3D Bioprinting Tissue Scaffolds with Living Cells for Tissue Engineering Applications
In tissue engineering, tissue scaffolds are used as temporary supports to promote regeneration of dysfunctional tissues. Of the available strategies, scaffolds produced from hydrogels and living cells show the great potential for their enhanced biological properties. To produce such scaffolds, three-dimensional (3D) bioprinting has evolved and is showing promise as a fabrication technique. However, its applications for fabricating customized hydrogel scaffolds containing living cells is still in its infancy. The major challenge with this approach is to print scaffolds while preserving cell viability and functionality as well as ensuring the structural integrity of the scaffold. To overcome this challenge, the present thesis aims to investigate the influences of hydrogel properties and the bioprinting process on cell viability and functionality, while also ensuing structural integrity, and on this basis, to develop bioprinting processes to produce tissue scaffolds with living cells for potential tissue engineering applications. This thesis first examined the influence of the mechanical properties of hydrogel on cell viability and functionality, utilizing alginate hydrogels and Schwann cells (the major glial cells of peripheral nervous system). Due to its poor cell adhesion, the alginate hydrogel was modified in this study with cell-adhesion supplements, including fibronectin, poly-l-lysine (PLL), and RGD (Arg-Gly-Asp) peptides. The RGD-modified alginate substrates were prepared with varying alginate concentrations in order to alter the mechanical properties of hydrogels, which were then seeded and encapsulated with Schwann cells. Cell viability and functionality, including proliferation, morphology, and expression of the extracellular matrix protein, were examined and correlated to the hydrogel mechanical properties. The results demonstrate that the viability and functionality of Schwann cells within alginate-based hydrogel vary with hydrogel mechanical properties, thus highlighting the importance of regulating the mechanical properties of hydrogel for improved cell viability and functionality in scaffold bioprinting. During the bioprinting process, cells are subject to process-induced forces, such as shear and extensional stresses, which can result in cell damage and therefore loss of cell function and even cell death. A method was developed to study the cell damage introduced by the shear and extensional stresses in the bioprinting process. A plate-and-cone rheometer was adopted to examine the effect of shear stress on cell damage. In these experiments, the relationship of cell damage to the shear stress was examined and quantified, which was then applied to identify the cell damage attributed to shear stress in bioprinting. On this basis, the damage to cells caused by extensional stress was inferred from the difference between the total cell damage occurring during the bioprinting process and the cell damage attributed to shear stress. This developed method allowed a relationship to be established between cell damage and both shear and extensional stresses during bioprinting. The experiments on this method provide insight into both the cell damage that occurs during bioprinting and the effect on cell viability and proliferative ability thereafter, which can be used to optimize the bioprinting process so as to preserve cell functionality. Based on the previous investigations, bioprinting processes were developed to fabricate tissue scaffolds containing Schwann cells for potential applications in nerve tissue engineering. Composite hydrogels consisting of alginate, fibrin, hyaluronic acid, and RGD peptide were prepared, and their hydrogel microstructures, mechanical stiffness after gelation, and capability to support the Schwann cell spreading were examined for identifying appropriate composite hydrogel for bioprinting processes. The flow behavior of composite hydrogel solutions and bioprinting process parameters (e.g., dispensing pressure, dispensing head speed, crosslinking process) were then examined with regard to their influence on the structure of the printed scaffolds and on this basis, bioprinting process were developed to fabricate scaffolds with Schwann cells. The functionality of Schwann cells within the printed scaffolds were assessed in terms of cell viability, proliferation, morphology, orientation, and protein expression, demonstrating that the printed scaffolds have potential for nerve tissue engineering applications. This thesis presents a comprehensive study on the bioprinting of scaffolds with living cells. The method developed and the study results will pave the way to fabricate scaffolds with living cells for more tissue engineering applications.
Tissue engineering, 3D bioprinting, Scaffold
Doctor of Philosophy (Ph.D.)