Biofunctional hydrogels based on elastin-like recombinamers as extracellular matrix analogues

Uno de los objetivos principales de esta tesis consiste en la formación de hidrogeles mediante un proceso citocompatible que permita encapsular células dentro de dichos geles en el momento de su formación y que simulen las propiedades de las matrices extracelulares naturales para su utilización en ingeniería de tejidos. Dichos hidrogeles se obtendrán utilizando Polímeros Recombinantes tipo Elastina (ELRs) por presentar unas excelentes propiedades de partida como son su elevada biocompatibilidad o la posibilidad de incorporar diferentes bioactividades entre otras. Tambien Se quiere desarrollar un sistema fractal de formación de nanogeles mediante tecnología click sin cobre, y comprobar la influencia de la temperatura en dicha fractalidad durante el proceso de formación de estos geles. Se pretende evaluar y caracterizar, mediante el estudio de las dimensiones así como de las propiedades microreológicas y eléctricas, las estructuras formadas para tener un mejor conocimiento de su validez como sistemas de dosificación de fármacos. Por otro lado, se investigará la capacidad de estos hidrogeles para ser utilizados como recubrimiento de distintos materiales como poliestireno, vidrio y titanio mediante la tecnología “capa a capa” (layer by layer); siendo el titanio de especial interés debido a su creciente utilización como implante en procesos quirúrgicos. Se pretende obtener un sistema de formación de capas rápido, reproducible y escalable. Además dicho recubrimiento debe ser totalmente citocompatible y que nos permita incluir diferentes agentes terapéuticos que puedan ser de interés en futuras aplicaciones tanto en ingeniería de tejidos como en dosificación de fármacos. Por último, otro objetivo fundamental de la tesis consistió en demostrar la eficacia de los hidrogeles formados por ELRs y obtenidos mediante tecnología click sin cobre, como “scaffolds” en ingeniería de tejidos y más concretamente en el tratamiento de enfermedades cardiovasculares. Las características, tanto mecánicas como biológicas, de estos hidrogeles podrían convertirlos en biomateriales especialmente útiles en el tratamiento de ciertas afecciones del sistema circulatorio.Departamento de Química AnalíticaDoctorado en Arquitectur

Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure

Producción CientíficaControl over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a 3D-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.European Commission (NMP-2014-646075, PITNGA-2012-317306)Ministerio de Economía, Industria y Competitividad ( grants PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R, MAT2016-79435-R)Junta de Castilla y León (VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels

Producción CientíficaThe development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.European Commission (NMP-2014-646075, PITN-GA-2012-317306)Ministerio de Economía, Industria y Competitividad (Projects PCIN-2015-010, MAT2015- 68901-R, MAT2016-78903-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Bioactive scaffolds based on elastin-like materials for wound healing

Producción CientíficaWound healing is a complex process that, in healthy tissues, starts immediately after the injury. Even though it is a natural well-orchestrated process, large trauma wounds, or injuries caused by acids or other chemicals, usually produce a non-elastic deformed tissue that not only have biological reduced properties but a clear aesthetic effect. One of the main drawbacks of the scaffolds used for wound dressing is the lack of elasticity, driving to non-elastic and contracted tissues. In the last decades, elastin based materials have gained in importance as biomaterials for tissue engineering applications due to their good cyto- and bio-compatibility, their ease handling and design, production and modification. Synthetic elastin or elastin like-peptides (ELPs) are the two main families of biomaterials that try to mimic the outstanding properties of natural elastin, elasticity amongst others; although there are no in vivo studies that clearly support that these two families of elastin based materials improve the elasticity of the artificial scaffolds and of the regenerated skin. Within the next pages a review of the different forms (coacervates, fibres, hydrogels and biofunctionalized surfaces) in which these two families of biomaterials can be processed to be applied in the wound healing field have been done. Here, we explore the mechanical and biological properties of these scaffolds as well as the different in vivo approaches in which these scaffolds have been used.Ministerio de Economía, Industria y Competitividad (Projects MAT2015-68901-R, MAT2016-78903-R, PCIN-2015-010)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)European Commission (ELASTISLET N. 646075

