Effects of topographical guidance cues on osteoblast cell migration

Abstract

Cell migration is a fundamental process that is crucial for many biological functions in the body such as immune responses and tissue regeneration. Dysregulation of this process is associated with cancer metastasis. In this study, polydimethylsiloxane platforms with various topographical features were engineered to explore the influence of guiding patterns on MC3T3-E1 osteoblast cell migration. Focusing on the guiding effects of grating patterns, variations such as etch depth, pattern discontinuity, and bending angles were investigated. In all experiments, MC3T3-E1 cells on patterned surfaces demonstrated a higher migration speed and alignment when compared to flat surfaces. The study revealed that an increase in etch depth from 150 nm to 4.5 μm enhanced cell alignment and elongation along the grating patterns. In the presence of discontinuous elements, cell migration speed was accelerated when compared to gratings of the same etch depth. These results indicated that cell directionality preference was influenced by a high level of pattern discontinuity. On patterns with bends, cells were more inclined to reverse on 45° bends, with 69% of cells reversing at least once, compared to 54% on 135° bends. These results are attributed to cell morphology and motility mechanisms that are associated with surface topography, where actin filament structures such as filopodia and lamellipodia are essential in sensing the surrounding environment and controlling cell displacement. Knowledge of geometric guidance cues could provide a better understanding on how cell migration is influenced by extracellular matrix topography in vivo.

Figure 1

Fabrication of patterned PDMS platforms. (a) Patterns were transferred onto Si using photolithography and replicated onto PDMS by soft lithography. O₂ plasma was used to bond PDMS to cell culture dish. Micrographs of (b) grating pattern with 6 μm ridges, 4 μm grooves, 4.5 μm depth and (c) bend density with 1 and 2 consecutive bends; 2 μm ridges and 4.5 μm depth.

Figure 2

Cell migration trajectories of MC3T3-E1 cells showing the guiding effect of gratings with different groove depths on (a) flat surface, (b) 150 nm, (c) 500 nm, (d) 1.0 μm, and (e) 4.5 μm.

Figure 3

(a) Cell migration speed of gratings with different depths. (b) Cell migration speed of gratings in x and y directions with different groove depths. All error bars are mean ± SEM. One-way ANOVA and Tukey’s post hoc test were performed to test for statistical significance (***p < 0.001).

Figure 4

Aspect ratio of MC3T3-E1 cells on gratings with different depths. Cell shape was observed at 0, 4, 8, 12, and 16 h and the mean aspect ratio was calculated. One-way ANOVA and Tukey’s post hoc test with **p < 0.01 and ***p < 0.001. Micrographs represent MC3T3-E1 cell elongation on different gratings.

Figure 5

(a) Migration speed of MC3T3-E1 cells on patterns with different levels of discontinuity. Micrographs present different patterns with ridges of 4 μm ridges, 5 μm spacing; ridges & pillars of 6 μm ridges, 5 μm spacing; and oblongs of 5 μm width, 10 μm length, 5 μm spacing. The depth of all patterns is 1 μm (One-way ANOVA and Tukey’s post hoc test with *p < 0.05 and ***p < 0.001). (b) RMS of displacement distance for MC3T3-E1 cells on discontinuous guiding patterns.

Figure 6

Cells were stained to observe nuclei, actin filaments, and cytoskeleton under fluorescent imaging with cells on (a) ridges, (b) ridges & pillars, and (c) oblongs. (d) Comparison of MC3T3-E1 cell migration speed in x and y directions on discontinuous guiding patterns.

Figure 7

(a) Cell migration speed of MC3T3-E1 cells on bent guiding patterns with bent angles of 135° and 45°. Micrographs present different bent patterns of gratings of 6 μm ridges and 4 μm grooves; 135° bend of 4 μm ridges, 1 μm grooves, 100 μm length and 25 μm bend length; bend density of 2 μm ridges, 3 μm grooves, 200 μm length, and 50 μm bend length; and 45° bend of 4 μm ridges, 1 μm grooves, 100 μm length, and 75 μm bend length. The depth of all patterns is 4.5 μm. One-way ANOVA and Tukey’s post hoc test with *p < 0.05 and **p < 0.01. (b) Directionality ratio of patterns with bends over 16 h time-lapse.

Figure 8

Minimum percentage of cell reversals of MC3T3-E1 cells on patterns with 45° bends (N = 65) and 135° bends (N = 53). Cell reversals were calculated by number of cells that reversed by 180° divided by total number of cells on patterned platforms. 2-way ANOVA and Sidak’s post hoc test indicated no significant differences between number of reversals and bending angle (p > 0.05).