Force-induced fibronectin assembly and matrix remodeling in a 3D microtissue model of tissue morphogenesis

Encapsulations of cells in type-I collagen matrices are widely used three-dimensional (3D) in vitro models of wound healing and tissue morphogenesis and are common constructs for drug delivery and for in vivo implantation. As cells remodel the exogenous collagen scaffold, they also assemble a dense fibronectin (Fn) matrix that aids in tissue compaction; however, the spatio-temporal (re) organization of Fn and collagen in this setting has yet to be quantitatively investigated. Here, we utilized microfabricated tissue gauges (mu TUGs) to guide the contraction of microscale encapsulations of fibroblasts within collagen gels. We combined this system with a Foerster Radius Energy Transfer (FRET) labeled biosensor of Fn conformation to probe the organization, conformation and remodeling of both the exogenous collagen and the cell-assembled Fn matrices. We show that within hours, compact Fn from culture media adsorbed to the collagen scaffold. Over the course of tissue remodeling, this Fn-coated collagen scaffold was compacted into a thin, sparsely populated core around which cells assembled a dense fibrillar Fn shell that was rich in both cell and plasma derived Fn. This resulted in two separate Fn populations with different conformations (compact/adsorbed and extended/fibrillar) in microtissues. Cell contractility and microtissue geometry cooperated to remodel these two populations, resulting in spatial gradients in Fn conformation. Together, these results highlight an important spatio-temporal interplay between two prominent extracellular matrix (ECM) molecules (Fn and collagen) and cellular traction forces, and will have implications for future studies of the force-mediated remodeling events that occur within collagen scaffolds either in 3D in vitro models or within surgical implants in vivo.