Sex-based differences in cell migration on aligned topographies

Abstract

Sexual dimorphism has been observed in many physiological and pathological responses, yet few studies incorporate both female and male experimental groups for preclinical work. For the development of biomaterial devices, in vitro studies are essential for design and optimization, and quantitative comparison of female and male cell migratory behavior is a crucial design consideration. In this work, we thoroughly examined sex-based migration on flat controls and aligned nanofiber scaffolds of various diameters using anomalous and random walk models. Male and female cells exhibited significantly different migration on flat substrates, with female cells having increased speed while male cells had higher persistence. Persistence increased with the introduction of aligned fiber topography for female cells, but only affected male cells on the highest fiber diameter. Speed along the axis of alignment differed between sexes on 1.2 and 1.8 µm fibers. Morphological analysis confirmed cell shape was a function of both sex and fiber size. These results provided critical information regarding sex-based cell migration, highlighting the importance of sex within in vitro studies for clinical device design.

Keywords: Cell migration; Nanofibers; Schwann cells; Sex-based differences.

Fig. 1

Touch-spinning was used to fabricate highly aligned nanofiber scaffolds of different diameters. In touch-spinning, a platform spins at a defined and tunable rate, allowing for a needle/rod to (A) come into contact with a droplet of the polymer solution, (B, C) draw the droplet into a fiber mechanically, and (D) collect the fiber on a stationary center substrate (i.e., coverslip). Middle row: Representative SEM images of touch-spun, aligned nanofibers of diameters (E) 0.9 (F) 1.2 (G) 1.8 µm are shown. The scale bar represents 20 µm. (H) Fiber diameter as a function of spin rate. By increasing the spin rate of the rotating platform, fibers of smaller diameters were yielded. Significance was calculated using one-way ANOVA with Tukey as a post hoc test. Error bar: standard deviation. Bottom row: Fibers are highly aligned, with alignment distribution of (I) 0.9 (J) 1.2 (K) 1.8 µm fiber scaffolds. Three independent polymer solutions were used for fabrication and diameter measurement (N = 3). At least 50 individual fibers (n) were measured (0.9 µm: n = 61; 1.2 µm: n = 60; 1.8 µm: n = 54) for each population.

Fig. 2

Schwann cell morphology was elongated on nanofibers. (A) Representative images of aligned actin filaments of male (left) and female (right) Schwann cells on (i) controls (ii) 0.9 µm (iii) 1.2 µm (iv) 1.8 µm diameter fibers. The direction of aligned fibers is indicated by the white arrow along the horizontal axis. Actin filaments (green) were labeled using phalloidin-Alexa Fluor 488 and the cell nuclei (blue) were labeled using Hoechst 33342. Scale bar: 50 µm. Images are of the closest data point to the mean in the third quartile of the violin plots. 7 images were taken of each substrate for a total of 21 images per biological replicate. (B) Percentage of actin alignment was measured using a MATLAB edge detection program. No significant differences were found between female and male cells within any experimental condition. Values reported represent mean ± standard deviation, where 100% indicates perfect alignment. (C) Whole-cell aspect ratio (AR) of individual female and male Schwann cells was measured. Whole-cell AR of male cells was statistically higher than female cells on flat substrates, while female cells are higher than males on 0.9 µm and 1.8 µm fibers. (D) Nuclear eccentricity ratio (ER) of individual female and male Schwann cells was separately examined. ER of female cells on 1.8 µm fibers was statistically higher than male cells. Three biological replicates were completed per sex (N = 3), and greater than 40 individual cells (n) were measured (control: n_male = 144, n_female = 68; 0.9 μm: n_male = 66, n_female = 40; 1.2 μm: n_male = 103, n_female = 63; 1.8 μm: n_male = 101, n_female = 52). Statistical analyses were calculated using two-tailed unpaired t-test (control) and two-way ANOVA test with Tukey as a post hoc test (fiber). Error bar: standard deviation.

Fig. 3

Female and male Schwann cells possess inherent sex-based differences on flat laminin-coated substrates. (A) Sample cell path with cellular morphology at several times (t). Both path and cell shape are noted, with shapes shown from each hour of the capture. (B, C) Cell paths of female and male cells, respectively, are plotted over the course of the 24 h, with starting points centered at 0,0. (D) Ensemble average MSD curves on a log–log scale indicated significant differences in fit parameters each model. (E) According to the anomalous migration model, male cells exhibited a greater α parameter (α = 1.41 ± 0.24) than females (α = 1.32 ± 0.21), although both sexes were superdiffusive (1 < α < 2). (F) In addition, male cells persisted in one direction longer than female cells as calculated by PR, and (G) female Schwann cells had higher average instantaneous speeds than male Schwann cells ( _female = 0.51 ± 0.31 µm/min; _male = 0.33 ± 0.14 µm/min). Three biological replicates (N = 3) were completed per sex, where at least 100 individual cells were included (n_male = 113, n_female = 121). Statistical analyses for the α parameters and average speeds were calculated using two-tailed unpaired t-test. Error bar: standard deviation.

Fig. 4

Schwann cell migration on fibers reduced sex-based differences. (A) Cells paths indicate the influence of aligned fibers, with migration primarily along the x-axis of fiber alignment. Columns are separated by fiber diameter, with male (i, iii and v) in black and female (ii, iv and vi) in pink. (B) MSD was calculated and the MSD curve for 1.8 µm fibers was lower for females compared to other fibers and controls, but males showed no differences. Each graph has the MSD for the appropriate fiber group and its flat control. (C) The fit of α for anomalous migration was found to increase over controls but showed no differences between the sexes. The increase in α for fibers indicated further increased persistence. Within each sex, α parameters statistically increased on fibers compared to flat substrates. (D) The velocity vector indicated that the movement was primarily in the x-direction, as expected, with cells moving back and forth along the fiber, averaging to a 0 overall velocity. (E) The PR was again calculated for the fibers and while it increased from controls, minimal differences were found between the sexes except on 1.8 µm fibers, where male cells were more persistent than female cells. (F) The differences in speed (along fiber axis or x axis for controls) between sex was reduced with the addition of fibers at 0.9 µm, but the female cells showed increased speed with the larger fibers over male cells. Three biological replicates (N = 3) were completed per sex, where at least 100 individual cells were included (0.9 µm - n_male = 124, n_female = 124; 1.2 µm - n_male = 138, n_female = 134); 1.8 µm - n_male = 169, n_female = 139). Statistical analyses were calculated using two-tailed unpaired t-test (control) and two-way ANOVA test with Tukey as a post hoc test (fiber). Error bar: standard deviation.

Fig. 5

APRW model fit to Schwann cell migration on aligned fibers. (A) The presence of fibers increased the persistence time of female cells along the fibers (primary direction, p) compared to controls, although it diminished the differences in persistence time seen between the sexes. Only the highest fiber diameters further increase the persistence time of male cells compared to controls. (B) The speed of both male and female cells on fibers (in the primary direction) is statistically decreased compared to sex-based controls, although the speed of female cells remains higher compared to male cells on the larger fibers. (C and D) Persistence time and speed fit for APRW model in the perpendicular direction (non-primary, np), with decreased persistence times and speed with the addition of fibers for both male and female cells. Three biological replicates (N = 3) were completed per sex, where at least 100 individual cells were included (0.9 µm - n_male = 124, n_female = 124; 1.2 µm - n_male = 138, n_female = 134); 1.8 µm - n_male = 169, n_female = 139). Statistical analyses were calculated using two-tailed unpaired t-test (control) and two-way ANOVA test with Tukey as a post hoc test (fibers). Error bar: standard deviation.