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Wound healing proceeds via fibroblast migration along three dimensional fibrillar substrates with multiple angles between fibers. We have developed a technique for preparation of three dimensional fibrillar scaffolds with where the fiber diameters and the angles between adjacent fiber layers could be precisely controlled. Using the agarose droplet method we were able to make accurate determinations of the dependence of the migration speed, focal adhesion distribution, and nuclear deformation on the fiber diameter, fiber spacing, and angle between adjacent fiber layers. We found that on oriented single fiber layers, whose diameters exceeded 1 μm, large focal adhesion complexes formed in a linear arrangement along the fiber axis and cell motion was highly correlated. On multi layered scaffolds most of the focal adhesion sites reformed at the junction points and the migration speed was determined by the angle between adjacent fiber layers, which followed a parabolic function with a minimum at 30°. On these surfaces we observed a 25% increase in the number of focal adhesion points and a similar decrease in the degree of nuclear deformation, both phenomena associated with decreased mobility. These results underscore the importance of substrate morphology on the en-mass migration dynamics., Statement of Significance: En-mass fibroblast migration is an essential component of the wound healing process which can determine rate and scar formation. Yet, most publications on this topic have focused on single cell functions. Here we describe a new apparatus where we designed three dimensional fibrillar scaffolds with well controlled angles between junction points and highly oriented fiber geometries. We show that the motion of fibroblasts undergoing en-mass migration on these scaffolds can be controlled by the substrate topography. Significant differences in cell morphology and focal adhesions was found to exist between cells migrating on flat versus fibrillar scaffolds where the migration speed was found to be a function of the angle between fibers, the fiber diameter, and the distance between fibers., (Copyright © 2015. Published by Elsevier Ltd.)

En masse cell migration is more relevant compared with single-cell migration in physiological processes of tissue formation, such as embryogenesis, morphogenesis, and wound healing. In these situations, cells are influenced by the proximity of other cells including interactions facilitated by substrate mechanics. Here, we found that when fibroblasts migrated en masse over a hydrogel, they established a well-defined deformation field by traction forces and migrated along a trajectory defined by field gradients. The mechanics of the hydrogel determined the magnitude of the gradient. For materials stiff enough to withstand deformation related to cellular traction forces, such patterns did not form. Furthermore, migration patterns functioned poorly on very soft matrices where only a minimal traction gradient could be established. The largest degree of alignment and migration velocity occurred on the gels with the largest gradients. Granulation tissue formation in punch wounds of juvenile pigs was correlated strongly with the modulus of the implanted gel, in agreement with in vitro en masse cell migration studies. These findings provide basic insight into the biomechanical influences on fibroblast movement in early wounds and relevant design criteria for the development of tissue-engineered constructs that aim to stimulate en masse cell recruitment for rapid wound healing.

We have shown that en masse cell migration of fibroblasts on the planar surface results in a radial outward trajectory, and a spatially dependent velocity distribution that decreases exponentially in time towards the single cell value. If the cells are plated on the surface of aligned electrospun fibers above 1 microm in diameter, they become polarized along the fiber, expressing integrin receptors which follow closely the contours of the fibers. The velocity of the cells on the fibrous scaffold is lower than that on the planar surface, and does not depend on the degree of orientation. Cells on fiber smaller than 1 microm migrate more slowly than on the planar surface, since they appear to have a large concentration of receptors. True three-dimensional migration can be observed when plating the droplet on a scaffold comprises of at least three layers. The cells still continue to migrate on the fibers surfaces, as they diffuse into the lower layers of the fibrous scaffold.

Cellular traction forces, resulting in cell-substrate physical interactions, are generated by actin-myosin complexes and transmitted to the extracellular matrix through focal adhesions. These processes are highly dynamic under physiological conditions and modulate cell migration. To better understand the precise dynamics of cell migration, we measured the spatiotemporal redistribution of cellular traction stresses (force per area) during fibroblast migration at a submicron level and correlated it with nuclear translocation, an indicator of cell migration, on a physiologically relevant extracellular matrix mimic. We found that nuclear translocation occurred in pulses whose magnitude was larger on the low ligand density surfaces than on the high ligand density surfaces. Large nuclear translocations only occurred on low ligand density surfaces when the rear traction stresses completely relocated to a posterior nuclear location, whereas such relocation took much longer time on high ligand density surfaces, probably due to the greater magnitude of traction stresses. Nuclear distortion was also observed as the traction stresses redistributed. Our results suggest that the reinforcement of the traction stresses around the nucleus as well as the relaxation of nuclear deformation are critical steps during fibroblast migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. A traction gradient foreshortening model was proposed to explain how the relocation of rear traction stresses leads to pulsed fibroblast migration.