Development of an in vitro model to study tooth cystogenesis

To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development.

To form spheroid structures, various numbers (1 × 10⁴ , 5 × 10⁴ or 1 × 10⁵ ) of epithelial cells (HaCaT and Cal27) were seeded in 96-well plates previously coated with 1.5% low-melting agarose. After 24 h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24-well plates previously coated with polymerized collagen and kept for up to 21 days. Images of spheroids were captured at each time-point (1, 5, 9, 15 and 21 days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One-way Anova was used for statistical analysis.

Both epithelial cell lines were able to generate regular and circular spheroids after 24 h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P < 0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst.

This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance.