Publications by authors named "José Perez-Rigueiro"

36 Publications

Silk Fibroin: An Ancient Material for Repairing the Injured Nervous System.

Pharmaceutics 2021 Mar 23;13(3). Epub 2021 Mar 23.

Center for Biomedical Technology, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Spain.

Silk refers to a family of natural fibers spun by several species of invertebrates such as spiders and silkworms. In particular, silkworm silk, the silk spun by larvae, has been primarily used in the textile industry and in clinical settings as a main component of sutures for tissue repairing and wound ligation. The biocompatibility, remarkable mechanical performance, controllable degradation, and the possibility of producing silk-based materials in several formats, have laid the basic principles that have triggered and extended the use of this material in regenerative medicine. The field of neural soft tissue engineering is not an exception, as it has taken advantage of the properties of silk to promote neuronal growth and nerve guidance. In addition, silk has notable intrinsic properties and the by-products derived from its degradation show anti-inflammatory and antioxidant properties. Finally, this material can be employed for the controlled release of factors and drugs, as well as for the encapsulation and implantation of exogenous stem and progenitor cells with therapeutic capacity. In this article, we review the state of the art on manufacturing methodologies and properties of fiber-based and non-fiber-based formats, as well as the application of silk-based biomaterials to neuroprotect and regenerate the damaged nervous system. We review previous studies that strategically have used silk to enhance therapeutics dealing with highly prevalent central and peripheral disorders such as stroke, Alzheimer's disease, Parkinson's disease, and peripheral trauma. Finally, we discuss previous research focused on the modification of this biomaterial, through biofunctionalization techniques and/or the creation of novel composite formulations, that aim to transform silk, beyond its natural performance, into more efficient silk-based-polymers towards the clinical arena of neuroprotection and regeneration in nervous system diseases.
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http://dx.doi.org/10.3390/pharmaceutics13030429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004633PMC
March 2021

Basic Principles in the Design of Spider Silk Fibers.

Molecules 2021 Mar 23;26(6). Epub 2021 Mar 23.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Madrid, Spain.

The prominence of spider silk as a hallmark in biomimetics relies not only on its unrivalled mechanical properties, but also on how these properties are the result of a set of original design principles. In this sense, the study of spider silk summarizes most of the main topics relevant to the field and, consequently, offers a nice example on how these topics could be considered in other biomimetic systems. This review is intended to present a selection of some of the essential design principles that underlie the singular microstructure of major ampullate gland silk, as well as to show how the interplay between them leads to the outstanding tensile behavior of spider silk. Following this rationale, the mechanical behavior of the material is analyzed in detail and connected with its main microstructural features, specifically with those derived from the semicrystalline organization of the fibers. Establishing the relationship between mechanical properties and microstructure in spider silk not only offers a vivid image of the paths explored by nature in the search for high performance materials, but is also a valuable guide for the development of new artificial fibers inspired in their natural counterparts.
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http://dx.doi.org/10.3390/molecules26061794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004941PMC
March 2021

Biotechnology and Biomaterial-Based Therapeutic Strategies for Age-Related Macular Degeneration. Part II: Cell and Tissue Engineering Therapies.

Front Bioeng Biotechnol 2020 10;8:588014. Epub 2020 Dec 10.

Neuro-computing and Neuro-robotics Research Group, Complutense University of Madrid, Madrid, Spain.

Age-related Macular Degeneration (AMD) is an up-to-date untreatable chronic neurodegenerative eye disease of multifactorial origin, and the main causes of blindness in over 65 y.o. people. It is characterized by a slow progression and the presence of a multitude of factors, highlighting those related to diet, genetic heritage and environmental conditions, present throughout each of the stages of the illness. Current therapeutic approaches, mainly consisting on intraocular drug delivery, are only used for symptoms relief and/or to decelerate the progression of the disease. Furthermore, they are overly simplistic and ignore the complexity of the disease and the enormous differences in the symptomatology between patients. Due to the wide impact of the AMD and the up-to-date absence of clinical solutions, Due to the wide impact of the AMD and the up-to-date absence of clinical solutions, different treatment options have to be considered. Cell therapy is a very promising alternative to drug-based approaches for AMD treatment. Cells delivered to the affected tissue as a suspension have shown poor retention and low survival rate. A solution to these inconveniences has been the encapsulation of these cells on biomaterials, which contrive to their protection, gives them support, and favor their retention of the desired area. We offer a two-papers critical review of the available and under development AMD therapeutic approaches, from a biomaterials and biotechnological point of view. We highlight benefits and limitations and we forecast forthcoming alternatives based on novel biomaterials and biotechnology methods. In this second part we review the preclinical and clinical cell-replacement approaches aiming at the development of efficient AMD-therapies, the employed cell types, as well as the cell-encapsulation and cell-implant systems. We discuss their advantages and disadvantages and how they could improve the survival and integration of the implanted cells.
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http://dx.doi.org/10.3389/fbioe.2020.588014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758210PMC
December 2020

Regenerated Silk Fibers Obtained by Straining Flow Spinning for Guiding Axonal Elongation in Primary Cortical Neurons.

ACS Biomater Sci Eng 2020 12 27;6(12):6842-6852. Epub 2020 Oct 27.

Center for Biomedical Technology (CTB), Universidad Politécnica de Madrid, Pozuelo de Alarcón, 28223 Madrid, Spain.

The recovery of injured nervous tissue, one of the main goals for regenerative therapeutic approaches, is often hindered by the limited axonal regeneration ability of the central nervous system (CNS). In this regard, the identification of scaffolds that support the reconstruction of functional neuronal tissues and guide the alignment of regenerating neurons is a major challenge in tissue engineering. Ideally, the usage of such scaffolds would promote and guide the axonal growth, a crucial phase for the restoration of neuronal connections and, consequently, the nerve function. Among the materials proposed as scaffolds for CNS regeneration, silk has been used to exploit its outstanding features as a biomaterial to promote axonal regeneration. In this study, we explore, for the first time, the possibility of using high-performance regenerated silk fibers obtained by straining flow spinning (SFS) to serve as scaffolds for inducing and guiding the axonal growth. It is shown that SFS fibers promote the spontaneous organization of dissociated cortical primary cells into highly interconnected cellular spheroid-like tissue formations. Neuronal projections (i.e., axons) from these cellular spheroids span hundreds of microns along the SFS fibers that act as guides and allow the connection of distant spheroids. In addition, it is also shown that SFS fibers serve as scaffolds for neuronal migration covering short and long distances. As a consequence, the usage of high-performance SFS fibers appears as a promising basis for the development of novel therapies, leading to directed axonal regeneration.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00985DOI Listing
December 2020

Biomimetic Approaches for Separated Regeneration of Sensory and Motor Fibers in Amputee People: Necessary Conditions for Functional Integration of Sensory-Motor Prostheses With the Peripheral Nerves.

Front Bioeng Biotechnol 2020 3;8:584823. Epub 2020 Nov 3.

Neuro-computing and Neuro-robotics Research Group, Complutense University of Madrid, Madrid, Spain.

The regenerative capacity of the peripheral nervous system after an injury is limited, and a complete function is not recovered, mainly due to the loss of nerve tissue after the injury that causes a separation between the nerve ends and to the disorganized and intermingled growth of sensory and motor nerve fibers that cause erroneous reinnervations. Even though the development of biomaterials is a very promising field, today no significant results have been achieved. In this work, we study not only the characteristics that should have the support that will allow the growth of nerve fibers, but also the molecular profile necessary for a specific guidance. To do this, we carried out an exhaustive study of the molecular profile present during the regeneration of the sensory and motor fibers separately, as well as of the effect obtained by the administration and inhibition of different factors involved in the regeneration. In addition, we offer a complete design of the ideal characteristics of a biomaterial, which allows the growth of the sensory and motor neurons in a differentiated way, indicating (1) size and characteristics of the material; (2) necessity to act at the microlevel, on small groups of neurons; (3) combination of molecules and specific substrates; and (4) temporal profile of those molecules expression throughout the regeneration process. The importance of the design we offer is that it respects the complexity and characteristics of the regeneration process; it indicates the appropriate temporal conditions of molecular expression, in order to obtain a synergistic effect; it takes into account the importance of considering the process at the group of neuron level; and it gives an answer to the main limitations in the current studies.
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http://dx.doi.org/10.3389/fbioe.2020.584823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670549PMC
November 2020

Biotechnology and Biomaterial-Based Therapeutic Strategies for Age-Related Macular Degeneration. Part I: Biomaterials-Based Drug Delivery Devices.

Front Bioeng Biotechnol 2020 3;8:549089. Epub 2020 Nov 3.

Neuro-Computing and Neuro-Robotics Research Group, Complutense University of Madrid, Madrid, Spain.

Age-related Macular Degeneration (AMD) is an up-to-date untreatable chronic neurodegenerative eye disease of multifactorial origin, and the main causes of blindness in over 65 years old people. It is characterized by a slow progression and the presence of a multitude of factors, highlighting those related to diet, genetic heritage and environmental conditions, present throughout each of the stages of the illness. Current therapeutic approaches, mainly consisting of intraocular drug delivery, are only used for symptoms relief and/or to decelerate the progression of the disease. Furthermore, they are overly simplistic and ignore the complexity of the disease and the enormous differences in the symptomatology between patients. Due to the wide impact of the AMD and the up-to-date absence of clinical solutions, the development of biomaterials-based approaches for a personalized and controlled delivery of therapeutic drugs and biomolecules represents the main challenge for the defeat of this neurodegenerative disease. Here we present a critical review of the available and under development AMD therapeutic approaches, from a biomaterials and biotechnological point of view. We highlight benefits and limitations and we forecast forthcoming alternatives based on novel biomaterials and biotechnology methods. In the first part we expose the physiological and clinical aspects of the disease, focusing on the multiple factors that give origin to the disorder and highlighting the contribution of these factors to the triggering of each step of the disease. Then we analyze available and under development biomaterials-based drug-delivery devices (DDD), taking into account the anatomical and functional characteristics of the healthy and ill retinal tissue.
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http://dx.doi.org/10.3389/fbioe.2020.549089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670958PMC
November 2020

First steps for the development of silk fibroin-based 3D biohybrid retina for age-related macular degeneration (AMD).

J Neural Eng 2020 10 31;17(5):055003. Epub 2020 Oct 31.

Neuro-computing & Neuro-robotics Research Group, Complutense University of Madrid, Spain. Innovation Research Group, Institute for Health Research San Carlos Clinical Hospital (IdISSC), Madrid, Spain. These authors equally contributed to this article.

Age-related macular degeneration is an incurable chronic neurodegenerative disease, causing progressive loss of the central vision and even blindness. Up-to-date therapeutic approaches can only slow down he progression of the disease.

Objective: Feasibility study for a multilayered, silk fibroin-based, 3D biohybrid retina.

Approach: Fabrication of silk fibroin-based biofilms; culture of different types of cells: retinal pigment epithelium, retinal neurons, Müller and mesenchymal stem cells ; creation of a layered structure glued with silk fibroin hydrogel.

Main Results: In vitro evidence for the feasibility of layered 3D biohybrid retinas; primary culture neurons grow and develop neurites on silk fibroin biofilms, either alone or in presence of other cells cultivated on the same biomaterial; cell organization and cellular phenotypes are maintained in vitro for the seven days of the experiment.

Significance: 3D biohybrid retina can be built using silk silkworm fibroin films and hydrogels to be used in cell replacement therapy for AMD and similar retinal neurodegenerative diseases.
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http://dx.doi.org/10.1088/1741-2552/abb9c0DOI Listing
October 2020

Application of the Spider Silk Standardization Initiative (SI) methodology to the characterization of major ampullate gland silk fibers spun by spiders from Pantanos de Villa wetlands (Lima, Peru).

J Mech Behav Biomed Mater 2020 11 1;111:104023. Epub 2020 Aug 1.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223, Pozuelo de Alarcón (Madrid), Spain; Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040, Madrid, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain. Electronic address:

Spider silk is a natural material with unique properties and a great potential for engineering and biomedical applications. In spite of its simple composition and highly conserved and stereotypical production, spider silks show a wide range of variability in their mechanical properties which, for long, have defied their classification and standardization. Here we propose to launch the Spider Silk Standardization Initiative (SI), a methodology based on the definition of the α* parameter, in an attempt to define a systematic procedure to classify the tensile properties exhibited by major ampullate gland silk (MAS) spun by Entelegynae spiders. The α* parameter is calculated from the comparison of the true stress-true strain curve of any MAS fiber after being subjected to maximum supercontraction, with the true stress-true strain curve of the species Argiope aurantia, which is set as a reference curve. This work presents the details of the SI methodology and, as an example, shows its application to an assemblage of Entelegynae spiders from different families collected at the Pantanos de Villa wetlands (Lima, Peru). The systematic and objective classification of the tensile properties of MAS fibers allowed by the SI will offer insights into key aspects of the biological evolution of the material, and address questions such as how history and adaptation contributed to shape those properties. In addition, it will surely have far reaching consequences in fields such as Materials Science, and Molecular and Evolutionary Biology, by organizing the range of tensile properties exhibited by spider silk fibers.
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http://dx.doi.org/10.1016/j.jmbbm.2020.104023DOI Listing
November 2020

Biomaterials to Neuroprotect the Stroke Brain: A Large Opportunity for Narrow Time Windows.

Cells 2020 04 26;9(5). Epub 2020 Apr 26.

