(10g) Biomimetic Fibronectin As Scalable Three-Dimensional Networks Enable Precise Extracellular Matrix Engineering

Introduction: The extracellular matrix (ECM) is a complex amalgam of proteins and polysaccharides that actively influences cell fate via biochemical and biophysical cues [1]. Fibronectin (Fn) is the bedrock of many native tissues, existing as a fibrillar protein network that sequesters cell signaling factors and other ECM macromolecules [2]. Despite its ubiquity in mammalian biology, it remains an outstanding challenge to recapitulate critical aspects of Fn in native ECM, such as capturing its fibrillar morphology in a scalable, three-dimensional (3D) in vitro construct. Native Fn fibrillogensis occurs through non-covalent Fn-Fn interactions following integrin mediated stretching of solute Fn by cells. Our lab recently pioneered an in vitro technique that enables the creation of 3D fibrillar Fn networks suspended across hyper-porous polymer scaffolds on the millimeter length scale by mimicking natural Fn fibrillogenesis [3]. This is achieved by shearing a Fn solution across a polymeric scaffold at the solution/air interface, which promotes the formation of robust 3D networks without the use of crosslinkers or denaturants. These engineered extracellular matrices (EECMs) are readily compatible with conventional cell culture techniques and analysis modalities. Furthermore, by leveraging control over the fluid-shear interface, tuning the architecture and orientation of the fibrillar networks is possible. Recognizing the complexity of native tissues, these EECMs can be combined with other ECM macromolecules to create multi-component mixtures in an attempt to more closely model the healthy or diseased state of various tissue niches.

Materials and Methods: Polymer scaffolds were coated dynamically at 8 rotations per minute (RPM) with human fibronectin (Corning Inc, Corning, NY) that was diluted to a concentration of 111 µg/mL in calcium/magnesium free Dulbecco’s phosphate buffered saline (DPBS). Polymer scaffolds were comprised of either SU-8 (made via photolithography) or poly(lactic-co-glycolic acid) produced via a novel electro-jetwriting process [4]. COMSOL Multiphysics® 5.3a was used to model fluid flow patterns of the Fn solution across the tessellated polymer microstructures in order to understand and control fibril morphology. To create aEECMs, scaffolds with a rectangular pore shape and gap length of 497 µm ±5 µm and height of 112 µm ±1.7 µm were used. Confocal laser scanning microscopy (CLSM) and time-lapse epifluorescence microscopy were used to image the Fn networks and/or cells. NIH-3T3 fibroblasts were used for cell migration studies. Cell tracks were fit to an anisotropic persistent random walk model (APRW) published by Wu et al. to quantitatively assess motility parameters [5]. Asterisks indicate p ≤ 0.05 * , p ≤ 0.01 ** , p ≤ 0.001 *** assessed via the Kruskal-Wallis test followed by the Conover test.

Results and Discussion: The EECMs are insoluble and stable when subjected to 1% deoxycholate, which was classically used to characterize cell secreted Fn networks in vitro [6]. The EECMs also facilitated growth of various cell types, including patient derived tumor cells [3]. Additionally, this shear induced fibrillogenesis technique revealed type-III domains of Fn that were only conformationally active when the Fn was presented in a fibrillar EECM and not when statically adsorbed onto a synthetic fiber surface [3]. Furthermore, we have demonstrated that the fluid shear-interface can be engineered during fibril assembly to tailor fluid flow profiles, enabling the creation of precisely aligned EECMs (aEECMs), see Figure 1. These aEECMs significantly influenced fibroblast fate by guiding cell orientation, inducing cytoskeletal and nuclear polarity, and promoting a dramatic increase in directionally persistent cell motility. These intriguing findings indicate aligned fibrillar Fn plays a critical role in guiding cell migration, which collectively make up the basis of a recent manuscript submission [7]. By taking advantage of Fn’s native binding activity and by developing new chemical conjugation strategies, multi-component fibrillar EECMs have been produced to include collagen-I and polysaccharides such as hyaluronic acid. In these systems, we demonstrate retention of critical biological characteristics and fibrillar morphology.

Conclusions and Outlook: These EECMs mark a novel contribution to tissue engineering by producing insoluble, 3D fibrillar Fn networks with controlled alignment across millimeter length scales without the use of solvents or crosslinkers. This is achieved by inducing Fn-Fn interactions from fluid shearing. These EECMs have been used to expand many established cell lines and primary patient cells that failed to expand using traditional methods. Additionally, aligned Fn networks can be engineered to guide cellular migration and model the aligned architecture present in many native tissue niches. With further work, these EECMs may lead to novel in vitro models for studying cell behavior with application in the fields of regenerative engineering, tumor microenvironment engineering, and drug discovery.

References:

[1] Frantz, C., Stewart, K. M. & Weaver, V. M. The extracellular matrix at a glance. J. Cell Sci. 123, 4195–4200 (2010)

[2] Zollinger, A. J. & Smith, M. L. Fibronectin, the extracellular glue. Matrix Biology 60–61, 27–37 (2017)

[3] Jordahl, S. et al. Engineered Fibrillar Fibronectin Networks as Three‐Dimensional Tissue Scaffolds. Adv. Mater. 31, 1904580 (2019)

[4] Jordahl, J. H. et al. 3D Jet Writing: Functional Microtissues Based on Tessellated Scaffold Architectures. Adv. Mater. 30, 1707196 (2018)

[5] Wu, P.-H., Giri, A. & Wirtz, D. Statistical analysis of cell migration in 3D using the anisotropic persistent random walk model. Nat. Protoc. 10, 517–527 (2015)

[6] McKeown-Longo, P. J. & Mosher, D. F. Binding of plasma fibronectin to cell layers of human skin fibroblasts. J. Cell Biol. 97, 466–72 (1983)

[7] Neale, D. B. et al. Precisely aligned networks of fibrillar fibronectin govern cellular orientation and motility. In submission (expected: 2020).