Migration of periodontal ligament fibroblasts on nanometric topographical patterns: influence of filopodia and focal adhesions on contact guidance

Abstract

Considered to be the "holy grail" of dentistry, regeneration of the periodontal ligament in humans remains a major clinical problem. Removal of bacterial biofilms is commonly achieved using EDTA gels or lasers. One side effect of these treatment regimens is the etching of nanotopographies on the surface of the tooth. However, the response of periodontal ligament fibroblasts to such features has received very little attention. Using laser interference lithography, we fabricated precisely defined topographies with continuous or discontinuous nanogrooves to assess the adhesion, spreading and migration of PDL fibroblasts. PDL fibroblasts adhered to and spread on all tested surfaces, with initial spreading and focal adhesion formation slower on discontinuous nanogrooves. Cells had a significantly smaller planar area on both continuous and discontinuous nanogrooves in comparison with cells on non-patterned controls. At 24 h post seeding, cells on both types of nanogrooves were highly elongated parallel to the groove long axis. Time-lapse video microscopy revealed that PDL fibroblast movement was guided on both types of grooves, but migration velocity was not significantly different from cells cultured on non-patterned controls. Analysis of filopodia formation using time-lapse video microscopy and labeling of vinculin and F-actin revealed that on nanogrooves, filopodia were highly aligned at both ends of the cell, but with increasing time filopodia and membrane protrusions developed at the side of the cell perpendicular to the cell long axis. We conclude that periodontal ligament fibroblasts are sensitive to nanotopographical depths of 85-100 µm, which could be utilized in regeneration of the periodontal ligament.

Figure 1. Characterization of the surfaces used in the study.

SEM images of the topographies employed in this investigation are shown in (A) nanogrooves, and (B) discontinuous nanogrooves. AFM measurement of (C) nanogrooves – average height 100 nm and (D) discontinuous nanogrooves – average height 85 nm. The pitch of the grooves in both instances was 500 nm. The features were considered to be of suitable dimensions used by previous studies, where 70–100 nm seemed to be a transition point .

Figure 2. Influence of surface topography on PDL fibroblast attachment, spreading and FA formation 30 min and 2 h after seeding on non-patterned, nanogrooves and discontinuous nanogrooves.

Cells were labelled for vinculin (green), F-actin (red) and nucleus (blue). Inset is vinculin staining from the same image to show FA formation alone.

Figure 3. Quantification of PDL fibroblast (A) adhesion, and (B) spreading, on non-patterned surfaces, nanogrooves, and discontinuous nanogrooves.

In (A). *  =  significantly different from non-patterned (p<0.05) and #  =  statistically significant from discontinuous nanogrooves (p<0.05).

Figure 4. Influence of continuous and discontinuous nanogrooves on alignment of focal adhesions in PDL fibroblasts.

(A) images of cells labeled for vinculin were thresholded and converted to binary images. Angle of alignment was then calculated relative to the vertical axis. (B) distribution of focal adhesion alignment.

Figure 5. Scanning electron micrographs of PDL fibroblasts cultured on (A) nanogrooves, and (B) discontinuous nanogrooves 24 h post seeding.

Inset shows PDL fibroblasts grown for 24 h on non-patterned surfaces.

Figure 6. Analysis of PDL fibroblast migration on (A) smooth, (B) nanogrooves, and (C) discontinuous nanogrooves (see also supplementary movies S1, S2, and S3).

The top panel in all images shows the cell tracking patterns for each movie. Arrow indicates orientation of the nanotopographies long axis. In (D) quantification of PDL fibroblast migration velocity on each tested topography, and in (E) influence of topography on directionality of the PDL fibroblast migration patterns. Data was analyzed using Axioimager software and statistical analysis was performed using one-way ANOVA with Bonferroni post-hoc test.

Figure 7. Sequential timelapse images of PDL fibroblast spreading on (A) non-patterned surfaces and (B) nanogrooves.

Images in (B) can be seen also in supplementary movie S4. In (C), scanning electron micrographs of filopodia formation in PDL fibroblasts cultured on nonpatterned, nanogrooves and discontinuous nanogrooves. In (A) arrowhead shows the direction of lamelipodia extension. In (B), arrows show filopodia and membrane protrusion extending along the length of the fibroblast during spreading.

Figure 8. Analysis of vinculin (green) and F-actin (red) localization in filopodia extended by PDL fibroblasts cultured on smooth surfaces and nanogrooves at (A) 2 h and (B) 24 h.

Arrows indicate direction of groove long axis.