Center for Biomedical Technology, Universidad Politécnica de Madrid, 28040 Madrid, Spain.

Ischemic stroke represents one of the most prevalent pathologies in humans and is a leading cause of death and disability. Anti-thrombolytic therapy with tissue plasminogen activator (t-PA) and surgical thrombectomy are the primary treatments to recanalize occluded vessels and normalize the blood flow in ischemic and peri-ischemic regions. A large majority of stroke patients are refractory to treatment or are not eligible due to the narrow time window of therapeutic efficacy. In recent decades, we have significantly increased our knowledge of the molecular and cellular mechanisms that inexorably lead to progressive damage in infarcted and peri-lesional brain areas. As a result, promising neuroprotective targets have been identified and exploited in several stroke models. However, these considerable advances have been unsuccessful in clinical contexts. This lack of clinical translatability and the emerging use of biomaterials in different biomedical disciplines have contributed to developing a new class of biomaterial-based systems for the better control of drug delivery in cerebral disorders. These systems are based on specific polymer formulations structured in nanoparticles and hydrogels that can be administered through different routes and, in general, bring the concentrations of drugs to therapeutic levels for prolonged times. In this review, we first provide the general context of the molecular and cellular mechanisms impaired by cerebral ischemia, highlighting the role of excitotoxicity, inflammation, oxidative stress, and depolarization waves as the main pathways and targets to promote neuroprotection avoiding neuronal dysfunction. In the second part, we discuss the versatile role played by distinct biomaterials and formats to support the sustained administration of particular compounds to neuroprotect the cerebral tissue at risk of damage.
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http://dx.doi.org/10.3390/cells9051074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291200PMC
April 2020

Mechanical and structural adaptations to migration in the flight feathers of a Palaearctic passerine.

J Evol Biol 2020 07 27;33(7):979-989. Epub 2020 Apr 27.

Evolution and Conservation Biology Research Group, Department of Biodiversity, Ecology and Evolution, Faculty of Biology, Universidad Complutense de Madrid, Madrid, Spain.

Current avian migration patterns in temperate regions have been developed during the glacial retreat and subsequent colonization of the ice-free areas during the Holocene. This process resulted in a geographic gradient of greater seasonality as latitude increased that favoured migration-related morphological and physiological (co)adaptations. Most evidence of avian morphological adaptations to migration comes from the analysis of variation in the length and shape of the wings, but the existence of intra-feather structural adjustments has been greatly overlooked despite their potential to be under natural selection. To shed some light on this question, we used data from European robins Erithacus rubecula overwintering in Campo de Gibraltar (Southern Iberia), where sedentary robins coexist during winter with conspecifics showing a broad range of breeding origins and, hence, migration distances. We explicitly explored how wing length and shape, as well as several functional (bending stiffness), developmental (feather growth rate) and structural (size and complexity of feather components) characteristics of flight feathers, varied in relation to migration distance, which was estimated from the hydrogen stable isotope ratios of the summer-produced tail feathers. Our results revealed that migration distance not only favoured longer and more concave wings, but also promoted primaries with a thicker dorsoventral rachis and shorter barb lengths, which, in turn, conferred more bending stiffness to these feathers. We suggest that these intra-feather structural adjustments could be an additional, largely unnoticed, adaptation within the avian migratory syndrome that might have the potential to evolve relatively quickly to facilitate the occupation of seasonal environments.
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http://dx.doi.org/10.1111/jeb.13630DOI Listing
July 2020

Structure-Function Relationship of Artificial Spider Silk Fibers Produced by Straining Flow Spinning.

Biomacromolecules 2020 06 10;21(6):2116-2124. Epub 2020 Apr 10.

Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, Centre for Veterinary Medicine and Animal Science, Box 7045, 756 51 Uppsala, Sweden.

The production of large quantities of artificial spider silk fibers that match the mechanical properties of the native material has turned out to be challenging. Recent advancements in the field make biomimetic spinning approaches an attractive way forward since they allow the spider silk proteins to assemble into the secondary, tertiary, and quaternary structures that are characteristic of the native silk fiber. Straining flow spinning (SFS) is a newly developed and versatile method that allows production under a wide range of processing conditions. Here, we use a recombinant spider silk protein that shows unprecedented water solubility and that is capable of native-like assembly, and we spin it into fibers by the SFS technique. We show that fibers may be spun using different hydrodynamical and chemical conditions and conclude that these spinning conditions affect fiber mechanics. In particular, it was found that the addition of acetonitrile and polyethylene glycol to the collection bath results in fibers with increased β-sheet content and improved mechanical properties.
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http://dx.doi.org/10.1021/acs.biomac.0c00100DOI Listing
June 2020

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

Int J Biol Macromol 2020 Apr 16;148:378-390. Epub 2020 Jan 16.

