Fibronectin fibril alignment is established upon initiation of extracellular matrix assembly.

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the functioning of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned meshwork matrix form matrix assembly sites around the cell periphery, but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked, since we discovered that transforming growth factor beta (TGF-ß1) enhances matrix assembly site polarity and assembly of aligned fibrils independent of cell elongation. We conclude that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites that form during the initial stages of cell-FN interactions.

A multilayered scaffold for regeneration of smooth muscle and connective tissue layers.

Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here, we describe the creation of a novel multilayered scaffold with distinct fiber organizations-aligned to unaligned and dense to porous-to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine-derived terpolymer, yielding densely-packed, aligned fibers that transition into randomly-oriented fibers of increasing diameter and porosity. We demonstrate that differently-oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue-specific protein expression in the tube were similar to the flat-sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations.