Aligned Networks of Engineered Fibrillar Fibronectin Guide Cellular Orientation and Motility

Abstract

The extracellular matrix (ECM) influences biological processes associated with tissue development and disease progression. However, robust cell‐free techniques to control fiber alignment of naturally derived ECM proteins, such as fibronectin (Fn), remain elusive. It is demonstrated that controlled hydrodynamics of Fn solutions at the air/fluid interface of porous tessellated polymer scaffolds (TPSs) generates suspended 3D fibrillar networks with alignment across multiple length scales (<1, 1–20 μm, extended to >1 mm). The direction of the fluid flow and the architecture of the polymeric supports influence protein solution flow profiles and, subsequently, the alignment of insoluble Fn fibrils. Aligned networks of fibrillar Fn characteristically alter fibroblast phenotype, indicated by increased directional orientation, enhanced nuclear and cytoskeletal polarity, and highly anisotropic and persistent cell motility when compared with nonaligned 3D networks and 2D substrates. Engineered extracellular matrices (EECMs) establish a critically needed tool for both fundamental and applied cell biology studies, with potential applications in diverse areas such as cancer biology and regenerative medicine.

Engineered extracellular matrices facilitate brain organoids from human pluripotent stem cells

“…3D jet writing is used to produce poly( d , l ‐lactide‐ co ‐glycolide, PLGA) scaffolds, as previously described 72 . Polymer PLGA scaffolds are mounted onto medical‐grade stainless steel and then this structure is placed into a fibronectin solution that undergoes rotation to induce fibrillogenesis and deposit an insoluble fibronectin (FN) matrix 20,22 . Human ESCs are then seeded onto EECMs.…”

“…Briefly, a highly porous polymer structure was generated by 3D jet writing and then placed between two medical‐grade stainless steel frames with window cut‐outs and secured with 1 μL of acrylate adhesive (Loctite, Rocky Hill, CT). To create EECMs of fibrillar fibronectin, this scaffolding was placed in a plasma fibronectin solution (Corning, Glendale, AZ) diluted in DPBS with no magnesium or calcium (Gibco, Grand Island, NY) to a concentration of 0.111 mg/mL and then tumbled in a microcentrifuge tube at 8 RPM in a 30°C chamber for at least 2 h. The air‐polymer‐protein interface induces fibrillogenesis of the fibronectin to convert the soluble protein to insoluble protein fibrils 20,22 . The resulting structure is an ultraporous polymer scaffold laden with a fibrillar fibronectin protein matrix, referred to collectively as EECM.…”

Fabrication of a Tailored, Hybrid Extracellular Matrix Composite

“…[25,30,31] On the other hand, in 2021 Neale et al demonstrated that shear-deployed FNfs could successfully induce an ordered and robust 3D network of FNfs, which can then be used to promote a directional, persistent migratory phenotype in fibroblasts. [32] Despite certain in vivo mimetic fibril structures and component hybridizations, they have yet to achieve the biologically complex structures and component configurations produced by the resident cells in a specific tissue. Therefore, it remains an important challenge to produce a tailored ECM scaffold that resembles native ECM through a simple and facile fabrication method.…”

An Extracellular Matrix‐Liposome Composite, a Novel Extracellular Matrix Delivery System for Accelerated Tissue Regeneration

“…[22] Most recently, Neale et al demonstrated that the aligned network of unfolded FN was critical for directing fibroblast orientation and migration. [23] These studies suggest that for higher wound-healing efficiency, FN used in tissue regeneration should undergo certain procedures to induce changes in its conformation. Some methods for doing so include physical binding on different substrates, modifying surfaces with chemical groups, or using gold nanoparticles.…”