Center for Biomaterials and Tissue Engineering, Universitat Politècnica de València, Cno. de Vera s/n, 46022 Valencia, Spain; Department of Medicine, Universitat Jaume I, Av. Vicent-Sos Baynat s/n, Castellón 12071, Spain.. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.01.149DOI Listing
April 2020

Evaluation of Neurosecretome from Mesenchymal Stem Cells Encapsulated in Silk Fibroin Hydrogels.

Sci Rep 2019 06 19;9(1):8801. Epub 2019 Jun 19.

Center for Biomedical Technology, Universidad Politécnica de Madrid, Madrid, Spain.

Physical and cognitive disabilities are hallmarks of a variety of neurological diseases. Stem cell-based therapies are promising solutions to neuroprotect and repair the injured brain and overcome the limited capacity of the central nervous system to recover from damage. It is widely accepted that most benefits of different exogenously transplanted stem cells rely on the secretion of different factors and biomolecules that modulate inflammation, cell death and repair processes in the damaged host tissue. However, few cells survive in cerebral tissue after transplantation, diminishing the therapeutic efficacy. As general rule, cell encapsulation in natural and artificial polymers increases the in vivo engraftment of the transplanted cells. However, we have ignored the consequences of such encapsulation on the secretory activity of these cells. In this study, we investigated the biological compatibility between silk fibroin hydrogels and stem cells of mesenchymal origin, a cell population that has gained increasing attention and popularity in regenerative medicine. Although the survival of mesenchymal stem cells was not affected inside hydrogels, this biomaterial format caused adhesion and proliferation deficits and impaired secretion of several angiogenic, chemoattractant and neurogenic factors while concurrently potentiating the anti-inflammatory capacity of this cell population through a massive release of TGF-Beta-1. Our results set a milestone for the exploration of engineering polymers to modulate the secretory activity of stem cell-based therapies for neurological disorders.
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http://dx.doi.org/10.1038/s41598-019-45238-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584675PMC
June 2019

Straining Flow Spinning of Artificial Silk Fibers: A Review.

Biomimetics (Basel) 2018 Oct 5;3(4). Epub 2018 Oct 5.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.

This work summarizes the main principles and some of the most significant results of straining flow spinning (SFS), a technology developed originally by the authors of this work. The principles on which the technology is based, inspired by the natural spinning system of silkworms and spiders, are presented, as well as some of the main achievements of the technique. Among these achievements, spinning under environmentally friendly conditions, obtaining high-performance fibers, and imparting the fibers with emerging properties such as supercontraction are discussed. Consequently, SFS appears as an efficient process that may represent one of the first realizations of a biomimetic technology with a significant impact at the production level.
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http://dx.doi.org/10.3390/biomimetics3040029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6352662PMC
October 2018

Comparison of cell mechanical measurements provided by Atomic Force Microscopy (AFM) and Micropipette Aspiration (MPA).

J Mech Behav Biomed Mater 2019 07 8;95:103-115. Epub 2019 Apr 8.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223, Pozuelo de Alarcón, Madrid, Spain; Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040, Madrid, Spain; CIBER-BBN, Biomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine, Spain.

A comparative analysis of T-lymphocyte mechanical data obtained from Micropipette Aspiration (MPA) and Atomic Force Microscopy (AFM) is presented. Results obtained by fitting the experimental data to simple Hertz and Theret models led to non-Gaussian distributions and significantly different values of the elastic moduli obtained by both techniques. The use of more refined models, taking into account the finite size of cells (simplified double contact and Zhou models) reduces the differences in the values calculated for the elastic moduli. Several possible sources for the discrepancy between the techniques are considered. The analysis suggests that the local nature of AFM measurements compared with the more general character of MPA measurements probably contributed to the differences observed.
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http://dx.doi.org/10.1016/j.jmbbm.2019.03.031DOI Listing
July 2019

Preparation and characterization of Nephila clavipes tubuliform silk gut.

Soft Matter 2019 Apr;15(14):2960-2970

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223, Pozuelo de Alarcón, Madrid, Spain.

Tubuliform silk glands were dissected from Nephila clavipes spiders, and silk gut fibers were produced by immersing the glands in a mild acid solution and subsequent stretching. The tensile properties of the as produced fibers were obtained through tensile tests, and the stress-strain curves were compared with those of naturally spun tubuliform silk fibers. The influence on the mechanical properties of the fibers after immersion in water and drying was also discerned. The microstructure of the silk guts was obtained by X-ray diffraction (XRD) and infrared spectroscopy (FTIR). It was found that the stress-strain curves of the stretched tubuliform silk guts concur with those of their natural counterparts (tubuliform silk fibers).
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http://dx.doi.org/10.1039/c9sm00212jDOI Listing
April 2019

Emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers.