Protease‐sensitive, VEGF‐mimetic peptide, and IKVAV laminin‐derived peptide sequences within elastin‐like recombinamer scaffolds provide spatiotemporally synchronized guidance of angiogenesis and neurogenesis

Producción CientíficaSpatiotemporal control of vascularization and innervation is a desiredhallmark in advanced tissue regeneration. For this purpose, we design a 3Dmodel scaffold, based on elastin-like recombinamer (ELR) hydrogels. Thiscontains two interior and well-defined areas, small cylinders, withdifferentiated bioactivities with respect to the bulk. Both are constructed on aprotease sensitive ELR with a fast-proteolyzed domain, but one bears aVEGF-mimetic peptide (QK) and the other a laminin-derived pentapeptide(IKVAV), to promote angiogenesis and neurogenesis, respectively. The outerbulk is based on a slow proteolytic sequence and RGD cell adhesion domains.In vitro studies show the effect of QK and IKVAV peptides on the promotion ofendothelial cell and axon spreading, respectively. The subcutaneousimplantation of the final 3D scaffold demonstrates the ability tospatiotemporally control angiogenesis and neurogenesis in vivo. Specifically,the inner small cylinder containing the QK peptide promotes fastendothelialization, whereas the one with IKVAV peptide promotes fastneurogenesis. Both, vascularization and innervation take place in advance ofthe bulk scaffold infiltration. This scaffold shows that it is possible to inducevascularization and innervation in predetermined areas of the scaffold wellahead to the bulk infiltration. That significantly increases the efficiency of theregenerative activity.Funding from the Spanish Government(Nos. RTI2018-096320-B-C22, FPU16-04015, PID2019-110709RB-I00, and PID2020-118669RA-I00)Interreg V España Portugal POCTEP (No.0624_2IQBIONEURO_6_E

Silk-ELR co-recombinamer covered stents obtained by electrospinning

Producción CientíficaIn the field of tissue engineering the choice of materials is of great importance given the possibility to use biocompatible polymers produced by means of biotechnology. A large number of synthetic and natural materials have been used to this purpose and processed into scaffolds using Electrospinning technique. Among materials that could be used for the fabrication of scaffold and degradable membranes, natural polymers such as collagen, elastin or fibroin offer the possibility to design structures strictly similar to the extracellular matrix (ECM). Biotechnology and genetic engineering made possible the advent of a new class of biopolymers called protein-based polymers. One example is represented by the silk-elastin-proteins that combine the elasticity and resilience of elastin with the high tensile strength of silk-fibroin and display engineered bioactive sequences. In this work, we use electrospinning technique to produce a fibrous scaffold made of the corecombinamer Silk-ELR. Obtained fibres have been characterized from the morphological point of view. Homogeneity and morphology have been explored using Scanning Electron Microscopy. A thorough study regarding the influence of Voltage, flow rate and distance have been carried out to determine the appropriate parameters to obtain the fibrous mats without defects and with a good distribution of diameters. Cytocompatibility has also been in vitro tested. For the first time we use the co-recombinamer Silk-ELR for the fabrication of a 2.5 angioplasty balloon coating. This structure could be useful as a coated scaffold for the regeneration of intima layer of vessels.“THE GRAIL” (Tissue in Host Engineering Guided Regeneration of Arterial Intima Layer) projectEuropean Union’s ‘Seventh Framework’ Programme for research, technological development and demonstration (grant HEALTH.2011.1.4-2-278557)European Commission (NMP-2014- 646075, MSCA-ITN-2014-642687)Ministerio de Economía, Industria y Competitividad (grant PCIN-2015-010, MAT2015-68901-R, MAT2016- 78903-R)Junta de Castilla y León (VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Spatial control and cell adhesion selectivity on model gold surfaces grafted with elastin-like recombinamers

