Fibronectin as a key regulator of macromolecular crowding-enhanced extracellular matrix assembly

Abstract

Tissue engineers seek to replace damaged and diseased tissues in the body with in vitro-grown tissue substitutes. This involves seeding cells onto an engineered scaffold and providing the right chemical and physical cues to promote cell-mediated tissue assembly and tissue-specific remodeling. One of the key factors limiting the success of tissue engineering is the long culture time required for cells to assemble matrix in the highly dilute cell culture medium. However, the in vivo environment where matrix assembly naturally occurs is not dilute like cell culture medium, but rather highly crowded by macromolecules. Studies have shown that adding soluble macromolecules to cell culture medium to mimic the natural macromolecular crowding enables cells to more effectively build matrix by promoting molecular interactions. Most mechanistic studies of how crowding enhances matrix assembly focus on the impact of crowding on collagen fiber assembly and largely ignore the highly abundant provisional matrix protein fibronectin. Fibronectin is the first matrix protein assembled by cells and it serves as a template to nucleate collagen fibers. Collagen cannot be assembled in the absence of fibronectin, and the tensional state of fibronectin impacts collagen binding. In this work we asked for the first time what role fibronectin plays in the underpinning mechanism of crowding-enhanced matrix assembly. First, we took a close look at fibronectin and collagen I assembly over time and showed that the assembly of both is increased by the neutral crowding molecule Ficoll and the two are colocalized in the early stages of matrix assembly. We then asked how crowding enhances fibronectin assembly, since this process is cell tension-mediated and does not rely on enzymatic cleavage like collagen assembly does. We found that there were no changes in cell mechanical behavior, including contractility, that could explain the increased assembly. We then tuned to the fibronectin and found that Ficoll doubles the amount of surface adherent fibronectin, which can be readily harvested by fibroblasts and speed up fibrillogenesis, thus providing evidence of the first mechanism for crowding enhanced fibronectin assembly. Next we asked what role fibronectin plays in the assembly of collagen in the presence of crowding. We used our well-validated Fn-FRET probe to reveal that Ficoll crowding upregulates the total amount of fibronectin fibers in a low-tension state through upregulating fibronectin assembly. Since unstretched fibronectin fibers have more collagen binding sites to nucleate the onset of collagen fibrillogenesis, our data suggests that the Ficoll-induced upregulation of low-tension fibronectin fibers contributes to enhanced collagen assembly in crowded conditions. We then manipulated the fibronectin assembly process by cross-linking fibronectin to the glass surface and found that fibroblasts could no longer harvest the coating to assemble early fibers. Remarkably, this suppressed the Ficoll-induced upregulation of collagen assembly, demonstrating that a fibronectin fiber template is still necessary, even when crowding is there to drive collagen processing and assembly. Finally, we asked how our findings could be useful to tissue engineers. We showed that adding a preadsorbed fibronectin coating to a material doubles the early matrix assembly in the presence of crowding. This is highly significant because the long culture time required to produce a tissue substitute in vitro is a key factor limiting the clinical success of tissue engineering. In summary, we showed that the accelerated collagen I assembly as induced by crowding is regulated by cell access to fibronectin and we provided a preadsorbed fibronectin coating as a new tool for tissue engineers to better harness the power of macromolecular crowding to increase the speed of matrix assembly.