Sci Rep 2019 02 20;9(1):2398. Epub 2019 Feb 20.

Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo, 184-8588, Japan.

The conditions required for the emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers are assessed through an experimental approach that combines the spinning of regenerated fibers with controlled properties and their characterization by C solid-state nuclear magnetic resonance (NMR). Both supercontracting and non-supercontracting regenerated fibers are produced using the straining flow spinning (SFS) technique from C labeled cocoons. The short-range microstructure of the fibers is assessed through C CP/MAS in air and C DD/MAS in water, and the main microstructural features are identified and quantified. The mechanical properties of the regenerated fibers and their microstructures are compared with those of natural silkworm silk. The combined analysis highlights two possible key elements as responsible for the emergence of supercontraction: (1) the existence of an upper and a lower limit of the amorphous phase compatible with supercontraction, and (2) the existence of two ordered phases, β-sheet A and B, which correspond to different packing arrangements of the protein chains.
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http://dx.doi.org/10.1038/s41598-019-38712-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382804PMC
February 2019

Potential use of silkworm gut fiber braids as scaffolds for tendon and ligament tissue engineering.

J Biomed Mater Res B Appl Biomater 2019 10 24;107(7):2209-2215. Epub 2019 Jan 24.

Department of Biotechnology, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Murcia, Spain.

Tendon and ligament tissue engineering require scaffolds for the treatment of various conditions in the medical field. These must meet requirements such as high tensile strength, biocompatibility, fast and stable repair and a rate of degradation that allows the repair of the damaged tissue. In this work, we propose the use of silkworm gut fiber braids as materials to temporarily replace and repair this type of tissues. The mechanical characterization of the braids made with different number of silk gut fibers is provided, as well as a descriptive analysis of the proliferation and adhesion of cultures of adult human mesenchymal stem cells from bone marrow and fibroblasts (L929) on the braids. As expected, the breaking force increases linearly in the scaffold with the number of fibers, thus being a parameter adaptable to the specific requirements of the tissue to repair and the animal model of study. On the other hand, in all of the cases studied, the values obtained for the elastic modulus of the hydrated fibers were in the range of the ones reported for various human tendons and ligaments. Moreover, the scaffold demonstrated excellent biocompatibility in vitro, allowing the adhesion and proliferation, in the same culture conditions, of the two cell types studied, therefore posing as an ideal candidate to be employed in future in vivo studies that allow elucidating its behavior in the articular environment or extra-articular tendinous areas. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: 2019. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2209-2215, 2019.
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http://dx.doi.org/10.1002/jbm.b.34300DOI Listing
October 2019

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

Front Cell Neurosci 2018 6;12:296. Epub 2018 Sep 6.

Center for Biomedical Technology, Universidad Politécnica de Madrid, Madrid, Spain.

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 (FLs) and caudal motor (cFLm) representations and an emergent sensory activity in peri-lesional areas belonging to cFLm. 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.
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http://dx.doi.org/10.3389/fncel.2018.00296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135908PMC
September 2018

Enhanced Biological Response of AVS-Functionalized Ti-6Al-4V Alloy through Covalent Immobilization of Collagen.

Sci Rep 2018 02 20;8(1):3337. Epub 2018 Feb 20.

Centro de Tecnología Biomédica. Universidad Politécnica de Madrid, 28223, Pozuelo de Alarcón (Madrid), Spain.

This study presents the development of an efficient procedure for covalently immobilizing collagen molecules on AVS-functionalized Ti-6Al-4V samples, and the assessment of the survival and proliferation of cells cultured on these substrates. Activated Vapor Silanization (AVS) is a versatile functionalization technique that allows obtaining a high density of active amine groups on the surface. A procedure is presented to covalently bind collagen to the functional layer using EDC/NHS as cross-linker. The covalently bound collagen proteins are characterized by fluorescence microscopy and atomic force microscopy and their stability is tested. The effect of the cross-linker concentration on the process is assessed. The concentration of the cross-linker is optimized and a reliable cleaning protocol is developed for the removal of the excess of carbodiimide from the samples. The results demonstrate that the covalent immobilization of collagen type I on Ti-6Al-4V substrates, using the optimized protocol, increases the number of viable cells present on the material. Consequently, AVS in combination with the carbodiimide chemistry appears as a robust method for the immobilization of proteins and, for the first time, it is shown that it can be used to enhance the biological response to the material.
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http://dx.doi.org/10.1038/s41598-018-21685-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820288PMC
February 2018

Hydrogels-Assisted Cell Engraftment for Repairing the Stroke-Damaged Brain: Chimera or Reality.

