Novel Hydrogel-forming elastin-like recombinamers for biomedical applications

There is an increasing interest in developing advanced biomaterials with improved biocompatibility and functionality that might find uses in the field of biomedicine, for example in tissue engineering and regenerative medicine (TERM). Nowadays, recombinant polypeptides or polymers are one of the most prominent types of biomaterials, due to their nature. They are obtained through recombinant DNA technology, which allows the controlled biosynthesis of tailored polypeptides that may be designed to include combinations of polymeric amino acid sequences and/or bioactive domains. Within this type of biomaterials we can find elastin-like polypeptides (ELPs), which have also been recently termed elastin-like recombinamers (ELRs), according to their recombinant origin. These ELRs are composed of repetitions of the VPGXG (Val-Pro-Gly-X-Gly) pentapeptide, in which X (guest residue) can be any amino acid expect L-Proline. This composition confers the ELRs a smart behaviour of thermoresponsiveness defined by the so-called Inverse Temperature Transition (ITT) occurring above the Transition Temperature (Tt), which implies a phase transition of the dissolved ELR when this Tt is reached. Therefore, this thermal response is of great interest, since it has been shown to promote the formation of different structures, such as hydrogels that mimic the extracellular matrix, at the physiological temperature, whereas an ELR solution can be easily handled (e.g. injected in vivo) below the Tt. Furthermore, the Tt can be modulated depending on the polarity of the side chain of the amino acid chosen as guest residue. Moreover, if this residue contains functional goups, it can also be used for further chemical modifications in order to achieve, for instance, chemically (covalently) cross-linked hydrogels.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

FRET-Paired Hydrogel Forming Silk-Elastin-Like Recombinamers by Recombinant Conjugation of Fluorescent Proteins

Producción CientíficaIn the last decades, recombinant structural proteins have become very promising in addressing different issues such as the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, such as hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e., the green Aequorea coerulescens enhanced green fluorescent protein and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G′ = 60–4000 Pa) by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-Aequorea coerulescens enhanced green fluorescent protein. Hence, this enables Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods, and a FRET efficiency of 0.06–0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.Este trabajo forma parte de Proyectos de Investigación financiados por la Comisión Europea (NMP-2014-646075, HEALTH-F4-2011-278557, PITN-GA-2012-317306 and MSCA-ITN-2014-642687), del MINECO (MAT2016-7R8903-R, MAT2016-79435-R, MAT2013-42473-R, MAT2013-41723- and MAT2012-38043), la Junta de Castilla y León (VA244U13 and VA313U14) y el Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

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

Elastin-like recombinamers as smart drug delivery systems

Drug delivery systems that are able to control the release of bioactive molecules and designed to carry drugs to target sites are of particular interest for tissue therapy. Moreover, systems comprising materials that can respond to environmental stimuli and promote self-assembly and higher order supramolecular organization are especially useful in the biomedical field. Suitable biomaterials for this purpose include elastin-like recombinamers (ELRs), a class of proteinaceous polymers bioinspired by natural elastin and designed usingrecombinant technologies. The self-assembly and thermoresponsive behaviour of these systems, along with their biodegradability, biocompatibility and well-defined composition as a result of their tailor-made design, make them particularly attractive for drug delivery.Este trabajo forma parte de Proyectos de Investigación financiados por la Comisión Europea a través del Fondo Social Europeo (FSE) y el Fondo Europeo de Desarrollo Regional (ERDF), por el del MINECO (MAT2013-41723R, MAT2013-42473-R, MAT2012-38043 y PRI-PIBAR-2011-1403), la Junta de Castilla y León (VA049A11, VA152A12 y VA155A12) y el Instituto de Salud Carlos III bajo el Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

hLMSC Secretome Affects Macrophage Activity Differentially Depending on Lung-Mimetic Environments

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs

Influence of the Thermodynamic and Kinetic Control of Self‐Assembly on the Microstructure Evolution of Silk‐Elastin‐Like Recombinamer Hydrogels

Complex recombinant biomaterials that merge the self-assembling properties of different (poly)peptides provide a powerful tool for the achievement of specific structures, such as hydrogel networks, by tuning the thermodynamics and kinetics of the system through a tailored molecular design. In this work, elastin-like (EL) and silk-like (SL) polypeptides are combined to obtain a silk-elastin-like recombinamer (SELR) with dual selfassembly. First, EL domains force the molecule to undergo a phase transition above a precise temperature, which is driven by entropy and occurs very fast. Then, SL motifs interact through the slow formation of β-sheets, stabilized by H-bonds, creating an energy barrier that opposes phase separation. Both events lead to the development of a dynamic microstructure that evolves over time (until a pore size of 49.9 ± 12.7 µm) and to a delayed hydrogel formation (obtained after 2.6 h). Eventually, the network is arrested due to an increase in β-sheet secondary structures (up to 71.8 ± 0.8%) within SL motifs. This gives a high bond strength that prevents the complete segregation of the SELR from water, which results in a fixed metastable microarchitecture. These porous hydrogels are preliminarily tested as biomimetic niches for the isolation of cells in 3D cultures.Este trabajo forma parte de los proyectos de investigación MAT2016-78903-R, MAT2016-79435-R, RTI2018-096320-B-C22 y DTS19/00162 del Ministerio de Ciencia e Innovación, del proyecto VA317P18 de la Junta de Castilla y León, del proyecto 0624_2IQBIONEURO_6_E del programa Interreg V A España Portugal POCTEP y del Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels

Producción CientíficaOver the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.Ministerio de Economía, Industria y Competitividad (Project (MAT2016-78903-R, MAT2016-9 79435-R, MAT2013-42473-R, MAT2013-41723-R and MAT2012-38043)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and GRS/516/A/10

Biocompatibility of two model elastin‐like recombinamer‐based hydrogels formed through physical or chemical cross‐linking for various applications in tissue engineering and regenerative medicine

Producción CientíficaBiocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein‐based ones, such as elastin‐like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix‐like hydrogels through either physical or chemical cross‐linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase‐expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR‐based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL‐1β, IL‐4, IL‐6, and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine‐related applications.European Commission (NMP-2014-646075, HEALTH-F4-2011-278557, PITN-GA-2012-317306 and MSCA-ITN-2014-642687)Ministerio de Economía, Industria y Competitividad (Projects MAT2016-78903-R, MAT2016-79435-R, MAT2013-42473-R, MAT2013-41723-R and MAT2012-38043)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref.VA244U13 and VA313U14)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y LeónInstituto de Salud Carlos III (grant RD12/0019/0017 )Fundação para a Ciência e Tecnologia (SFRH/BD/86451/ 2012