doi:10.1021/bm800686hVenugopal, J. R., Prabhakaran, M. P., Mukherjee, S., Ravichandran, R., Dan, K., & Ramakrishna, S. (2011). Biomaterial strategies for alleviation of myocardial infarction. Journal of The Royal Society Interface, 9(66), 1-19. doi:10.1098/rsif.2011.0301Nair, L. S., & Laurencin, C. T. (2007). Biodegradable polymers as biomaterials. Progress in Polymer Science, 32(8-9), 762-798. doi:10.1016/j.progpolymsci.2007.05.017Shoichet, M. S. (2010). Polymer Scaffolds for Biomaterials Applications. Macromolecules, 43(2), 581-591. doi:10.1021/ma901530rCharlton, D. C., Peterson, M. G. E., Spiller, K., Lowman, A., Torzilli, P. A., & Maher, S. A. (2008). Semi-Degradable Scaffold for Articular Cartilage Replacement. Tissue Engineering Part A, 14(1), 207-213. doi:10.1089/ten.a.2006.0344Spiller, K. L., Holloway, J. L., Gribb, M. E., & Lowman, A. M. (2010). Design of semi-degradable hydrogels based on poly(vinyl alcohol) and poly(lactic-co-glycolic acid) for cartilage tissue engineering. Journal of Tissue Engineering and Regenerative Medicine, 5(8), 636-647. doi:10.1002/term.356Scholten, P. M., Ng, K. W., Joh, K., Serino, L. P., Warren, R. F., Torzilli, P. A., & Maher, S. A. (2011). A semi-degradable composite scaffold for articular cartilage defects. Journal of Biomedical Materials Research Part A, 97A(1), 8-15. doi:10.1002/jbm.a.33005Pérez Olmedilla, M., Garcia-Giralt, N., Pradas, M. M., Ruiz, P. B., Gómez Ribelles, J. L., Palou, E. C., & García, J. C. M. (2006). Response of human chondrocytes to a non-uniform distribution of hydrophilic domains on poly (ethyl acrylate-co-hydroxyethyl methacrylate) copolymers. Biomaterials, 27(7), 1003-1012. doi:10.1016/j.biomaterials.2005.07.030Rico, P., Hernández, J. C. R., Moratal, D., Altankov, G., Pradas, M. M., & Salmerón-Sánchez, M. (2009). Substrate-Induced Assembly of Fibronectin into Networks: Influence of Surface Chemistry and Effect on Osteoblast Adhesion. Tissue Engineering Part A, 15(11), 3271-3281. doi:10.1089/ten.tea.2009.0141Soria, J. M., Sancho-Tello, M., Esparza, M. A. G., Mirabet, V., Bagan, J. V., Monleón, M., & Carda, C. (2011). Biomaterials coated by dental pulp cells as substrate for neural stem cell differentiation. Journal of Biomedical Materials Research Part A, 97A(1), 85-92. doi:10.1002/jbm.a.33032Campillo-Fernandez, A. J., Pastor, S., Abad-Collado, M., Bataille, L., Gomez-Ribelles, J. L., Meseguer-Dueñas, J. M., … Ruiz-Moreno, J. M. (2007). Future Design of a New Keratoprosthesis. Physical and Biological Analysis of Polymeric Substrates for Epithelial Cell Growth. Biomacromolecules, 8(8), 2429-2436. doi:10.1021/bm0703012Campillo-Fernández, A. J., Unger, R. E., Peters, K., Halstenberg, S., Santos, M., Sánchez, M. S., … Kirkpatrick, C. J. (2009). Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation. Tissue Engineering Part A, 15(6), 1331-1341. doi:10.1089/ten.tea.2008.0146Veiga, D. D., Antunes, J. C., Gómez, R. G., Mano, J. F., Ribelles, J. L. G., & Soria, J. M. (2010). Three-Dimensional Scaffolds as a Model System for Neural and Endothelial ‘In Vitro’ Culture. Journal of Biomaterials Applications, 26(3), 293-310. doi:10.1177/0885328210365005Soria, J. M., Martínez Ramos, C., Salmerón Sánchez, M., Benavent, V., Campillo Fernández, A., Gómez Ribelles, J. L., … Barcia, J. A. (2006). Survival