Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis

• Published in: PloS one 2016-08, Vol.11 (8), p.e0160369-e0160369
• Main Authors: Foolen, Jasper, Shiu, Jau-Ye, Mitsi, Maria, Zhang, Yang, Chen, Christopher S, Vogel, Viola
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Binding sites; Biology and Life Sciences; Calibration; Cancer; Cell adhesion & migration; Cell density; Cell Line; Cell migration; Cell Movement - drug effects; Collagen; Collagen - metabolism; Contractility; Cues; Dietary fiber; Embryo fibroblasts; Embryos; Engineering; Engineering and Technology; Extracellular matrix; Extracellular Matrix - metabolism; Fibers; Fibroblasts; Fibroblasts - cytology; Fibroblasts - metabolism; Fibronectin; Fibronectins; Fibronectins - metabolism; Fluorescence Resonance Energy Transfer; Gels; Genetic aspects; Growth factors; Health sciences; Imaging, Three-Dimensional; Kinases; Laboratories; Medicine and Health Sciences; Mice; Mice, Knockout; Morphogenesis; Morphogenesis - drug effects; Oligonucleotide Array Sequence Analysis; Physical Sciences; Physiological aspects; Plasma; Research and Analysis Methods; Software; Spatial distribution; Surface boundary layer; Three dimensional models; Tissue Engineering; Tissues; Vitronectin; Wound healing
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0160369

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of single fibronectin fibers enhanced the non-persistent migration of both Fnf/f and Fn-/- MEFs, the migration speed was increased for Fn-/- MEFs on plasma fibronectin fibers compared to Fnf/f MEFs. In contrast, the average speed was the same for all cells on collagen-coated Fn fibers. A Fn-FRET sensor revealed that fibronectin on average was more extended on the microtissue surface compared to fibronectin in the core. Gradients of collagen-to-fibronectin ratios and of the fraction of collagen-adsorbed