Bioprinting of Chondrocyte-Laden Hydrogel Constructs and their In-Vitro Characterization for Cartilage Tissue Engineering

Abstract Articular cartilage lines the ends of bones, provides low friction and load bearing, and allows for efficient joint movement. Once damaged, articular cartilage has difficulty of repairing itself due to lack of blood and nerve supply. Cartilage tissue engineering (CTE) aims to provide solutions to cartilage defects and involves the use of cells, scaffolds, and stimulating factors, alone or in combination. Hydrogel, a crosslinked polymeric network containing large amounts of water, is regarded as the ideal scaffolding material for CTE due to its structural similarity to native cartilage. Encapsulating chondrocytes in hydrogels is a promising approach to provide high cell seeding density, uniform cell distribution and a suitable microenvironment for encapsulated chondrocytes. However, fabrication of hydrogel scaffolds with desired microstructure/ internal structure and living cells is the key issue, which limits hydrogel’s applications in cartilage tissue engineering. To address these issues, this thesis aimed to bioprint cartilage constructs that incorporate living cells and characterize them in vitro for CTE. This aim was achieved via pursuing the following three specific objectives. The first objective was to fabricate CTE scaffolds based on the bioprinting technique and to study the influence of scaffold design on the mechanical performance. Gelatin and alginate mixtures were synthesized and printed into porous hydrogel scaffolds with the help of thermal/submerged ionic crosslinking process. The scaffold geometries, including stand orientation and the spacing between them, were adjusted for bioprinting and their influence on the scaffold mechanical properties were investigated. Results showed that there was a significant influence of internal design on the mechanical performance of printed hydrogel scaffolds and porosity, contact area between strands and spacing variation were three key factors that influence the mechanical performance of scaffolds. The second objective was to develop a 3D Bioplotting technique or process supplemented with the submerged cross-linking mechanism to fabricate alginate hydrogel constructs with living cells. In vitro biological performance of the printed alginate constructs was evaluated in terms of cell viability, proliferation and secretion of sulfated glycosaminoglycan (GAG) and Collagen type II. Chondrocytes were homogeneously distributed in the bioprinted hydrogels and cell viabilities were around 80%. Cartilage extracellular matrix (ECM) including glycosaminoglycan (GAG) and Collagen type II were synthesized by embedded chondrocytes, demonstrating the promising biocompatibility of this bioprinting technique. The third objective was to test the hypothesis that homogeneously dispersed hydroxyapatite in alginate hydrogel promotes the formation of calcified cartilage matrix. Cell growth, extracellular matrix (ECM) production, and mineralization potential were evaluated in the presence or absence of hydroxyapatite particles for comparison. The hydroxyapatite (HAP) phase was evenly dispersed into alginate hydrogel with the addition of a surfactant-sodium citrate (SC). Chondrocytes embedded in this composite hydrogel demonstrated expression of alkaline phosphatase (ALP) after 14 days of culture. Characteristic ECM in calcified cartilage such as minerals and Collagen type X showed a significantly higher synthesis in composite hydrogels with pre-incorporated HAP than that of alginate hydrogels. These results provided researchers with a facile technique to bioprint porous chondrocyte-laden hydrogel constructs for application in CTE and demonstrated a technique of inducing chondrocytes to synthesize calcified cartilage matrix by simply mixing HAP into hydrogel. Taken all together, this thesis presented the techniques/methods developed to bioprint cartilage constructs with living cells and would bring forward the fabrication of constructs for the repair of cartilage defects