title: Engineered fibronectin-based hydrogels as novel three-dimensional microenvironments to promote microvasculature growth
Creators: name: Trujillo-Muñoz, Sara
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Hydrogels, fibronectin, tissue engineering, biomedical engineering, growth factors, VEGF, BMP-2, bone, angiogenesis.

description: Hydrogel systems are of growing interest as extracellular matrix (ECM) mimetics due to their intrinsic and controllable properties (e.g. water content, stiffness and/or architecture) (Chaudhuri et al., 2016; Tse &amp; Engler, 2010). Moreover, these systems can be further tailored with biologically active ligands (e.g. cell-adhesion motifs, protease-degradable peptides) and they can be used to instruct cell behaviour (DeVolder &amp; Kong, 2012; Lutolf &amp; Hubbell, 2005). In order to sophisticate hydrogels as ECM mimetics, significant efforts have been made to incorporate proteins or protein fragments into a hydrogel backbone (Almany &amp; Seliktar, 2005; Francisco et al., 2014; Mikaël M. Martino et al., 2011; Seidlits et al., 2011; Watarai et al., 2015). By incorporating full-length proteins, hydrogels could present binding domains for different molecules, which traditional peptide ligands lack. To this end, our work focuses on the formulation of hydrogels based on one of the major constituents of the ECM, fibronectin (FN). Fibronectin is a glycoprotein that presents binding sites for heparin, collagen, other fibronectin molecules and growth factors, amongst others (Pankov, 2002). In addition, it has been shown that the exploitation of growth factor-fibronectin synergistic interactions can alter cell behaviour (e.g. improve cell migration, proliferation or differentiation) (Llopis-hernández et al., 2016; M. M. Martino &amp; Hubbell, 2010). In this work, we have developed and optimised two strategies to covalently link fibronectin to synthetic (polyethylene glycol, PEG) and natural (hyaluronic acid, HA) polymers to form three-dimensional microenvironments to promote vascularisation. FNPEG hydrogels were formed using a Michael-type addition reaction that takes place at physiological pH and temperature. Using this approach, fibronectin was incorporated up to one mg·mL-1. The mechanical properties of this system were characterised together with the degradation profile when using protease-sensitive crosslinkers. Cytocompatibility was also studied using murine myoblasts and human endothelial cells. In addition, the interaction between vascular endothelial growth factor (VEGF) and fibronectin within FNPEG hydrogels was also explored, carrying out release and binding experiments. Fibronectin-VEGF interactions were investigated with endothelial cells, carrying out experiments of endothelial cell sprouting (i.e. angiogenesis) and endothelial cell reorganisation into multicellular structures (i.e. vasculogenesis assays). FNHA hydrogels were also fabricated, using a norbornene-modified HA and a ultraviolet (UV)-initiated thiol-ene chemistry. In this case, fibronectin was tethered to the HA backbone at different concentrations and up to two mg·mL-1. The mechanical properties of these hydrogels were characterised using different amounts of fibronectin. The morphology and yes associated protein (YAP) localisation of mesenchymal stem cells (MSCs) were studied using this system in two-dimensional (2D) cultures. Also, cytocompatibility of the hydrogels with MSCs was assessed in a three-dimensional (3D) culture system. In conclusion, this thesis presents a new family of ECM mimetics that incorporate fibronectin covalently bound to the PEG or HA backbone for the 3D encapsulation of cells and molecules. Moreover, the interaction between fibronectin and VEGF was studied with the intention to use these fibronectin-based hydrogels as efficient 3D proangiogenic microenvironments.