Hydrazone Crosslinked Polysaccharide-based Hydrogels for Soft Tissue Engineering

Soft tissue engineering (TE) aims to generate new soft tissue by combining cells with a porous scaffold in order to replace or repair lost or diseased tissues. Soft TE and stem cell therapy can be used, for example, to replace damaged corneal stroma or to treat a damaged central nervous system (CNS). Blindness due to corneal dysfunction is currently treated by the transplantation of a donor cornea. However, alternative treatment methods are needed due to the limitations of the procedure. In order to mimic the functions of the cornea, scaffold material should at the minimum provide protection, transparency, and adequate refractive power. The human CNS suffers from low inbuilt regenerative capacity. Therefore, healing the damaged CNS with traditional medicine is insufficient and better treatment options, such as cell therapy combined with a supportive biomaterial scaffold, are needed. In addition to the general requirements, brain-mimicking mechanical properties and injectability, which enables minimally invasive surgery, are desired properties for neural applications. Hydrogels are considered to be the most attractive soft TE scaffolds due to their functional and structural similarities with the natural extracellular matrix (ECM). Hydrogels crosslinked with hydrazone chemistry are especially promising due to their advantageous characteristics, such as mild reaction conditions, versatility, simplicity, reversibility, and absence of toxic reagents and side products. The main aim of this thesis was to find an optimal hydrazone crosslinked hydrogel material for soft TE, especially for corneal and neural applications, and to characterize the hydrogels thoroughly using several methods. Different polysaccharides, i.e., hyaluronan (HA), alginate (AL), and gellan gum (GG), and synthetic polymer polyvinyl alcohol (PVA) were modified with complementary reactive aldehydeand hydrazide-groups to enable hydrazone crosslinking. Different HA-PVA-, AL-PVA-, GG-HA-, and HA-HA-based hydrogels were fabricated by varying the gel parameters, i.e., the degree of substitution and molecular weight of the gel components, the ratio of components, and the polymer concentration of the hydrogels. The mechanical, rheological, swelling/deswelling, enzymatic degradation, and diffusion properties of the hydrogels were characterized. Since microstructure plays an essential role in the control of hydrogel properties, the microstructure of the hydrogels was also characterized using rheologyand diffusion (fluorescence recovery after photobleaching, FRAP)-based methods. More precisely, the structural parameters, i.e., the mesh size, the average molecular weight of the polymer chain between neighboring crosslinks, and crosslinking density were evaluated. HA-HA-based hydrogels were intended for the regeneration of the corneal stroma. Therefore, their suitability for the delivery of human adipose stem cells (hASCs) was studied. The addition of collagen I, which is the main ECM component in the corneal stroma, was also tested. In addition, the suitability of HA-PVAand AL-PVA-based hydrogels to serve as 3D supportive and biomimicking materials for human pluripotent stem cell-derived neuronal cells was tested. GG-HA-based