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ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Carnegie Mellon University

Natural biopolymers such as alginate have become important materials for a variety of biotechnology applications including drug delivery, cell encapsulation and tissue engineering. This expanding use has spurred the development of new approaches to engineer these materials at the nano- and microscales to better control cell interactions. Here we describe a method to fabricate freestanding alginate-based microfibers and microstructures with tunable geometries down to approximately 3 µm. To do this, a polydimethylsiloxane stamp is used to micromold alginate or alginate-fibrin blends onto a sacrificial layer of thermally-sensitive poly(N-isopropylacrylamide) (PIPAAm). A warm calcium chloride solution is then used to crosslink the alginate and, upon cooling below the lower critical solution temperature (∼32 °C), the PIPAAm layer dissolves and releases the alginate or alginate-fibrin as freestanding microfibers and microstructures. Proof-of-concept experiments demonstrate that C2C12 myoblasts seeded onto the alginate-fibrin microfibers polarize along the fiber length forming interconnected cell strands. Thus, we have developed the ability to engineer alginate-based microstructured materials that can selectively bind cells and direct cellular assembly.

Fabrication of freestanding alginate microfibers and microstructures for tissue engineering applications.

Our Patterning on Topography (PoT) printing technique enables fibronectin, laminin and other proteins to be applied to biomaterial surfaces in complex geometries that are inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface. Here, we used this method to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment.

Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces.

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

John M. Szymanski

Department of Biomedical Engineering

Carnegie Mellon University

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

John M. Szymanski

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Carnegie Mellon University

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Department of Biomedical Engineering