Speaker
Description
"Introduction
The fabrication of three-dimensional (3D) scaffolds able to promote a spatiotemporal guidance of cell infiltration, vascularization and innervation are of great interest in tissue engineering and regenerative medicine (TERM) applications. To this end, peptide sequences displaying fast and slow proteolytic rates towards urokinase plasminogen activator uPA, namely GTAR and DRIR, the QK vascular endothelial growth factor mimetic peptide and the IKVAV laminin-devired peptide for neuronal adhesion and proliferation have been described in literature as promising candidates [1,2]. Hydrogels based on Elastin-like recombinamers (ELRs) also have shown applicability in this regard, as their tailorable recombinant nature allow the genetic encoding of proteolytic sequences into their backbone and their peptide sequence enable the covalent tethering of bioactive residues such as the QK peptide or IKVAV.
Methodology
The ELRs prepared in this work were decorated with the GTAR or DRIR proteolytic sequences, the RGD cell-adhesion domain, the QK pro-angiogenic peptide or the IKVAV pro-innervation peptide. Furthermore, click catalyst-free crosslinkable domains were attached to the ELRs to produce the intended hydrogels. In vitro studies allow to determine the effect of IKVAV peptide over C6 glial cells adhesion, whereas the porous structure of the prepared hydrogels was evaluated by microscopic techniques. To assess the ability of ELRs to promote angiogenesis and neurogenesis, we fabricated a 3D construct containing two different cylindrical ELR hydrogels. In detail, the first cylinder contains the QK peptide with the GTAR fast-proteolytic sequence, whereas the second cylinder contains the IKVAV peptide with the GTAR fast-proteolytic sequence, in order to evaluate both bioactivities. In contrast, the outer part lack or display the DRIR slow-proteolytic sequence. Cell infiltration, vascularization and innervation were analyzed by histology and immunohistochemistry (IHC) upon subcutaneous implantation in Swiss CDR-1 mice with time.
Results
Microscope analysis showed the porous structure of the fabricated hydrogels. In vitro studies confirmed the effect of the IKVAV peptide over the cell-adhesion of C6 glial cells. Furthermore, in vivo studies of 3D ELR models revealed a marked increase in cell colonization in the interior tubes containing fast-proteolytic sequences, when compared to the outer part lacking or bearing slow-proteolytic sequences. Histology and IHC results showed the effect of the QK peptide triggering angiogenesis, and the effect of the IKVAV peptide triggering innervation in the pre-design orientation
Conclusions
The combination of proteolytic-sensitive sequences, the QK pro-angiogenic peptide and the IKVAV pro-innervation peptide into 3D ELR hydrogels confirmed the ability to spatiotemporally control angiogenesis and innervation in vivo. Specifically, the cylinder displaying the QK peptide promote a faster endothelialization, whereas the cylinder displaying the IKVAV peptide promote a faster innervation, following the pre-designed orientation. These results set the basis for the development of ELR-based scaffolds for TERM applications where the spatiotemporal control of vascularization and innervation play an important role.
References
1. González-Pérez, F. et al., Acta Biomater. 130 149–160 (2021).
2. Farrukh, A. et al., ACS Appl. Mater. Interfaces 10, 41129−41137 (2018)."
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