and differentiation of embryonic neural explants on different biomaterials. Journal of Biomedical Materials Research Part A, 79A(3), 495-502. doi:10.1002/jbm.a.30803Soria, J. M., Martínez Ramos, C., Bahamonde, O., García Cruz, D. M., Salmerón Sánchez, M., García Esparza, M. A., … Barcia, J. A. (2007). Influence of the substrate’s hydrophilicity on thein vitro Schwann cells viability. Journal of Biomedical Materials Research Part A, 83A(2), 463-470. doi:10.1002/jbm.a.31297Martínez-Ramos, C., Vallés-Lluch, A., Verdugo, J. M. G., Ribelles, J. L. G., Barcia Albacar, J. A., Orts, A. B., … Pradas, M. M. (2012). Channeled scaffolds implanted in adult rat brain. Journal of Biomedical Materials Research Part A, 100A(12), 3276-3286. doi:10.1002/jbm.a.34273Woodruff, M. A., & Hutmacher, D. W. (2010). The return of a forgotten polymer—Polycaprolactone in the 21st century. Progress in Polymer Science, 35(10), 1217-1256. doi:10.1016/j.progpolymsci.2010.04.002Dash, T. K., & Konkimalla, V. B. (2012). Poly-є-caprolactone based formulations for drug delivery and tissue engineering: A review. Journal of Controlled Release, 158(1), 15-33. doi:10.1016/j.jconrel.2011.09.064Ulery, B. D., Nair, L. S., & Laurencin, C. T. (2011). Biomedical applications of biodegradable polymers. Journal of Polymer Science Part B: Polymer Physics, 49(12), 832-864. doi:10.1002/polb.22259Middleton, J. C., & Tipton, A. J. (2000). Synthetic biodegradable polymers as orthopedic devices. Biomaterials, 21(23), 2335-2346. doi:10.1016/s0142-9612(00)00101-0Sperling, L. H. (1981). Interpenetrating Polymer Networks and Related Materials. doi:10.1007/978-1-4684-3830-7Más Estellés, J., Vidaurre, A., Meseguer Dueñas, J. M., & Castilla Cortázar, I. (2007). Physical characterization of polycaprolactone scaffolds. Journal of Materials Science: Materials in Medicine, 19(1), 189-195. doi:10.1007/s10856-006-0101-2Shafy, A., Fink, T., Zachar, V., Lila, N., Carpentier, A., & Chachques, J. C. (2012). Development of cardiac support bioprostheses for ventricular restoration and myocardial regeneration. European Journal of Cardio-Thoracic Surgery, 43(6), 1211-1219. doi:10.1093/ejcts/ezs480Sarac, A. S. (1999). Redox polymerization. Progress in Polymer Science, 24(8), 1149-1204. doi:10.1016/s0079-6700(99)00026-xYagci, C., & Yildiz, U. (2005). Redox polymerization of methyl methacrylate with allyl alcohol 1,2-butoxylate-block-ethoxylate initiated by Ce(IV)/HNO3 redox system. European Polymer Journal, 41(1), 177-184. doi:10.1016/j.eurpolymj.2004.08.008Diego, R. B., Olmedilla, M. P., Aroca, A. S., Ribelles, J. L. G., Pradas, M. M., Ferrer, G. G., & Sánchez, M. S. (2005). Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering. Journal of Materials Science: Materials in Medicine, 16(8), 693-698. doi:10.1007/s10856-005-2604-7García, A. J. (s. f.). Interfaces to Control Cell-Biomaterial Adhesive Interactions. Advances in Polymer Science, 171-190. doi:10.1007/12_071Dado, D., & Levenberg, S. (2009). Cell–scaffold mechanical interplay within engineered tissue. Seminars in Cell & Developmental Biology, 20(6), 656-664. doi:10.1016/j.semcdb.2009.02.001Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (2006). Matrix Elasticity Directs Stem Cell Lineage Specification. Cell, 126(4), 677-689. doi:10.1016/j.cell.2006.06.04