Polymers (Basel) 2018 Feb 13;10(2). Epub 2018 Feb 13.

Neurocomputing and Neurorobotics Research Group: Faculty of Biology and Faculty of Optics, Universidad Complutense de Madrid, 28040 Madrid, Spain.

The use of advanced biomaterials as a structural and functional support for stem cells-based therapeutic implants has boosted the development of tissue engineering applications in multiple clinical fields. In relation to neurological disorders, we are still far from the clinical reality of restoring normal brain function in neurodegenerative diseases and cerebrovascular disorders. Hydrogel polymers show unique mechanical stiffness properties in the range of living soft tissues such as nervous tissue. Furthermore, the use of these polymers drastically enhances the engraftment of stem cells as well as their capacity to produce and deliver neuroprotective and neuroregenerative factors in the host tissue. Along this article, we review past and current trends in experimental and translational research to understand the opportunities, benefits, and types of tentative hydrogel-based applications for the treatment of cerebral disorders. Although the use of hydrogels for brain disorders has been restricted to the experimental area, the current level of knowledge anticipates an intense development of this field to reach clinics in forthcoming years.
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http://dx.doi.org/10.3390/polym10020184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415003PMC
February 2018

Production of High Performance Bioinspired Silk Fibers by Straining Flow Spinning.

Biomacromolecules 2017 04 3;18(4):1127-1133. Epub 2017 Mar 3.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid , 28223 Pozuelo de Alarcón (Madrid), Spain.

In the last years, there has been an increasing interest in bioinspired approaches for different applications, including the spinning of high performance silk fibers. Bioinspired spinning is based on the natural spinning system of spiders and worms and requires combining changes in the chemical environment of the proteins with the application of mechanical stresses. Here we present the novel straining flow spinning (SFS) process and prove its ability to produce high performance fibers under mild, environmentally friendly conditions, from aqueous protein dopes. SFS is shown to be an extremely versatile technique which allows controlling a large number of processing parameters. This ample set of parameters allows fine-tuning the microstructure and mechanical behavior of the fibers, which opens the possibility of adapting the fibers to their intended uses.
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http://dx.doi.org/10.1021/acs.biomac.6b01757DOI Listing
April 2017

Improved Measurement of Elastic Properties of Cells by Micropipette Aspiration and Its Application to Lymphocytes.

Ann Biomed Eng 2017 05 17;45(5):1375-1385. Epub 2017 Jan 17.

Center for Biomedical Technology. Universidad Politécnica de Madrid, 28223, Pozuelo de Alarcón, Spain.

Mechanical deformability of cells is an important property for their function and development, as well as a useful marker of cell state. The classical technique of micropipette aspiration allows single-cell studies and we provide here a method to measure the two basic mechanical parameters, elastic modulus and Poisson's ratio. The proposed method, developed from finite-element analysis of micropipette aspiration experiments, may be implemented in future technologies for the automated measurement of mechanical properties of cells, based on the micropipette aspiration technique or on the cell transit through flow constrictions. We applied this method to measure the elastic parameters of lymphocytes, in which the mechanical properties depend on their activation state. Additionally, we discuss in this work the accuracy of previous models to estimate the elastic modulus of cells, in particular the analytical model by Theret et al., widely used in the field. We show the necessity of using an improved model, taking into account the finite size of the cells, to obtain new insights that may remain hidden otherwise.
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http://dx.doi.org/10.1007/s10439-017-1795-7DOI Listing
May 2017

Safety and tolerability of silk fibroin hydrogels implanted into the mouse brain.

Acta Biomater 2016 11 2;45:262-275. Epub 2016 Sep 2.

Center for Biomedical Technology, Universidad Politécnica, Madrid, Spain; Biomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain. Electronic address:

At present, effective therapies to repair the central nervous system do not exist. Biomaterials might represent a new frontier for the development of neurorestorative therapies after brain injury and degeneration. In this study, an in situ gelling silk fibroin hydrogel was developed via the sonication-induced gelation of regenerated silk fibroin solutions. An adequate timeframe for the integration of the biomaterial into the brain tissue was obtained by controlling the intensity and time of sonication. After the intrastriatal injection of silk fibroin the inflammation and cell death in the implantation area were transient. We did not detect considerable cognitive or sensorimotor deficits, either as examined by different behavioral tests or an electrophysiological analysis. The sleep and wakefulness states studied by chronic electroencephalogram recordings and the fitness of thalamocortical projections and the somatosensory cortex explored by evoked potentials were in the range of normality. The methodology used in this study might serve to assess the biological safety of other biomaterials implanted into the rodent brain. Our study highlights the biocompatibility of native silk with brain tissue and extends the current dogma of the innocuousness of this biomaterial for therapeutic applications, which has repercussion in regenerative neuroscience.

