A biophysical study of the mechanotransduction of cells in controlled multi-dimensional microenvironments

Cells are mechanically coupled to the surrounding microenvironment which acts as local physical cues for triggering cells to carry fundamental activities. In the quest of understanding mechanotransduction of cells, it is essential to reveal the biocomplexity paradigm underlying cellular behaviors which are believed to be derived from the collective interactions among multiple signaling pathways. However, the study of cellular biomechanics is significantly impaired due to the great difficulties in developing characterization methods for probing mechanical responses in the sub-cellular level. Therefore the main objective of my thesis is to develop biophysical techniques for probing the biomechanical responses of cells in different culture systems and for facilitating the development of innovative and robust tissue-engineered platforms. Cell traction force microscopy (CTFM) has been recently developed to measure traction force generated by cell towards a deformable support. Cell traction force is a direct representative parameter of intracellular contractility established by the dynamic force equilibrium between cell and microenvironment. The implementation of micro-characterization by atomic force microscopy, Mooney-Rivilin constitutive law as the material model, and finite element method (FEM) in the force computation allowed us to analyze the three-dimensional (3D) stresses with higher accuracy and efficiency compared with previously reported methods. The shape of the vascular smooth muscle cells (SMCs) have been known as the fundamental characteristics of the vasoactivity in cardiovascular physiology. In order to investigate the effects of cells shape on cell traction force distribution, micro-contact printing technique and microchanneled scaffolds were introduced in the fabrication of highly controlled cell culture systems. It was found that the cell spreading area was linearly related to the overall intensity of traction force, whereas the cell elongation factor was governing the orientation of traction force. SMCs with high aspect ratio exhibited highly regulated force orientation along the cell long axis due to the organization of stress fiber and the preferential membrane extension along the cell long axis. SMCs seeded on microchanneled scaffold were first appeared as synthetic and proliferative and adopted contractile phenotypes upon confluence. Due to the microtopographic features, SMCs seeded at the edges of microchannels adopted elongated morphology and established significant traction force due to the extra dimensionality provided by microwalls. The forces were found to be propagated from the edge to the centre of microchannels via the cell-cell interactions to promote contractile phenotype and cell alignment at confluence. The effects of microtopographic features on the collective traction force in the formation of aligned cell sheet were first demonstrated to study the biophysical interactions among multiple cells and microenvironment. Lastly, the encapsulated chondrocytes in the 3D hydrogel were observed to exhibit abundant neo-tissue outgrowth at the edge of the hydrogel, this phenomenon was thus termed as “edge flourish” (EF). In order to fully elucidate EF phenomenon in the cartilage tissue engineering, in-depth biomechanical characterizations of such phenomenon are needed. Histological staining, 3D multiple-particle tracking assay, and newly developed surface tension characterization assay were introduced to elucidate the mechanobiological activities of EF phenomenon. This phenomenon was shown to be driven by the oriented outgrowth of chondrocytic isogenous groups located along the edge of hydrogel. The isogenous groups exhibited directed outgrowth towards the surface of the hydrogel and gradually generated substantial surface tension on the interface of hydrogel and medium. Ultimately, the encapsulated chondrocytes closest to the hydrogel/medium interface sprouted out of the hydrogel spontaneously to form a layer of rich proliferative and chondrocytic extracellular matrix secretive chondrocytes at the surface of hydrogel. The in-depth understandings of neo-tissue formation in 3D microenvironment are expected to inspire the innovative hydrogel scaffold for cartilage or bone tissue engineering.DOCTOR OF PHILOSOPHY (SCBE