• Karvinen, Jennika

Abstract

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.<br/><br/>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 aldehyde- and 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 rheology- and 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-PVA- and 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 hydrogels were not intended for any specific application, but their potential applicability as scaffold material for soft TE was tested in terms of their material properties.<br/><br/>Results showed that the fabrication of hydrazone crosslinked HA-PVA-, AL-PVA-, GGHA-, and HA-HA-based hydrogels from complementary reactive polymers was successful. The traditional crosslinking methods of GG were replaced with this method, and variable mechanical and physical properties were obtained by varying the gel parameters described earlier. These GG-HA-based hydrogels showed ionic nature of deswelling in the presence of cations. This means that the physical properties of the hydrogels can be controlled in different solution environments. They also showed that their stiffness was similar to that of soft tissues at low strains. It should be noted that due to the non-linear elastic behavior of the hydrogels (and tissues), the stiffness was also presented as a function of strain, instead of only giving the second-order elastic constants. Overall, the properties and injectability of GG-HA-based hydrogels supported their potential use in soft TE.<br/><br/>For the fabrication of the HA-HA-based hydrogels, two different types of aldehydeand hydrazide-modifications were tested. In order to promote hASC attachment and survival, collagen I was added to the hydrogel with better stability. This led to a reduced swelling ratio and increased hydrogel stiffness. Good optical properties (transparency and refractive index) were obtained with both gel types. However, even though all HAHA-based hydrogels showed good hASC survival directly after encapsulation, only in the collagen-containing hydrogel were cells with elongated morphology found. Furthermore, the cornea orga...

Topics

• porous
• density
• impedance spectroscopy
• microstructure
• polymer
• reactive
• molecular weight
• alcohol
• aldehyde