Conversión de la porina OmpF de Escherichia coli en una adhesina específica de péptidos amiloidogénicos

1 p.Las proteínas amiloides están presentes en entornos naturales, bien sea debido a su secreción por microorganismos o a las excretas de mamíferos afectados por enfermedades priónicas, suscitando preocupación creciente por su potencial infectividad y toxicidad en contextos como la microbiota intestinal o los suelos. Nuestro objetivo es diseñar, a partir de proteínas de la membrana externa de bacterias Gram-negativas, biosensores y adhesinas sintéticos específicos de amiloides, con vistas a su posible neutralización. La inserción de un segmento amiloidogénico del amiloide funcional bacteriano RepA-WH1 (1) en uno de los lazos extracelulares de la (abundante y no esencial) porina OmpF de Escherichia coli permitió a las bacterias unir en su superficie esa misma secuencia cuando se suministra en forma de péptido marcado con rodamina. Esta interacción homotípica es característica de los ensamblajes amiloides, y se asemeja a la responsable de la formación de pares RepA-RepA, mediados por el dominio amiloidogénico WH1, que actúan como inhibidores de la replicación prematura de plásmidos (2). Polifenoles conocidos por su actividad como inhibidores del ensamblaje de diversos amiloides impidieron el reconocimiento del péptido por parte de la porina sintética. Cuando la proteína RepA-WH1 completa (etiquetada en su extremo C-terminal con mCherry) se inmovilizó sobre portaobjetos de vidrio activados con N-hidroxisuccinimida, las bacterias que expresan la variante sintética de OmpF se unieron a dicha superficie funcionalizada, demostrando así el potencial de la amiloidogénesis como mediadora de la adhesión celular.1.Giraldo R. mSystems (2020) 5:e00553-20.2. Molina-García L, et al. Sci Rep (2016) 6:25425.AEI (RTI2018-094549-B-I00)Peer reviewe

Conversion of the OmpF porin into a device to gather amyloids on the E. coli outer membrane

13 p,.5 fig.-1 graph. abst.Protein amyloids are ubiquitous in natural environments. They typically originate from microbial secretions or spillages from mammals infected by prions, currently raising concerns about their infectivity and toxicity in contexts such as gut microbiota or soils. Exploiting the self-assembly potential of amyloids for their scavenging, here, we report the insertion of an amyloidogenic sequence stretch from a bacterial prion-like protein (RepA-WH1) in one of the extracellular loops (L5) of the abundant Escherichia coli outer membrane porin OmpF. The expression of this grafted porin enables bacterial cells to trap on their envelopes the same amyloidogenic sequence when provided as an extracellular free peptide. Conversely, when immobilized on a surface as bait, the full-length prion-like protein including the amyloidogenic peptide can catch bacteria displaying the L5-grafted OmpF. Polyphenolic molecules known to inhibit amyloid assembly interfere with peptide recognition by the engineered OmpF, indicating that this is compatible with the kind of homotypic interactions expected for amyloid assembly. Our study suggests that synthetic porins may provide suitable scaffolds for engineering biosensor and clearance devices to tackle the threat posed by pathogenic amyloids.This work has been financed with grant RTI2018-094549-B-I00 from the Spanish MCIN/AEI (10.13039/501100011033 and FEDER “A way to make Europe”) to R.G.Peer reviewe

Strategies for the Biofunctionalization of Straining Flow Spinning Regenerated Bombyx mori Fibers

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).High-performance regenerated silkworm (Bombyx mori) silk fibers can be produced efficiently through the straining flow spinning (SFS) technique. In addition to an enhanced biocompat-ibility that results from the removal of contaminants during the processing of the material, regenerated silk fibers may be functionalized conveniently by using a range of different strategies. In this work, the possibility of implementing various functionalization techniques is explored, including the production of fluorescent fibers that may be tracked when implanted, the combination of the fibers with enzymes to yield fibers with catalytic properties, and the functionalization of the fibers with cell-adhesion motifs to modulate the adherence of different cell lineages to the material. When considered globally, all these techniques are a strong indication not only of the high versatility of-fered by the functionalization of regenerated fibers in terms of the different chemistries that can be employed, but also on the wide range of applications that can be covered with these functionalized fibers

Axonal Guidance Using Biofunctionalized Straining Flow Spinning Regenerated Silk Fibroin Fibers as Scaffold

After an injury, the limited regenerative capacity of the central nervous system makes the reconnection and functional recovery of the affected nervous tissue almost impossible. To address this problem, biomaterials appear as a promising option for the design of scaffolds that promote and guide this regenerative process. Based on previous seminal works on the ability of regenerated silk fibroin fibers spun through the straining flow spinning (SFS) technique, this study is intended to show that the usage of functionalized SFS fibers allows an enhancement of the guidance ability of the material when compared with the control (nonfunctionalized) fibers. It is shown that the axons of the neurons not only tend to follow the path marked by the fibers, in contrast to the isotropic growth observed on conventional culture plates, but also that this guidance can be further modulated through the biofunctionalization of the material with adhesion peptides. Establishing the guidance ability of these fibers opens the possibility of their use as implants for spinal cord injuries, so that they may represent the core of a therapy that would allow the reconnection of the injured ends of the spinal cord