Graduation Year

2011

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Mechanical Engineering

Major Professor

Nathan Gallant, Ph.D.

Committee Member

Alex Volinsky, Ph.D.

Committee Member

Craig Lusk, Ph.D.

Committee Member

Ryan Toomey, Ph.D.

Committee Member

Jaya Padmanabhan, Ph.D.

Keywords

Micropatterning, Cell Traction, Integrin, Biomaterial, Tissue Engineering

Abstract

Cell adhesion to extracellular matrix (ECM) is critical to various cellular processes like cell spreading, migration, growth and apoptosis. At the tissue level, cell adhesion is important in the pathological and physiological processes that regulate the tissue morphogenesis. Cell adhesion to the ECM is primarily mediated by the integrin family of receptors. The receptors that are recruited to the surface are reinforced by structural and signaling proteins at the adhesive sites forming focal adhesions that connect the cytoskeleton to further stabilize the adhesions. The functional roles of these focal adhesions extend beyond stabilizing adhesions and transduce mechanical signals at the cell-ECM interface in various signaling events. The objective of this research is to analyze the role of the spatial distribution of the focal adhesions in stabilizing the cell adhesion to the ECM in relation to cell's internal force balance.

The central hypothesis was that peripheral focal adhesions stabilize cell adhesion to ECM by providing for maximum mechanical advantage for resisting detachment as explained by the membrane peeling mechanism. Micropatterning techniques combined with robust hydrodynamic shear assay were employed to test our hypothesis. However, technical difficulties in microcontact printing stamps with small and sparse features made it challenging to analyze the role of peripheral focal adhesions in stabilizing cell adhesion. To overcome this limitation, the roof collapse phenomenon in stamps with small and sparse features (low fill factor stamps) that was detrimental to the reproduction of the adhesive geometries required to test the hypothesis was analyzed. This analysis lead to the valuable insight that the non-uniform pressure distribution during initial contact caused by parallelism error during manual microcontact printing prevented accurate replication of features on the substrate. To this end, the template of the stamp was modified so that it included an annular column around the pattern zone that acted as a collapse barrier and prevented roof collapse propagation into the pattern zone. Employing this modified stamp, the required geometries for the cell adhesion analysis were successfully reproduced on the substrates with high throughput.

Adhesive areas were engineered with circular and annular patterns to discern the contribution of peripheral focal adhesions towards cell adhesion strength. The patterns were engineered such that two distinct geometries with either constant adhesive area or constant spreading area were obtained. The significance of annular patterns is that for the same total adhesive area as the circular pattern, the annular pattern provided for greater cell spreading thereby increasing the distance of the focal adhesions from the cell's center. The adhesion strength analysis was accomplished by utilizing hydrodynamic shear flow in a spinning disk device that was previously developed. The results indicate that for a constant total adhesive area, the annular patterns provide for greater adhesion strength by enhancing cell spreading area and providing for greater moment arm in resisting detachment due to shear.

The next examination was the effect of the cell's internal force balance in stabilizing the cell adhesion. The working hypothesis was that microtubules provide the necessary forces to resist the tensile forces expressed by the cell contractile machinery, thereby stabilizing cell adhesion. Since microtubule disruption is known to enhance cell contractility, its effect on the cell adhesion strength was examined. Moreover, the force balance in cells was altered by engineering adhesive areas so that the cells were either spherical or completely spread and then disrupted microtubules to understand the significance of the force balance in modulating the cell adhesion strength. The results indicated that disruption of microtubules in cells on adhesive islands resulted in a 10 fold decrease in adhesion strength compared to untreated controls whereas no significant change was observed in completely spread cells between treated and untreated controls. This is in surprising contrast to the previous contractility inhibition studies which indicate a less pronounced regulation of adhesion strength for both micropatterned and spread cells. Taken together, these findings suggest that the internal force balance regulated by cell shape strongly modulates the adhesion strength though the microtubule network.

In summary, this project elucidates the role of peripheral focal adhesions in regulating the cell adhesion strength. Furthermore, this study also establishes the importance of the internal force balance towards stabilizing the cell adhesion to the ECM through the microtubule network.

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