In this study, we have used FN-null MEFs adherent to native collagen gels to investigate the functional role of fibronectin fibrillogenesis on cell proliferation, cellular self-assembly, and microtissue morphology. Our data demonstrate that fibronectin specifically stimulates proliferation and self-assembly of cells adherent to native collagen fibrils into tissue-like structures by a mechanism that requires fibronectin matrix assembly. These responses were specific to intact fibronectin, as neither fibronectin fragments nor other ECM proteins triggered microtissue formation. Fibronectin-induced tissue body formation occurred in the absence of cell proliferation, indicating that proliferation and cellular self-assembly are independent responses to fibronectin matrix assembly.
Unlike tissue spheroids, in which balls of compacted cells form floating aggregates,15
cells at the collagen gel interface of fibronectin-induced tissue bodies were adherent and well spread on the collagen substrate. Altering either fibronectin or collagen concentration influenced the extent of cell proliferation as well as the ultimate shape of the microtissue. Increasing fibronectin concentrations induced a monotonic increase in cell proliferation, but had a biphasic effect on microtissue height, where higher fibronectin concentrations produced flatter, sheet-like structures with broad bases. In contrast, increasing collagen concentrations resulted in a dose-dependent inhibition of fibronectin-induced cell proliferation, but increased tissue body height, producing monolith-like structures with small bases. Taken together, these data indicate that the relative proportion of collagen and fibronectin fibrils polymerized into the ECM influences the extent of cell proliferation and the final shape of microtissues.
Cellular self-assembly in response to matrix fibronectin occurred only on polymeric collagen gels and not on monomeric collagen substrates, implying that the macromolecular organization of collagen fibrils plays a permissive role in fibronectin-induced microtissue formation. Indeed, results from our study provide support for the concept that cellular self-assembly may be controlled through local differences in adhesive and anti-adhesive forces that occur between cells and their substratum. A similar phenomenon was reported recently for bovine aortic endothelial cells wherein decreased cell–substrate adhesivity corresponded to increased cell–cell adhesion and endothelial network formation.18
Others have demonstrated that decreasing substrate rigidity permits the concurrent development of cell–matrix and cell–cell adhesions, while increasing substrate rigidity favors cell–matrix adhesions.19,35
As such, the biphasic effect of fibronectin on tissue body height observed in the current study may be a consequence of enhanced rigidity or adhesivity in response to increased fibronectin fibril formation that, in turn, increased cell–matrix adhesions and decreased cell–cell adhesions, leading to flatter structures.
In contrast to the biphasic effect of fibronectin concentration on tissue body morphology (), increasing the collagen concentration of the polymerized substrate reduced fibronectin-mediated cell spreading and increased tissue body height, leading to more compact structures (). Increasing the collagen concentration of polymerized type I collagen gels increases both the stiffness of the gel and the density of the collagen fibrils, but does not affect collagen fibril diameter.34
Others have shown that increasing substrate stiffness while maintaining ligand density promotes cell spreading and reduces cellular self-assembly.19
On the other hand, peak cell spreading on compliant collagen gels occurs at intermediate collagen densities36
and is reduced at high collagen fibril densities.37
Hence, in the current study, the reduction in tissue body area that occurred in response to increasing collagen concentrations was likely due to effects of increasing collagen fibril density, which in our model, predominated over the expected cellular response to increasing substrate stiffness. Importantly, these data indicate that cellular self-organization is an integrated response to chemical and mechanical signals arising from both fibronectin and collagen fibrils in the ECM. As such, it may be possible to direct the formation and shape of engineered tissues by spatially and/or temporally manipulating the composition and organization of the supporting ECM.
An extensive fibronectin matrix was polymerized by cells throughout the microtissues and was not limited to areas in direct contact with the collagen substrate or to well-spread cells. At least two distinct morphologies of fibronectin matrices were associated with tissue bodies. Elongated, fibrillar fibronectin appeared localized to outer regions of the tissue body and co-localized with proliferating cells. Fibrillar fibronectin staining was also observed in tracks leading away from tissue bodies, suggesting active matrix polymerization by cells during migration to the central structure. In addition, a pericellular form of the fibronectin matrix localized to central regions of the tissue bodies and was associated with nonproliferating cells. We hypothesize that regional variations in the organizational patterns of fibronectin fibrils, due in part to local variations in tension or rigidity, may give rise to distinct fibronectin matrices and, in turn, distinct cell behaviors.
Smooth muscle cells and fibroblasts seeded on native collagen fibrils fail to spread and display a reduced proliferative capacity associated with increased expression of growth inhibitory signaling molecules.38,39
Similarly, in the present study, FN-null MEFs seeded on polymeric collagen gels in the absence of fibronectin failed to spread and did not proliferate. In contrast, initiation of cell-mediated fibronectin matrix polymerization overcame the inhibitory effect of polymerized collagen and promoted cell proliferation. The mechanisms by which fibronectin matrix polymerization initiates cell proliferation on fibrillar collagen are not known. Growth-promoting intracellular signaling events initiated by cell adhesion to fibronectin fibrils40
may simply override growth-inhibitory signals induced by fibrillar collagen. Alternatively, binding of fibronectin to collagen41
may physically insulate growth-inhibitory epitopes of fibrillar collagen from cells or, conversely, form a pro-migratory complex that permits cell migration over the collagen substrate8
to initiate cell aggregation.
Several cell types, including endothelial cells, fibroblasts, and myocytes, sense and respond to substrate rigidity. For example, fibroblasts adherent to flexible collagen-coated polyacrylamide gels show reduced cell spreading and higher rates of motility than cells on more rigid collagen-coated substrates, which spread and form stable focal contacts.33
Further, substrate rigidity and intracellular cytoskeletal tension generation strongly influence fibronectin fibril formation.32,42
In turn, the formation of a fibronectin matrix enhances actin cytoskeletal tension13,43
and increases the mechanical tensile properties of cell-embedded collagen gels.14
In the current study, the relative proportion of collagen and fibronectin fibrils polymerized into the ECM influenced the formation and shape of microtissues, with increasing fibronectin levels leading to progressively flatter structures. These studies indicate that the local balance of ECM-derived forces, influenced in part by the extent of fibronectin fibril formation, contributes to the microenvironment of the cell to locally influence cellular behaviors essential for tissue morphogenesis.