Resumen de Engineering responsive and biomimetic material based on elastin-like recombinamers for biomedical application
Regenerative Medicine is a well-established field of science that aims to replace, engineer and regenerate human cells, damaged tissues or organs to restore their normal function. This branch of translational research finds a deep interest in the Science of Biomaterials; indeed, the knowledge acquired in that field goes proportionally with the development of novel biomaterials. There is a great need in developing advanced biomaterials capable to fulfil the requirements of stability and bioactivity for their application in biomedicine. Moreover, considering the complexity of the human body, this system needs a certain rate of versatility in order to be tailored to a specific area of application. For all these reasons, recombinant proteins are an interesting approach, in which, elastin-like recombinamers (ELRs) represent one of the most promising biomaterials. ELRs are obtained through DNA recombinant technology, which allows the precise control at the genetic level, affording exquisite control over final protein functionality. ELRs are protein-based polypeptides that comprise repetitive units of the Val−Pro−Gly−X−Gly (VPGXG)n pentapeptide, in which X (guest residue) could be any amino acid except L-proline. In terms of biomaterial design, ELRs show several outstanding properties. ELRs are inspired by elastin, which is a component of natural extracellular matrix (ECM), showing excellent biocompatibility. One of the most important features of ELRs is that they exhibit thermo-responsiveness; this is due to the change of protein conformation above the so-called transition temperature (Tt), which depends on the amino acid composition of the polymer. Moreover, according to the ELRs design, they can be processed as several supramolecular structures, such as micelles, nanoparticles, films, and hydrogels. The large variety of ELRs, both in terms of structures and bioactivity, permits the application of these protein-based biomaterials to diverse biomedical applications.
This Thesis represents a sort of journey towards the exploration of the evolution of ELRs as a powerful tool with great potential in the biomedical field. The first part of this Thesis is dedicated to the description of the history of ELRs from their ancient chemical origin as ELPs (Elastin-like Polypeptides) to the most cutting‐edge bioproduction techniques becoming into ELRs. Moreover, it is reported an exhaustive explanation of how ELRs can be processed in many forms (aggregates, fibers, layers, nanoparticles, or hydrogels), giving examples of their great potential in many fields, including drug delivery, tissue engineering, protein purification, anticancer gene therapies, and nanovaccines. Moreover, considering their large interest, one chapter of this Thesis is dedicated to the ELRs as biomaterial forming hydrogels for tissue regeneration and repair. The different mechanisms of gelation are reported, and it is given an overview of the possible applications in tissue engineering, such as osteochondral application, (cardio-)vascular tissue regeneration, and ocular prostheses.
The first experimental work of this Thesis is dedicated to the development of novel ELRs-based hydrogel for cartilage repair. Tissue 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. In this work, it has been 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 study, it was set up and optimized a pre-annealing treatment based on the evolution of silk motifs into β-sheet structures in order to fulfil the required mechanical properties of hydrogels for cartilage repair. The new pre-annealed 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 glycosaminoglycans and collagen. Finally, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage, which leads to the successful regeneration of hyaline cartilage in an ex vivo model.
Not only the physically cross-linked hydrogels have been investigated; indeed, an in situ chemically cross-linked hydrogels have been developed for osteochondral repair. Moreover, another bioactive composition of this biomaterial has been tested; this ELRs-based hydrogel has been designed containing bioactive sequences, such as the well knows adhesion sequences RGD and REDV, and the elastase target domain VGVAPG that provides proteolytic sensitivity to the biomaterial. Compared to the previous study reported in this Thesis, where the ex vivo platform was used, the regeneration properties of the chemically cross-linked ELRs hydrogel were evaluated with an in vivo study. Furthermore, it has been made a comparison between the usage of that biomaterial itself, and the biomaterial embedded with cells (tissue engineering). Both the ELR-based hydrogel alone and the ELR-based hydrogel embedded with rabbit Mesenchymal Stem Cells (rMSCs) were tested for the regeneration of critical subchondral defects in 10 New Zealand rabbits. Thus, cylindrical osteochondral defects were filled with an aqueous solution of ELRs. The animals were sacrificed at 4 months for histological and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality and evaluation of the new matrix in the defects. Although both groups helped cartilage regeneration, the results suggest that the specific composition of the rMSCs-containing hydrogel permitted adequate bone regeneration, whereas the ELR-based hydrogel alone led to an excellent regeneration of hyaline cartilage. In conclusion, the ELR cross-linker solution can be easily delivered and forms a stable, well-integrated hydrogel that supports infiltration and de novo matrix synthesis.
