Contact guidance, the tendency of a cell to orient and migrate in response to topographical features such as parallel grooves, is a fundamental cell behavior key to many physiological processes and tissue engineering strategies. The goal of this project is to identify the signal that cells sense when interacting with aligned fibrils in a fibrin gel and exhibit contact guidance. The project will specifically test the hypotheses that the directional dependences of stiffness and adhesion of the network of aligned fibrils are the major contributing signals. This will be accomplished by systematically changing only the stiffness directional components of the fibrin network and observing whether or not the contact guidance response of fibroblasts in the fibrin gel changes, and then conducting similar studies with blocking antibodies to modulate adhesion and thereby modulate a potential adhesion directional dependent signal. Beyond the fundamental value in answering the question regarding what signal cells sense when detecting aligned fibrils, the research should lead to improved engineered tissues by providing a rationale for designing fibrillar scaffolds that yield the desired cell alignment and the associated tissue mechanical function.
Contact guidance is a fundamental cell behavior key to multiple physiological processes and tissue engineering strategies. While it has been highly studied on planar substrata, and even related to the cell cytoskeleton for certain geometries, it has been little studied in aligned fibrillar networks such as native tissues or the reconstituted collagen and fibrin gels that are used as tissue models. Despite its importance, the mechanism underlying cell contact guidance in an aligned fibrillar network has defied elucidation due to multiple interdependent signals that such a network presents to cells, including anisotropy of adhesion, porosity, and stiffness. The proposed research combines several key technologies along with appropriate experimental design to test the hypothesis that contact guidance is primarily driven by sensing of anisotropic stiffness or adhesion in the local fibrillar network. By forming magnetically-aligned fibrin gels with the same alignment strength, but crosslinked to different extents, the anisotropic stiffness and anisotropic adhesion hypotheses of contact guidance will be tested. Cell orientation behavior and the dynamic pattern of cell protrusions will be evaluated for fibroblasts seeded on the gel post-crosslinking, which preliminary data show increases stiffness anisotropy and yields stronger contact guidance. Crosslinking is achieved using a ruthenium-catalyzed photocrosslinking method that forms dityrosine bonds in tyrosine-rich fibrin fibrils. Experiments will be conducted to confirm preliminary findings that the crosslinking only affects stiffness anisotropy and does not affect anisotropy of adhesion or porosity, which are confounding effects in reported contact guidance studies to date. The effects of modifying cell adhesion using antibodies to binding sites on fibronectin and cognate b1 integrins will similarly be evaluated in these contact guidance experiments, which can be related to the anisotropic adhesion hypothesis. Adhesion will be quantified with a centrifugation assay of fibroblasts seeded on fibrin gels and porosity (more generally network microstructure) will be quantified from analysis of 3D reconstructions from confocal image stacks. Beyond the fundamental value of the proposed studies in answering the question--What signal do cells sense when detecting aligned fibrils?-- the research should lead to improved engineered tissues by providing a rationale for design of fibrillar scaffolds so as to yield the desired strength of cell alignment and the associated tissue mechanical function. Educational impact is achieved through enhanced courses, access to a shared imaging system that will facilitate interactions between students and engineers from local industries and collaborations expected with cell biologists.
