Fibronectin

Three-dimensional culture of IHGE cells was performed as described previously [22 (link)] with some modification. Briefly, IHGE cells collected by centrifugation after trypsinization were alternatively incubated for 1 min with 0.2 mg mL^-1 fibronectin (Sigma-Aldrich) and 0.1% (w/v) gelatin (Nacalai Tesque) dissolved in phosphate-buffered saline (PBS). After each procedure, cells were washed with PBS (pH = 7.4) by centrifugation at 180 g for 2 min to remove unabsorbed polymers. After nine steps of immersion, fibronectin/gelatin nano-films were coated onto single-cell surfaces. A total of 2 × 10⁶ cells coated with fibronectin/gelatin nano-films were seeded on coverslips coated with 0.2 mg mL^-1 fibronectin dissolved in PBS in 24-well plates (Iwaki). After 36 h of incubation, tissues were subjected to experiments, fixed, and analyzed on a confocal microscope (TCS SP8; Leica Microsystems). For infection of gingival epithelial tissues, the density of P. gingivalis cells in culture media was adjusted to OD (600 nm) of 0.12.

Matrigel invasion assay was performed as described previously [17 (link)]. Briefly, membrane filters (Whatman, Maidstone, UK) were attached to Transwell chambers (Costar, Cambridge, MA, USA) and the lower surface was pre-coated with 1.25 µg fibronectin (Iwaki, Tokyo, Japan) and the upper surface was pre-coated with 5 µg Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Pre-treated cells (3 × 10⁴ cells/100 µl in RPMI 1640 Medium with 0.1% bovine serum albumin) were added to the upper compartment of the chamber. After incubation for 6 h, the invaded cells were stained by hematoxylin and eosin and counted manually under a microscope at 50x magnification.