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

Abstract

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 (μTUGs) 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.

Fig. 1

Fabrication and seeding of microfabricated tissue gauge (µTUG) molds. Process flow diagram for the creation of µTUG arrays. After replicating the rigid photoresist structures with a PDMS elastomer, the mold is immersed within a prepolymer solution of cells and type-I collagen. The entire assembly is then centrifuged to drive the cells into the templates. Excess solution is removed and the matrix is polymerized (by increasing the temperature to 37 °C) prior to adding media.

Fig. 2

3D averaged density maps of ECM protein in microtissues. (a–d) Immunofluorescent images showing DAPI, collagen I and plasma Fn within microtissues fixed after 24, 48 and 72 hours of remodeling, or after 72 hours of remodeling with acute (2 hours prior to fixing) incubation with 50 µM blebbistatin. Density maps represent the DNA or protein density, respectively, at a given location averaged over the axis orthogonal to the image plane. In order to register images between tissues of different thickness, cross-sections (xz sect.) are plotted from the uppermost to the lower most surface of the tissues (i.e. normalized by tissue thickness). All density maps are the average of 10 individual microtissues from each condition. Color scales are normalized to depict the min and max values for each stain. Thus, the intensities measured at different time points for the same stain are comparable, but the intensities of different protein stains are not.

Fig. 3

Fn structure and Fn–DA FRET labeling. (a) Cartoon of a Fn monomer depicting multiple cell and ECM binding sites (adapted from Vogel, Annu. Rev., 2006). Yellow sphere represents the 12 nm radius of potential energy transfer to acceptor labeled cysteines (twice the Förster radius). Donors are randomly labeled at amines at an average of 7 donors and 4 acceptors per molecule. (b) Probability density functions (PDFs) of the FRET ratios from Fn–DA in solution with increasing concentrations of the denaturant GdnHCl. (c) Mean values for the histograms depicted in b. Fn begins to lose its secondary structure at or below 1 M GdnHCl (I_a/I_d = 0.40) and is completely denatured at 4 M GdnHCl (I_a/I_d = 0.29). (b, c) The average (±SD) of 5 fields of view under each condition.

Fig. 4

FRET measurement of Fn conformation in microtissues. (a) PDFs of total Fn–DA measured in microtissues fixed after 24, 48 or 72 hours of remodeling, or after 72 hours of remodeling with acute (2 hours prior to fixing) incubation with 50 µM blebbistatin. Corresponding values from Fn–DA in solutions of 0 M, 1 M, 2 M and 4 M GdnHCl denaturant are indicated. (b) Box and whiskers plot of the median FRET values within microtissues depicted in a. (c) Scaled PDFs showing separate populations of collagen-colocalized Fn–DA and non-collagen-colocalized Fn–DA. PDFs are scaled such that the areas under each curve equal the percent of either collagen-colocalized Fn–DA or non-collagen-colocalized Fn–DA at each time point. (d) Box and whiskers plot of the median FRET values within microtissues depicted in c showing either collagen-colocalized Fn–DA or non-collagen-colocalized Fn–DA. (e) Percent of Fn–DA within microtissues fixed at each time point that is either colocalized with collagen, or non-colocalized with collagen. (f) Percent of either collagen-colocalized, non-collagen-colocalized, or total Fn–DA present in microtissues with a loss in secondary structure (FRET ratios below that of Fn in 1 M GdnHCl). (g) Average density maps of the FRET ratios (I_a/I_d) within microtissues from a–f. Corresponding denaturation points are indicated on the colorbar. All data and images were computed from 10 microtissues within each condition. Data in a–f were computed from the raw experimental data and not from the density maps. Data from a and c are the compiled PDFs for all microtissues within a given condition. Data from e are mean values ± SD.

Fig. 5

Increases in tissue stress occur concurrently with the assembly of a progressively unfolded fibrillar Fn matrix. (a) Schematic of 2 post µTUG molds generated from multilayer SU-8 photolithography. (b) Calibration of cantilever spring constants and corresponding transmitted light images of cap deflection. A representative plot of force vs. cap displacement is shown. Inset: calculated spring constant (k = 148 ± 35 nN µm⁻¹ (SD) for n = 15 measurements, 5 cantilevers each across 3 substrates). (c) Representative top down and cross-section views for microtissues showing Fn (green), collagen (pink) and DAPI (blue) after 48 hours of remodeling. Dashed boxes indicate regions for calculation of cross-sectional area, cross-sectional stress and Fn–DA FRET. Scale bar=100 µm. (d) Box and whiskers plot of tissue tension for 2-post microtissues fixed after 24, 48 and 72 hours of remodeling, or after 72 hours of remodeling with acute (2 hours prior to fixing) incubation with 50 µM blebbistatin. (e) Box and whiskers plot of cross-sectional area for tissues under the same conditions as d. (f) Box and whiskers plot of cross-sectional stress for tissues under the same conditions as d. (g) Scatter plot for the median FRET ratio (I_a/I_d) vs. cross-sectional stress for tissues under the same conditions as d. (d—g) Computed from 25 individual microtissues under each condition.

Fig. 6

Fn–DA pulse-chase experiments and colocalization analysis for 4-post microtissues. Fn–DA (yellow boxes) or unlabeled Fn (grey boxes) was present in culture for specified windows during tissue remodeling. Microtissues were then fixed at the indicated times and FRET signals recorded. (a) Box and whiskers plot of the median FRET ratios (I_a/I_d) for microtissues fixed after 72 hours in which Fn–DA was present between either 0–24, 24–48, or 48–72 hours or for microtissues fixed immediately after the first 24 hours of remodeling. (b) Box and whiskers plot of the median FRET ratios (I_a/I_d) showing either collagen-colocalized Fn–DA or non-collagen-colocalized Fn–DA for the same conditions as a. (c) Percent of Fn–DA within microtissues assembled at the indicated increments that is either colocalized with collagen or non-colocalized with collagen. Data from a–c are from 10 microtissues in each condition. Data from b are mean values ± SD.