Statement Of Significance: The increasingly use of sophisticated biomaterials to encapsulate stem cells has changed the comprehensive overview of potential strategies for repairing the nervous system. Silk fibroin (SF) meets with most of the standards of a biomaterial suitable to enhance stem cell survival and function. However, a proof-of-principle of the in vivo safety and tolerability of SF implanted into the brain tissue is needed. In this study we have examined the tissue bioresponse and brain function after implantation of SF hydrogels. We have demonstrated the benign coexistence of silk with the complex neuronal circuitry that governs sensorimotor coordination and mechanisms such as learning and memory. Our results have repercussion in the development of advances strategies using this biomaterial in regenerative neuroscience.
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http://dx.doi.org/10.1016/j.actbio.2016.09.003DOI Listing
November 2016

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

Sci Rep 2016 Jan 12;6:18991. Epub 2016 Jan 12.

Centro de Tecnología Biomédica. Universidad Politécnica de Madrid. 28223 Pozuelo de Alarcón (Madrid). Spain.

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 β-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.
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http://dx.doi.org/10.1038/srep18991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709512PMC
January 2016

Mechanical behaviour and formation process of silkworm silk gut.

Soft Matter 2015 Dec;11(46):8981-91

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón (Madrid), Spain and Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040, Madrid, Spain.

High performance silk fibers were produced directly from the silk glands of silkworms (Bombyx mori) following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors' best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber.
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http://dx.doi.org/10.1039/c5sm01877cDOI Listing
December 2015

Unexpected behavior of irradiated spider silk links conformational freedom to mechanical performance.

Soft Matter 2015 Jun 21;11(24):4868-78. Epub 2015 May 21.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.

Silk fibers from Argiope trifasciata and Nephila inaurata orb-web weaving spiders were UV irradiated to modify the molecular weight of the constituent proteins. Fibers were characterized either as forcibly silked or after being subjected to maximum supercontraction. The effect of irradiation on supercontraction was also studied, both in terms of the percentage of supercontraction and the tensile properties exhibited by irradiated and subsequently supercontracted fibers. The effects of UV exposure at the molecular level were assessed by polyacrylamide gel electrophoresis and mass spectrometry. It is shown that UV-irradiated fibers show a steady decrease in their main tensile parameters, most notably, tensile strength and strain. The combination of the mechanical and biochemical data suggests that the restricted conformational freedom of the proteins after UV irradiation is critical in the reduction of these properties. Consequently, an adequate topological organization of the protein chains emerges as a critical design principle in the performance of spider silk.
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http://dx.doi.org/10.1039/c5sm00395dDOI Listing
June 2015

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes.

Phys Biol 2015 Mar 19;12(2):026005. Epub 2015 Mar 19.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, E-28223 Pozuelo de Alarcón (Madrid), Spain. Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, E-28040, Madrid, Spain.

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens.
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http://dx.doi.org/10.1088/1478-3975/12/2/026005DOI Listing
March 2015

Spider silk gut: development and characterization of a novel strong spider silk fiber.

Sci Rep 2014 Dec 5;4:7326. Epub 2014 Dec 5.

1] Centro de Tecnología Biomédica. Universidad Politécnica de Madrid. 28223 Pozuelo de Alarcón (Madrid). Spain [2] Departamento de Ciencia de Materiales. ETSI Caminos, Canales y Puertos. Universidad Politécnica de Madrid. 28040. Madrid. Spain.

Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering.
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http://dx.doi.org/10.1038/srep07326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256644PMC
December 2014

The variability and interdependence of spider viscid line tensile properties.

J Exp Biol 2013 Dec 26;216(Pt 24):4722-8. Epub 2013 Sep 26.

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.

True stress-true strain curves of naturally spun viscid line fibres 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 fibres and a technique that allows the accurate measurement of their cross-sectional area. Comparison of the tensile behaviour of different samples indicated 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 the identification of two independent parameters that underlie the variability and characterize the observed range of true stress-true strain curves. The 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.
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http://dx.doi.org/10.1242/jeb.094011DOI Listing
December 2013
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