As it has been reported above, the aim of this Thesis is to explore the possibilities of ELRs as a powerful tool capable of containing various bioactivities with great potential in the biomedical field. As a further step in the evolutional process towards advanced bioactive ELRs, one objective of this Thesis is to combine these two diametrically opposed approaches in a new hybrid biomaterial. Biomaterial design in tissue engineering aims to identify appropriate cellular microenvironments in which cells can grow and guide new tissue formation. Despite the large diversity of synthetic polymers available for regenerative medicine, most of them fail to fully match the functional properties of their native counterparts. In this work, we have combined the strategy of synthetic peptides with the DNA recombinant techniques generating a new hybrid biomaterial. Human umbilical vein endothelial cells (HUVECs) adhesion and proliferation were studied over the ELRs covalently functionalized with each three high-affinity and selectivity αvβ3- and α5β1-binding bicyclic RGD peptides. Next, to the bicycles, ELRs were also functionalized with various integrin-binding benchmark peptides, i.e. knottin-RGD, cyclo-[KRGDf] and GRGDS, allowing for better classification of the obtained results. Covalent functionalization with the RGD peptides, as validated by MALDI-TOF analysis, guarantees flexibility and a minimal steric hindrance for interactions with cellular integrins. In addition to the covalently modified RGD-ELRs, it was also synthesized another benchmark ELR comprising RGD as part of the backbone. HUVECs adhesion and proliferation analysis using the PicoGreen® assay revealed a higher short-term adhesion and proliferative capacity of cells on ELR surfaces functionalized with high affinity, integrin-binding bicyclic RGD-peptides compared with the ELRs containing RGD in the backbone.
Finally, in order to move forward the evolution of ELRs as advanced biomaterial showing multiple modular behaviours, it has been developed a new smart ELRs with the aim of targeting complex biomedical system. Taking advantages by the recombinant DNA techniques, it has been developed a smart biomaterial based on Elastin-like Recombinamers with allosteric control of RNase A activity. The ELRs design comprised bioactive sequences sensible to external stimuli; It was designed containing ten consensus sequence phosphorylation sites regularly distributed along the ELR, and by the Ribonuclease A active sequence (RNase A). According to the position of RNase A relative to the ELR backbone, several variants of the smart-ELR have been produced. The smart-ELRs were further characterized by several experimental techniques (SDS-PAGE, FTIR and HPLC-HR-MS), showing the capacity to be fully phosphorylated and further de-phosphorylated. This reversible system was then investigated by turbidity analysis, demonstrating an evident shift in Temperature transition (Tt) value due to the (de-)phosphorylation. Finally, the allosteric control of the RNase A catalytic activity was evaluated for all the different variants of the smart-ELR. The allosteric control of RNase A activity by the selective phosphorylation was demonstrated. Moreover, the different designs of the smart-ELRs exhibited different catalytic activity, showing the importance of the RNase A position according to the ELR backbone.
In summary, the works reported in this Thesis provides an overview of the ELRs as engineering responsive and biomimetic material for the biomedical application. Specifically, it describes in the first part the base knowledge of this class of recombinant protein, focussing on the different structures that can be formed and their great potential in many biomedical fields. Furthermore, special interest has been dedicated to the potential of ELRs as biomaterial forming hydrogels for tissue regeneration and repair. Following this trend, for osteochondral repair application, two types of ELRs based hydrogel showing different bioactivities and gelation mechanisms have been developed and tested with an ex vivo and an in vivo study. Finally, taking advantages from the DNA recombinant technology which allows the precise control at the genetic level, news advanced ELRs have been developed. A new class of hybrid ELRs combining the synthetic synthesis of peptides with the DNA recombinant techniques has been designed, and a new generation of smart-ELRs with allosteric control has been obtained.
