| Summary: | Engineering functional blood vessels play an important role in both tissue regeneration and wound healing. However, engineered blood vessel equivalent remains to be far from clinical realization due to the difficulty in reconstituting the complex three-layer capillary histology. Among all major challenges, the elucidation on the intricate mechanisms involved in tissue morphogenesis of functional blood vessels engineering in vivo significantly hinders the development of engineered blood vessel equivalent in vitro. Thus the search for a highly biocompatible material for vascular tissue culture is inevitable in the design of artificial blood vessel. During the past several decades, novel biomaterial scaffold for cell attachment and culture have been developed for applications in tissue engineering, biosensing and regeneration medicine.
In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of the tunica media consisted of vascular smooth muscle cells (SMCs) which is known to be critical for triggering vasoconstriction and vasodilatation in circulatory system. To simulate the physiological functions of blood vessel, the establishment of hyperplasticity of SMCs in vitro has been focused in several previous studies to produce viable synthetic SMCs in the initial stage and develop into quiescent contractile SMCs with highly aligned orientation by the use of microchannel arrays. However, the mechanism of such hyperplastic transformation occurring inside a microchannel to produce highly regulated orientation and phenotype of SMCs sheet remained to be elucidated.
Complex mechanotransduction between cells and the surrounding microenvironment such as the neighboring cells, the extracellular matrix (ECM) and biomaterial scaffold, plays an important role in the regulation of various physiological processes. Among these interactions, cell traction force (CTF) is a crucial representative parameter in cellular functions. The conventional traction force assay for single cell measurement was extended herein for applications in three dimensional cell aggregates. Generally, the cooperative multiple cell-cell and cell-microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite element modeling. Therefore, the main objective of my thesis is to develop different types of micropatterned cell culture platforms for carrying new cell-microenvironment mechanotransduction study in-vitro. At the same time, conventional cell traction force microscopy (CTFM) has been further developed for executing biophysical study of collective SMCs grown in the micropatterned scaffolds as mentioned above.
In tissue regeneration, the geometrical and mechanical properties of the microenvironment surrounding cells play an important role in cell physiology including migration, proliferation, differentiation and apoptosis. To mimic physiological conditions in-vivo, micropatterning of cells by applying soft lithography provides new possibilities for conducting experimental studies on cell mechanotransduction. In my thesis, I have designed and developed several types of biomaterial scaffolds with various unique features such as parallel discontinuous microwalls, concentric microwalls and bifurcate microgrooves by applying microfabrication technology and soft lithography. First of all, such microfabricated arrays were coated by polyacrylamide gel with embedded fluorescence microbeads in order to probe the mechanotransduction of multi-cell system such as smooth muscle cell layer. Secondly, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of polyacrylamide gel coating on the microfabricated arrays. In the first part of my thesis, a novel microfabricated array of discontinuous microwall was developed for stimulating the formation of highly aligned layer of SMCs. At the same time, the cell shape, density and alignment orientation of SMCs during cell alignment and cell layer formation were probed with CTFM. Secondly, the collective mechanotransduction of vascular tissue during circumferential alignment was studied with the use concentric micro-walls and CTFM. Finally, the morphological switch and orientated adaptation process of SMCs within the bifurcate microgrooves were monitored to reveal the influences of different bifurcation angles on the mechanotransduction of SMC layer.
In the first part of my thesis, our results from discontinuous microwalls showed that the traction forces of highly aligned cells lying in the middle region between the two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. In the second part of my work, it was found that the mechanotransduction of SMC layer grown between the circular microwalls was significantly altered compared to the SMC layer grown on a flat or planar substrate. The results indicated the presence of an optimal region for formation of circumferentially aligned SMC layer verified by both quantitative analysis and biomechanical study. Besides, the distributions of traction forces and von Mises stresses during cell alignment were affected by the cellular mechanotransduction from the extracellular physical constraints and cell-cell interactions. In the last part of my thesis, the relatively low stress zone occurred around 90° bifurcation provided a novel explanation for the early localization and development of atherosclerosis.
In order to verify our previous postulation on the change of cytoskeleton structure of SMCs due to the formation of collective cell sheet and establish the biological differences between microwall-constrained SMCs and control SMCs, the 2D nano-LC-MS/MS and real time quantitative polymerase chain reaction (RT-qPCR) analyses were carried in addition to our biophysical studies. The online 2D LC-MS/MS analysis verified the modulation of focal adhesion formation under the influence of parallel microwalls through the regulation in the expression of three key cytoskeletal proteins. The real time quantitative polymerase chain reaction (RT-qPCR) analysis of actin further revealed the relation between the monolayer-generated traction forces and the direction of circumferential aligned actin filaments. Also, the immunofluorescence staining of SMC sheet demonstrated that the collective mechanotransduction induced by 3D topographic cues was correlated with the variation of actin and vinculin expression.
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