Producción CientíficaA simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out. After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof. Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV - 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic, hydrophobic and mechanical properties.2020-092020-09Comisión Europea (NMP-2014-646075, MSCA-ITN-2014-642687)Ministerio de Economía, Industria y Competitividad (PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R and MAT2016-79435-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. Project VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Nanogel Formation from Dilute Solutions of Clickable Elastin-like Recombinamers and its Dependence on Temperature: Two Fractal Gelation Modes

Producción CientíficaDiluted, complementary, click-reactive elastin-like recombinamer (ELR) solutions have been prepared and mixed at two different temperatures, one below and one above the characteristic transition temperature (Tt) of these chemically modified ELRs. FTIR measurements, size, aspect ratio, zeta potential, and microrheological measurements have been carried out on the nanostructures formed under these dilute conditions as a way to better understand the relationship between the final macroscopic properties of ELR-based hydrogels and the molecular conditions governing the initial stages of the chemical cross- linking process that occurs, especially its dependence on the preparation temperature relative to Tt. As a result, two different fractal modes of gel formation have been found at the two temperatures studied (above and below Tt). Thus, when the reaction mixture is prepared below Tt, essentially one-dimensional linear nanogels with a high aspect ratio are obtained. In contrast, 3D nanogels are formed above Tt, with spherical shapes predominating. These different structures seem to reflect the two molecular organizations of the single components of the mixture under these conditions, namely extended chains below Tt and a spherical arrangement above Tt. In addition to the interest in these nanogels as models for understanding the formation of microscopic structures and differential macroscopic properties under more conventional hydrogel-formation conditions, these nanogels are of interest because of their thermoresponsiveness and biocompatibility, which provide them with potential uses for drug delivery and other biomedical applications in living systems.Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13

Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering

Producción CientíficaHerein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications.Ministerio de Economía, Industria y Competitividad (Projects MAT2015-68901-R, MAT2016-78903-R, PCIN-2015-010)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)European Commission (ELASTISLET No 646075, BIOGEL No 642687

Cartilage Regeneration in Preannealed Silk Elastin-Like Co-Recombinamers Injectable Hydrogel Embedded with Mature Chondrocytes in an Ex Vivo Culture Platform

Producción CientíficaTissue engineering for cartilage repair requires biomaterials that show rapid gelation and adequate mechanical properties. Although the use of hydrogel is the most promising biomaterial, it often lacks in rigidity and anchorage of cells when they are surrounded by synovial fluid while they are subjected to heavy loads. We developed and produced the Silk Elastin-Like co-Recombinamer (SELR), which contains both the physical interaction from elastin motifs and from silk motifs. In the first part of this work, we set up and optimized a preannealing treatment based on the evolution of silk motifs into β-sheet structures in order to fulfill the required mechanical properties of hydrogels for cartilage repair. The new preannealed SELRs (pA(EIS)2-(I5R)6) were characterized with the combination of several experimental techniques (CD, TEM, SEM, and rheology) to provide a deep insight into the material features. Finally, the regeneration properties of the pA(EIS)2-(I5R)6 hydrogel embedded with chondrocytes were evaluated. After 4 weeks of culturing in a standardized and representative ex vivo model, the biochemical and histological analysis revealed the production of glycosaminglycans and collagen. Moreover, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage. Hence, we conclude that the pA(EIS)2-(I5R)6 hydrogel presents improved mechanical properties while conserving the injectability, which leads to successful regeneration of hyaline cartilage in an ex vivo model.2019-10-202019-10-20Ministerio de Economía, Industria y Competitividad (Proyects PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R,MAT2016-79435-RJunta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. Project VA015U16)European Union’sHorizon 2020 research and innovation programme under theMarie Sklodowska-Curie grant agreement No. 6426