RESUMEN
Intrinsically disordered protein polymers (IDPPs) have attracted a lot of attention in the development of bioengineered devices and for use as study models in molecular biology because of their biomechanical properties and stimuli-responsiveness. The present study aims to understand the effect of charge density on the self-assembly of IDPPs. To that end, a library of recombinant IDPPs based on an amphiphilic diblock design with different charge densities was bioproduced, and their supramolecular assembly was characterized on the nano-, meso-, and microscale. Although the phase transition was driven by the collapse of hydrophobic moieties, the hydrophilic block composition strongly affected hierarchical assembly and, therefore, enabled the production of new molecular architectures, thus leading to new dynamics that govern the liquid-gel transition. These results highlight the importance of electrostatic repulsion for the hierarchical assembly of IDPPs and provide insights into the manufacture of supramolecular protein-based materials.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Interacciones Hidrofóbicas e Hidrofílicas , Transición de Fase , Polímeros , Electricidad EstáticaRESUMEN
Despite the remarkable progress in the generation of recombinant elastin-like (ELR) hydrogels, further improvements are still required to enhance and control their viscoelasticity, as well as limit the use of expensive chemical reagents, time-consuming processes and several purification steps. To alleviate this issue, the reactivity of carboxylic groups from glutamic (E) acid distributed along the hydrophilic block of an amphiphilic ELR (coded as E50I60) with amine groups has been studied through a one-pot amidation reaction in aqueous solutions, for the first time. By means of this approach, immediate conjugation of E50I60 with molecules containing amine groups has been performed with a high yield, as demonstrated by the 1H NMR and MALDI-TOF spectroscopies. This has resulted in the preparation of viscoelastic irreversible hydrogels through the "in-situ" cross-linking of E50I60 with another ELR (coded as VKV24) containing amine groups from lysines (K). The rheology analysis demonstrated that the gelation process takes place following a dual mechanism dependent on the ELR concentration: physical cross-linking of I60 block through the hydrophobic interactions, and covalent cross-linking of E50I60 with VKV24 through the amidation reaction. While the chemical network formed between the hydrophilic E50 block and VKV24 ELR preserves the elasticity of ELR hydrogels, the self-assembly of the I60 block through the hydrophobic interactions provides a tunable physical network. The presented investigation serves as a basis for generating ELR hydrogels with tunable viscoelastic properties promising for tissue regeneration, through an ''in-situ", rapid, scalable, economically and feasible one-pot method.
RESUMEN
Elastin polypeptides based on -VPGVG- repeated motifs are widely used in the production of biomaterials because they are stimuli-responsive systems. On the other hand, glycine-rich sequences, mainly present in tropoelastin terminal domains, are responsible for the elastin self-assembly. In a previous study, we have recombinantly expressed a chimeric polypeptide, named resilin, elastin, and collagen (REC), inspired by glycine-rich motifs of elastin and containing resilin and collagen sequences as well. Herein, a three-block polypeptide, named (REC)3, was expressed starting from the previous monomer gene by introducing key modifications in the sequence. The choice was mandatory because the uneven distribution of the cross-linking sites in the monomer precluded the hydrogel production. In this work, the cross-linked polypeptide appeared as a soft hydrogel, as assessed by rheology, and the linear un-cross-linked trimer self-aggregated more rapidly than the REC monomer. The absence of cell-adhesive sequences did not affect cell viability, while it was functional to the production of a material presenting antiadhesive properties useful in the integration of synthetic devices in the body and preventing the invasion of cells.
Asunto(s)
Elastina , Hidrogeles , Colágeno , Elastina/genética , Péptidos , Tropoelastina/genéticaRESUMEN
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.
RESUMEN
Cells interact mechanically with their environment, exerting mechanical forces that probe the extracellular matrix (ECM). The mechanical properties of the ECM determine cell behavior and control cell differentiation both in 2D and 3D environments. Gelatin (Gel) is a soft hydrogel into which cells can be embedded. This study shows significant 3D Gel shrinking due to the high traction cellular forces exerted by the cells on the matrix, which prevents cell differentiation. To modulate this process, Gel with hyaluronic acid (HA) has been combined in an injectable crosslinked hydrogel with controlled Gel-HA ratio. HA increases matrix stiffness. The addition of small amounts of HA leads to a significant reduction in hydrogel shrinking after cell encapsulation (C2C12 myoblasts). We show that hydrogel stiffness counterbalanced traction forces of cells and this was decisive in promoting cell differentiation and myotube formation of C2C12 encapsulated in the hybrid hydrogels.