Novel polyethylene glycol hydrogels incorporating RADA16 peptides as in vitro tissue mimetics

Abstract

Event Abstract Back to Event Novel polyethylene glycol hydrogels incorporating RADA16 peptides as in vitro tissue mimetics Susanna Piluso1, 2, Alexandros K. Leonidakis2, 3 and Jennifer Patterson1, 2 1 KU Leuven, Department of Materials Engineering, Belgium 2 KU Leuven, Prometheus, Division of Skeletal Tissue Engineering, Belgium 3 KU Leuven, Department of Mechanical Engineering, Belgium Introduction: Hydrogels are attractive materials as scaffolds for tissue engineering applications due to their structural similarities to the natural extracellular matrix (ECM). However many tissues, such as nerve, bone or cartilage, have a heterogeneous structure and are composed of fibrous proteins that also provide structural cues to guide cell behavior[1],[2]. To recapitulate the spatial heterogeneity of the ECM, we developed a hybrid hydrogel system based on polyethylene glycol (PEG) and RADA16 peptides which combine chemical crosslinking to allow modulation of hydrogel mechanical properties with self-assembly to form nanofibers, providing a biomimetic signal for modulating cells’ behavior. Materials and Methods: PEG-RADA16 hydrogels were prepared by conjugating a cysteine-containing RADA16 peptide to one arm of a vinyl sulfone functionalized 4-arm PEG and then crosslinking with a dithiol crosslinker. Hydrogels were characterized by swelling and dynamic mechanical analysis. Their morphology was investigated by Congo red staining, SEM, fluorescence microscopy and FRAP. Cell adhesion to and viability on hydrogels were investigated using 10T1/2 fibroblasts and a Live/Dead assay. Results and Discussion: PEG-RADA16 hydrogels were prepared via a Michael-type addition reaction. The effective conjugation of the RADA16 peptide to the PEG macromer was confirmed by quantifying the free peptide left in solution. The swelling ratio and Young’s modulus of the PEG-RADA16 hydrogels were similar to that for PEG hydrogels which did not contain RADA16, suggesting that the incorporation of the peptide does not disrupt the chemically crosslinked network. To confirm the self-assembly of the RADA16 peptides within the PEG network, hydrogels were stained by Congo red, which binds to peptides forming cross β-sheet structures[3] (Fig. 1a). Moreover, a fluorescent (F)-RADA16 peptide without cysteine, which should self-assemble with the covalently bound RADA16, was incorporated in the hydrogels and resulted in localized fluorescence within the RADA16 gels but not in controls (Fig. 1b&c). A diffusion study using FRAP and a release study of the F-RADA16 from PEG-RADA16 hydrogels both indicated that the fluorescent peptide bound within the gel and showed slower diffusion than that of F-RADA16 incorporated in a PEG hydrogel without RADA16. The ability of these hydrogels to support cell adhesion was confirmed by seeding fibroblasts on the hydrogels also functionalized with RGD. After 24 hours, the cells exhibited a spread morphology, and the majority of cells were viable (Fig. 1d). Conclusion: We developed novel hydrogels incorporating fibrous structures to mimic in vitro the spatial heterogeneity of natural tissues. In addition, the self-assembling domains within the network enable the site-specific incorporation and controlled release of peptide-based compounds. Therefore, this system might be useful as a dynamic cell culture system for studying complex cell behavior in 2D and 3D. This work was supported by the special research fund of the KU Leuven, grant number CREA/13/017.; This work is part of Prometheus, the R&D Division of Skeletal Tissue Engineering of the KU Leuven: http://www.kuleuven.be/prometheus.