A Methodology to Study Cell Deformation Behaviour within Native and Tissue Engineered Cartilage Subjected to Incremental Compression Using Confocal Microscopy

In cartilage tissue engineering, it is vital that an appropriate amount of stress is received by the cells to favour for the promotion of a chondrocyte-like phenotype and induction of deposition of an appropriate matrix for tissue maturation. The aim of this thesis was to determine the effects of incremental tissue compression on cell deformation in native and tissue engineered cartilage, in an attempt to better understand Finlay et al. (2016)’s hypothesised mechanism in developing cartilage-like constructs. A novel compression device was developed to apply compressive tissue strains and simultaneous allow visualisation of cells within native and tissue engineered cartilage disks in real time. A dual staining method of Hoechst 33342 and CellMask Green plasma membrane stains with superior retention of fluorescence signal following the capture of sequential images was developed and used to visualise, track and image cell morphology in native and tissue engineered cartilage. Both were used to address the aim of this thesis. Significant changes in deformation of superficial zone chondrocytes within cartilage disks were observed under 10 % and 15 % compressive strains and it is believed that these changes were largely influenced by the local ECM environment as well as the pericellular matrix. For engineered constructs, limited amount of cartilage-like matrix was seen in contrast to Finlay et al. (2016). The reason for the differences could be related to the starting scaffold porosity used in the constructs, emphasising the need to understand the relationships between scaffold porosity, cell seeding density and time to cell differentiation. Limited change in synoviocyte morphology was observed within loaded constructs under the estimated strain experienced by the construct during mechanical loading and under 28 % compressive strain. The observed effects were likely be influenced by the microenvironment around the measured synoviocytes. It is concluded that a novel methodology capable to visualise, track, image and quantify changes in cell morphology within native cartilage disks and tissue engineered cartilage constructs under static compressive tissue strains has been achieved. This provides a platform upon which to build for future cell deformation experimental studies