title: Designing an in vitro model of the bone marrow niche using polyethylene (glycol) hydrogels and poly(ethyl acrylate) surfaces
Creators: name: Donnelly, Sam
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: Hydrogels, biomaterials, polymers, stem cells, mesenchymal stem cells, bone marrow niche.

description: Stem cells are of interest in many research areas due to their ability to selfrenew while also undergoing differentiation to regenerate the tissue it occupies (Weiss and Troyer, 2006). Stem cell fate is influenced by many different factors, these include stiffness, cell to cell interactions and secreted factors among others. The cells reside within unique microenvironments referred to as a stem cell niche where these factors are controlled to regulate the maintenance and differentiation of the stem cell population (Ferraro, Celso and Scadden, 2010). One such niche is that found in the bone marrow which hosts populations of haematopoietic stem cells (HSCs), responsible for haematopoiesis, and mesenchymal stem cells (MSCs), important for HSC regulation along with osteogenic, adipogenic and myogenic potential (Yin and Li, 2006; Pinho and Frenette, 2019). In this thesis we show the development of a model, combining both hydrogels and polymer surfaces, for MSC culture to produce a bone marrow like environment in vitro. Poly ethylene(glycol) (PEG) is a bioinert and biocompatible material that can be used to form hydrogels, for 3-dimensional cell culture (Raeber, Lutolf and Hubbell, 2005; Zhu, 2010). Models using hydrogels are becoming increasingly popular due to the control established over the physiochemical properties that are an important part of the in vivo ECM such as the water content and stiffness (Tse and Engler, 2010). These gels have also been developed to include bioactive ligands and even full length proteins, that would be found in the ECM, increasing cell interactions with the scaffold (Lutolf and Hubbell, 2005; Trujillo et al., 2020). In this thesis, we have utilised this PEG hydrogel system to produce a 3D scaffold that mimics the properties of the bone marrow stem cell niche. This includes tuning the stiffness of the gels to match that of the bone marrow and the introduction of fibronectin to enhance biological interactions with the gel. The gels were also shown to allow growth factor diffusion and retention an important property of the ECM in vivo. Poly(ethyl acrylate) (PEA) can be used to induce fibrillogenesis, where on contact with the polymer surface the spontaneous formation of fibronectin (FN) networks occurs (Salmerón-Sánchez et al., 2011; Cantini, González-García, et al., 2012; Llopis-Hernández et al., 2013). Formation of these protein networks exposes various cryptic domains, hidden in the globular formation, along the length of the protein which aid cell adhesion and allow the synergistic presentation of growth factors (Llopis-Hernández et al., 2016). In this thesis we investigate various methodologies, spin coating, plasma polymerisation, UV polymerisation and, surface initiated atomic transfer radical polymerisation, to introduce PEA into our model using different techniques to determine the success of each method. Once a PEA surface was established the interaction with FN was utilised to introduce growth factors into the model relevant to the bone marrow niche, BMP-2 and NGF. The final model was composed of PEA coated microcarrier polystyrene beads and degradable PEG hydrogels with full length FN incorporated. MSCs seeded onto the PEA beads were successfully encapsulated within the hydrogels and cultured for up to 3 weeks. By combining these two materials, PEG hydrogels and PEA, we can control physiochemical properties of the model progressing toward a more accurate in vitro representation of the bone marrow niche.