Influence of Micropatterning on Human Periodontal Ligament Cells' Behavior

Abstract

The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-μm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells' peripheral regions. Moreover, proliferation was promoted by 10- and 20-μm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells' stem cell markers. β-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration.

Figure 1

The effect of micropatterning on cell morphology and cytoskeleton rearrangement in PDL cells. (A) This panel shows the detection of the integrity of micropatterns by FITC-BSA (upper) and phase-contrast images of PDL cells cultured on control and micropatterned groups (lower). (B) This panel shows a schematic depicting the methodology used in cell angle calculation (left) and cell angles changed after micropatterning (n > 300). (C) CSI clearly decreased in micropatterned groups (n > 20). (D) The cell spreading area of PDL cells decreased significantly in micropatterned groups (n > 25, #p < 0.01). (E) F-actin was stained with Texas red isothiocyanate-conjugated phalloidin (red), and DAPI was stained for nucleus (blue) visualization. (F) The actin filaments were reorganized along the direction of micropatterned strips in micropatterned groups. To see this figure in color, go online.

Figure 2

The effect of micropatterning on nuclear morphology change. (A) Representative 3D reconstruction of PDL cell nuclei in control and micropatterned groups (scan bar = 10 μm) are shown. Nucleus shape index (B) and nuclear projected area (C) were decreased in 10- and 20-μm groups (n > 25, #p < 0.01). (D) Representative z-projected images of nuclear (D) and quantification of PDL cells’ nuclear height cultured on control and micropatterned groups (E) are shown. The nuclear height increased in 10- and 20-μm groups (n = 16, ^∗p < 0.05). (F) The nuclear volume of PDL cells showed no significant difference between control and micropatterned groups (n > 150). To see this figure in color, go online.

Figure 3

The proliferation of PDL cells increased in micropatterned groups. (A) Representative images of EdU-positive (green) PDL cells in control, 10-, and 20-μm groups are shown. Nuclei were stained with Hoechst 33342 (blue). (B) Quantification of the percentage of EdU-positive cells in each group is given. The proliferation of PDL cells increased in 10- and 20-μm groups compared with the control. Data represent mean ± SD of at least triplicate experiments (n > 1000, #p < 0.01). To see this figure in color, go online.

FIGURE 4

The effect of micropatterning on PDL cell differentiation. Gene expression of osteogenesis markers (ALP, RUNX2) (A) and adipogenic markers (PPARγ2, CEBPα) (B) was evaluated in control and micropatterned groups of PDL cells. Expression of GAPDH was used to normalize mRNA content. Data represent mean ± SD of at least triplicate experiments (^∗p < 0.05, #p < 0.01).

Figure 5

The effect of micropatterning on stemness of PDL cells. (A) Gene expression of stem cell markers (Sox2, Nanog, and Oct4) was evaluated in control and micropatterned groups of PDL cells. Expression of GAPDH was used to normalize mRNA content. (B) Sox2 (green), (C) Nanog (green), and (D) Oct4 (green) and nuclear (blue) staining in PDL cells of control or micropatterned groups are shown. Quantification of average fluorescence intensity is shown in the panel. Data represent mean ± SD of at least triplicate experiments (^∗p < 0.05, #p < 0.01). To see this figure in color, go online.

Figure 6

The effect of micropatterning on distribution of β-catenin in PDL cells. PDL cells were immunostained for β-catenin (red) and nucleolus with DAPI (blue) of control and micropatterned groups. The ratio of nuclear/total β-catenin fluorescence intensity is quantified in the lower panel. Data represent mean ± SD of at least triplicate experiments (^#p < 0.01). To see this figure in color, go online.

Figure 7

Micropatterning regulates YAP/TAZ nuclear localization and activity. (A) YAP/TAZ nuclear localization in PDL cells cultured on control and micropatterned groups is shown. The lower panel indicates nuclear YAP/TAZ fluorescence intensity in PDL cells (n > 370). (B) Real-time PCR analysis of PDL cells’ relative CTGF and ANKRD1 mRNA expression to measure YAP/TAZ transcriptional activity is shown. Data represent mean ± SD. ^∗ and # represent ^∗p < 0.05, #p < 0.01. To see this figure in color, go online.

Figure 8

The effect of micropatterning on extracellular matrix synthesis. (A) Collagen I (green) and nuclear (blue) staining in PDL cells of control or micropatterned groups is shown. Gene expression of MMP-1 (B), MMP-2 (C), TIMP-1 (D), and TIMP-2 (E) was evaluated in PDL cells from control and micropatterned groups. Expression of GAPDH was used to normalize mRNA content. Data represent mean ± SD of at least triplicate experiments (^∗p < 0.05, #p < 0.01). To see this figure in color, go online.