Development of biopolymer hydrogels as complex tissue engineering scaffolds (2016)

As global life expectancy increases, so does the demand for new technologies to address healthcare issues associated with disease and degradation of biological tissues and organs. Implantation is still a heavily relied upon method but patient demand is far greater than donor availability. Tissue engineering continues to show promise as a potential alternative to the reliance on donors and is fundamentally based on the concept of using a patient’s own cells to create new, healthy tissue. Strategies often include incorporation of cells into 3D culture scaffolds as a means of replicating in vivo culture environments in vitro, thus stimulating expression of more native cellular phenotypes. Biopolymer hydrogels are popular tools for this approach due to lack of cytotoxicity, high porosity and a capacity to both introduce chemical cues and tune mechanical properties. Research, however, often focuses on culturing a single cell type and scaffolds often only exhibit a single mechanical property. Additionally, there is difficulty in delivering different chemical cues to a single encapsulated population due to limitations in controlling diffusion of small molecules through hydrogel matrices. This places limitations on the capacity to fabricate scaffolds for repair of complex layered structures comprised of multiple matrix components and cell types. The work presented in this thesis focuses on development of biopolymer hydrogel culture systems for providing cells with multiple chemical and mechanical cues. This could provide a platform for creating scaffolds for regeneration of more complex, layered structures such as articular cartilage and osteochondral tissue.\n\nChapter 4 presents a study into using pulsed sonication to tune mechanical properties of hydrogel scaffolds of gellan gum (gellan). By applying different amplitudes of sonication, molecular weight was successfully tuned as evidenced by changes in intrinsic viscosity. This resulted in changes in both the dynamic viscosity of gellan solutions and the matrix stiffness and elasticity of gellan hydrogels. The impact on tuning mechanical properties of gellan hydrogels on cell behaviour was\ninvestigated using MC3T3 mouse pre-osteoblasts. A reduction in matrix stiffness via sonication coincided with a drop in expression of a key osteogenic marker, namely alkaline phosphatase. This demonstrated how tuning mechanical properties of gellan scaffolds with sonication could potentially be used to influence phenotype expression of many cell types with a possibility to influence cell differentiation.\n\nChapters 5 and 6 build on this concept of manipulating mechanical properties to influence cell behaviour in vitro. Fluid gels are presented as a material for supporting deposition of biopolymer solutions for additive layer manufacturing of tissue culture scaffolds. The aim was to use this system to fabricate scaffolds exhibiting multiple mechanical properties and multiple cell types. Chapter 5 presents development of the method with investigation into how fluid gel mechanical properties impacted on self-healing properties and a capacity to suspend gellan solutions. Furthermore, the effect of multiple deposition parameters (gellan viscosity, needle aperture and deposition speed) on the resolution of suspended structures was evaluated. Complex structures were fabricated including a mineralised gellan helix and layered, biphasic osteochondral-like scaffolds which were further investigated in Chapter 6. Cell-loaded, autologous osteochondral scaffolds were formed, implanted into human osteochondral tissue and cultured for 30 days. Analysis of mRNA expression revealed evidence of expression of chondrogenic and osteogenic phenotypes in the cartilage and bone regions of the scaffold. Moreover, there was evidence of an interface between both cell types and materials providing support to the conclusion that a 3D osteochondral culture model had been successfully generated.\n\nChapter 7 presents an alternative approach to creating gradient structures such as an osteochondral tissue culture scaffold. A fluidic hydrogel system is presented for controlled delivery of multiple chemical cues to a single rBMSC population. Delivery of osteogenic and chondrogenic differentiation cues was controlled by restricting diffusion of small molecules through the porous hydrogel network. After 6 weeks of culture, rBMSC’s displayed evidence of controlled differentiation down both osteogenic and chondrogenic lineages. Analysis of alkaline phosphatase activity paired with type I and II collagen mRNA synthesis revealed evidence of segregated populations of osteoblasts and chondrocytes. Additionally, there was evidence of an interface between the two, thus presenting another possible osteochondral culture model.