Genetically engineered hydrogels based on elastin-like recombinamers for cardiovascular applications
- Autores: Fernando González Pérez
- Directores de la Tesis: José Carlos Rodríguez Cabello (dir. tes.)
- Lectura: En la Universidad de Valladolid ( España ) en 2023
- Idioma: inglés
- Tribunal Calificador de la Tesis: Conrado José Aparicio Bádenas (presid.), Luis Quintanilla Sierra (secret.), Gastón Fuentes Estévez (voc.)
- Programa de doctorado: Programa de Doctorado en Física por la Universidad de Valladolid
- Materias:
- Enlaces
- Tesis en acceso abierto en: UVADOC
- Resumen
Tissue engineering and regenerative medicine (TERM) is a prominent field of research that aims to repair or replace damaged tissues or organs, by the development of scaffolds with essential features, such as biocompatibility and functionality. Nowadays, recombinant polypeptides arise as promising candidates due to their tunability at the genetic level, affording exquisite control over the final physico-chemical properties and bioactivities. In particular, elastin-like recombinamers (ELRs) are genetically engineered polypeptides based on the repetition of the pentapeptide Val-Pro-Gly-X-Gly, found in the hydrophobic domains of tropoelastin, where X can be any amino acid except L-proline. These, ELRs exhibit a reversible phase transition in aqueous environments and their recombinant nature allows the inclusion of specific epitopes, such as cell adhesion, proteolytic sequences, and biologically active molecules such as growth factors. Interestingly, they can be chemically modified to obtain covalently cross-linked hydrogels through orthogonal and cytocompatible “click chemistry” reactions.
The first chapter of this thesis is dedicated to the spatiotemporal control of angiogenesis, which has been proven essential for the correct integration and long-term stability of the implant. To this end, we designed a three-dimensional (3D) model consisting of a coaxial binary ELR tubular construct that displays proteolytic sequences with fast and slow cleavage kinetics towards the urokinase plasminogen activator protease on its inner and outer part respectively. The ELRs further included the universal cell-adhesion domain (RGD) and a VEGF-mimetic tethered peptide (QK) to induce angiogenesis. In vitro studies evidenced the effect of the QK peptide on endothelial cell extension and anastomosis. The subcutaneous implantation of the 3D models in mice showed a guided cell infiltration and capillary formation in the pre-designed spatiotemporal arrangement of the construct. Furthermore, the ELR hydrogels induced a mild macrophage response that resolved over time, supporting the potential integration of the resorbable scaffold within the host tissue.
The second chapter study the preferential guidance of angiogenesis and neurogenesis in a spatiotemporal manner. In particular, we designed a 3D model ELR scaffold comprising two internal cylinders, with the pro-angiogenic peptide (QK) in one of them, and the neuronal cell adhesive peptide (IKVAV) in the vicinal one, both covalently tethered. In addition, these cylinders contain proteolytic sequences with fast cleavage kinetics towards the urokinase plasminogen activator enzyme and RGD cell adhesive domains. On the other hand, the outer part displays a slow-resorbable or non-protease-sensitive ELR hydrogel. In vitro studies demonstrated the effect of IKVAV epitope on neurite extension. The subcutaneous implantation of the 3D model ELR constructs in mice showed a guided cell infiltration accompanied by preferential angiogenesis or innervation on the respective QK and IKVAV containing cylinders, with a faster integration within the host tissue for the slow-resorbable scaffold.
The third chapter describes the development of a ready-to-use bi-leaflet transcatheter venous valve for the treatment of chronic venous insufficiency (CVI), a leading worldwide vascular disease. For this purpose, we combined (i) ELRs, (ii) a textile mesh reinforcement and (iii) a bioabsorbable magnesium stent. Burst strength analysis demonstrated mechanical properties suitable for vascular pressures, whereas equibiaxial analysis confirmed the anisotropic performance equivalent to the native saphenous vein valves. In vitro studies identified the non-thrombogenic, minimal hemolysis and self-endothelialization properties endowed by the ELR hydrogel. The hydrodynamic testing under pulsatile conditions revealed minimal regurgitation (< 10%) and pressure drop (< 5 mmHg) in accordance with values stated for functional venous valves, and no stagnation points. Furthermore, in vitro simulated transcatheter delivery showed the ability to withstand the implantation procedure.
In summary, the thesis presented herein provide new insights in the design and development of novel ELR-forming hydrogels to be used in tissue engineering and regenerative medicine applications.
