1D). build predictive models of fibronectin-mediated microtissue fabrication on a variety of substrates. Keywords: fibronectin, extracellular matrix, self-assembly, cell adhesion, biomimetic material == 1 . Introduction == Modular tissue engineering is emerging as a bottom-up approach for the de novo fabrication of tissues and organs. In principle, the structural and functional features of complex tissues may be spatially recreated through the directed assembly of smaller, tissue-like building blocks [1]. Fabricating biomimetic units intended for modular assembly requires developing methods to reproducibly recreate the microstructural features of native tissue. Extracellular matrices (ECMs) are complex networks of proteins and polysaccharides that normally provide the structural and functional framework intended for cells to organize into three-dimensional (3D) tissue [2]. During embryonic development, cells spontaneously 2,2,2-Tribromoethanol organize into multicellular 3D structures in the absence of applied external forces by a process termed cellular self-assembly [3]. Inducing cells to self-assemble in vitro recapitulates many aspects of native tissue formation by allowing cells to organize into 3D structures using the ECM as a natural scaffold [4]. As such, engineering approaches that utilize cellular self-assembly to promote the 2,2,2-Tribromoethanol spontaneous organization of cells and ECM proteins into functional 3D microtissue units may provide a biologically inspired approach to modular tissue fabrication. A complex, integrated balance of cell-cell and cell-matrix adhesive interactions generates the mechanical causes and chemical signals necessary to control cell activity and tissue morphology during cellular self-assembly [5-8]. Fibronectin is a principal component of interstitial ECMs and plays a fundamental role in cell adhesion and tissue formation during embryonic development [9]. ECM fibronectin fibrils can localize to either cell-cell [5, 10-12] or cell-matrix [13] adhesions, where they stimulate Rho-mediated cell contractility [14, 15]. Fibronectin fibrils associated with intercellular adhesions support cellular self-assembly by promoting the cohesion of cells into tightly packed 3D clusters [5, 11, 16]. Conversely, fibronectin fibrils that localize basally to cell-matrix adhesions are associated with the disassembly of 2,2,2-Tribromoethanol cadherin-mediated cell-cell contacts [17], enhanced cell migration [18], and tumor cell metastasis [19], indicating a paradoxical role intended for fibronectin fibrils in cellular dyscohesion. As such, the global balance of causes and signals derived from fibronectin fibrils within either cell-cell or cell-matrix adhesions may be a central determinant of cellular self-assembly, tissue cohesion, and cell aggregate morphology. The insoluble, fibrillar form of fibronectin is the biologically active conformation of the molecule [20]. The polymerization of inactive, soluble fibronectin protomers into biologically active ECM fibrils occurs via a tightly controlled, cell-dependent process [20]. The bioactivity of ECM fibronectin has been localized, in part, to a matricryptic, heparin-binding site located within the first type III repeat of fibronectin (FNIII1) [21-23]. This bioactive site is inaccessible in soluble fibronectin, but becomes exposed upon polymerization into ECM fibrils [24]. Once assembled, highly elastic Rabbit Polyclonal to BRI3B ECM fibronectin fibrils can be further remodeled in response to cell and tissue-derived causes [23, 25, 26]. Compressive, tensile and/or shear stresses exerted on polymerized fibronectin fibrils may subsequently alter the spacing and/or availability of cell and protein binding sites within fibronectin fibrils [27], giving rise to a dynamic range of structurally and functionally distinct forms of ECM fibronectin. How different structural or conformational forms of fibrillar fibronectin influence cell and tissue function is only beginning to be understood. Recent studies provide evidence that relaxed fibronectin fibrils are associated with reduced 51 integrin activation [28], decreased cell proliferation [28], and increased cell cohesion [29]. In contrast, stretched/unfolded fibronectin fibrils are associated with enhanced cell proliferation [28] and migration [30], decreased adhesion [31], and enhanced growth factor secretion [31]. Further, manually stretched fibronectin fibers support increased osteogenic differentiation of mesenchymal stem cells [32]. To begin to understand how the balance of cement adhesive forces derived from conformationally-labile fibronectin fibrils impacts cellular self-assembly, we developed a series of recombinant fibronectin fragments that mimic different conformational states of ECM fibronectin. These fibronectin fragments were produced by directly coupling the open, bioactive heparin-binding fragment of FNIII1 (FNIII1H).