Elastin-like recombinamers: Deconstructing and recapitulating the functionality of extracellular matrix proteins using recombinant protein polymers

Producción CientíficaIn the development of tissue engineering strategies to replace, remodel, regenerate, or support damaged tissue, the development of bioinspired biomaterials that recapitulate the physicochemical characteristics of the extracellular matrix has received increased attention. Given the compositional heterogeneity and tissue-to-tissue variation of the extracellular matrix, the design, choice of polymer, crosslinking, and nature of the resulting biomaterials are normally depended on intended application. Generally, these biomaterials are usually made of degradable or nondegradable biomaterials that can be used as cell or drug carriers. In recent years, efforts to endow reciprocal biomaterial–cell interaction properties in scaffolds have inspired controlled synthesis, derivatization, and functionalization of the polymers used. In this regard, elastin-like recombinant proteins have generated interest and continue to be developed further owing to their modular design at a molecular level. In this review, the authors provide a summary of key extracellular matrix features relevant to biomaterials design and discuss current approaches in the development of extracellular matrix-inspired elastin-like recombinant protein based biomaterials.Comisión Europea (grants MSCA-ITN-2014-ETN-642687 and NMP-2014-646075)Gobierno de España (grants MAT2016-78903-R, MAT2015-68901-R, and RTI2018-096320-B-C22)Junta de Castilla y León (project VA317P18)"Interreg V A España Portugal" Programa operativo de cooperación transfronteriza España Portugal (POCTEP) (grant 0624_2IQBIONEURO_6_E

Recombinant AMP/Polypeptide Self-Assembled Monolayers with Synergistic Antimicrobial Properties for Bacterial Strains of Medical Relevance

Producción CientíficaNosocomial infections are one of the most frequent causes of indwelling biomedical device failure. In this regard, the use of antibiofilm nanocoatings based on antimicrobial peptides (AMPs) is a promising alternative to prevent multiresistant biofilm infections. However, the limitations of chemical production impede the large-scale development of advanced antimicrobial materials that improve the properties of AMPs. Herein, we present a multifunctional modular design for the recombinant coproduction of self-assembled monolayers (SAMs) based on AMPs and elastin-like recombinamers (ELRs), which combine the antimicrobial properties of a designer AMP, GL13K, and low-fouling activity of an ELR in a synergistic manner. The inclusion of a grafting domain intended for oriented tethering onto surfaces allowed the recombinant polymers to be covalently immobilized onto model gold surfaces. The antibiofilm properties against two of the bacterial strains most frequently responsible for indwelling medical device-associated infections, namely Staphylococcus epidermidis and Staphylococcus aureus, were then evaluated. GL13K peptide was found to provide antibiofilm properties to the surface, with these being synergistically enhanced by the antifouling effect of the ELR. This new design offers a promising tool for the development of advanced AMP-based nanocoatings for medical devices with powerful and enhanced features.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (project PCIN-2015-010 / MAT2015-68901-R / MAT2016-78903-R)Junta de Castilla y León (project VA317P18

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

Hybrid Protein-Glycosaminoglycan Hydrogels Promote Chondrogenic Stem Cell Differentiation

[EN] Gelatin-hyaluronic acid (Gel-HA) hybrid hydrogels have been proposed as matrices for tissue engineering because of their ability to mimic the architecture of the extracellular matrix. Our aim was to explore whether tyramine conjugates of Gel and HA, producing injectable hydrogels, are able to induce a particular phenotype of encapsulated human mesenchymal stem cells without the need for growth factors. While pure Gel allowed good cell adhesion without remarkable differentiation and pure HA triggered chondrogenic differentiation without cell spreading, the hybrids, especially those rich in HA, promoted chondrogenic differentiation as well as cell proliferation and adhesion. Secretion of chondrogenic markers such as aggrecan, SOX-9, collagen type II, and glycosaminoglycans was observed, whereas osteogenic, myogenic, and adipogenic markers (RUNX2, sarcomeric myosin, and lipoproteinlipase, respectively) were not present after 2 weeks in the growth medium. The most promising matrix for chondrogenesis seems to be a mixture containing 70% HA and 30% Gel as it is the material with the best mechanical properties from all compositions tested here, and at the same time, it provides an environment suitable for balanced cell adhesion and chondrogenic differentiation. Thus, it represents a system that has a high potential to be used as the injectable material for cartilage regeneration therapies.The authors are grateful for the financial support received from the Spanish Ministry through the MAT2016-76039-C4-1-R project (including the FEDER financial support), the BES-2011-046144, and the EEBB-I-14-08725 grants. The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. M.S.-S. acknowledges the European Research Council (ERC-HealInSynergy 306990) and the UK Engineering and Physical Sciences Research Council (EPSRC-EP/P001114/1)Moulisova, V.; Poveda-Reyes, S.; Sanmartin-Masia, E.; Quintanilla-Sierra, L.; Ferrer, G.; Salmerón Sánchez, M. (2017). Hybrid Protein-Glycosaminoglycan Hydrogels Promote Chondrogenic Stem Cell Differentiation. ACS Omega. 2(11):7609-7620. https://doi.org/10.1021/acsomega.7b01303S7609762021

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

Recombinant DNA technology and click chemistry: a powerful combination for generating a hybrid elastin-like-statherin hydrogel to control calcium phosphate mineralization

Producción CientíficaUnderstanding the mechanisms responsible for generating different phases and morphologies of calcium phosphate by elastin-like recombinamers is supreme for bioengineering of advanced multifunctional materials. The generation of such multifunctional hybrid materials depends on the properties of their counterparts and the way in which they are assembled. The success of this assembly depends on the different approaches used, such as recombinant DNA technology and click chemistry. In the present work, an elastin-like recombinamer bearing lysine amino acids distributed along the recombinamer chain has been cross-linked via Huisgen [2 + 3] cycloaddition. The recombinamer contains the SNA15 peptide domains inspired by salivary statherin, a peptide epitope known to specifically bind to and nucleate calcium phosphate. The benefit of using click chemistry is that the hybrid elastin-like-statherin recombinamers cross-link without losing their fibrillar structure. Mineralization of the resulting hybrid elastin-like-statherin recombinamer hydrogels with calcium phosphate is described. Thus, two different hydroxyapatite morphologies (cauliflower- and plate-like) have been formed. Overall, this study shows that crosslinking elastin-like recombinamers leads to interesting matrix materials for the generation of calcium phosphate composites with potential applications as biomaterials.Ministerio de Economía, Industria y Competitividad (Project MAT2013- 42473-R and MAT2013-41723-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and GRS/516/A/10

The Incorporation of etanercept into a porous tri-layer scaffold for restoring and repairing cartilage tissue

Producción CientíficaCartilage diseases currently affect a high percentage of the world’s population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this could be controlled with biomaterials that act as an anti-inflammatory delivery system, capable of dosing these drugs over time in a specific area. The objective of this study was to incorporate etanercept (ETA) into porous three-layer scaffolds to decrease the inflammatory process in this soft tissue. ETA is a blocker of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). For this reason, the scaffold was built based on natural polymers, including chitosan and type I collagen. The scaffold was grafted next to subchondral bone using hydroxyapatite as filler. One of the biomaterials obtained was also crosslinked to compare its mechanical properties with the non-treated one. Both samples’ physicochemical properties were studied with SEM, microCT and photoacoustic imaging, and their rheological properties were also compared. The cell viability and proliferation of the human chondrocyte C28/I2 cell line were studied in vitro. An in vitro and in vivo controlled release study was evaluated in both specimens. The ETA anti-inflammatory effect was also studied by in vitro TNF-α and IL-6 production. The crosslinked and non-treated scaffolds had rheological properties suitable for this application. They were non-cytotoxic and favoured the in vitro growth of chondrocytes. The in vitro and in vivo ETA release showed desirable results for a drug delivery system. The TNF-α and IL-6 production assay showed that this drug was effective as an anti-inflammatory agent. In an in vivo OA mice model, safranin-O and fast green staining was carried out. The OA cartilage tissue improved when the scaffold with ETA was grafted in the damaged area. These results demonstrate that this type of biomaterial has high potential for clinical applications in tissue engineering and as a controlled drug delivery system in OA articular cartilage.European Union through the Erasmus PLUS doctoral fellowship (project 2015-1-NL01-KA 107-008639)VIDI personal grant (project 723.012.110

Analisis estructural del polietilen tereftalato y de la poliamida 6,6 reforzados con fibra de vidrio mediante Pa-FTIR y DSC

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