In vitro development of a radicular cyst: A morphological investigation of a spheroid cyst-like model associated with fibroblasts

Objective

The aim of this study was to improve the in vitro model of tooth radicular cyst previously developed by incorporating stromal components and to describe histologic analysis.

Design

A radicular cystogenesis-like 3D model was generated using HaCaT cells (1 × 10⁵) to developing spheroid. After 24 h, spheroids were embedded in non-polymerized collagen in combination with 1 × 10⁵ fibroblast cells (HaCaT + 1 × 10⁵ hFIB) to mimic stromal microenvironment. Micrographs were taken to evaluate the cystic stability and dispersion area, while histological hematoxylin/eosin staining was used to measure the ratio of epithelial lining area. Analysis was conducted on days 1, 3, and 7 using ImageJ software. Statistical analyses were performed using GraphPad Prism 5 software.

Results

The model, with fibroblasts included, preserved the cystic structure and allowed cyst growth, with an increase in both the area and dispersion of the cystic structure throughout the experimental period (p < 0.05). Histological analysis of the cyst model revealed morphological similarities with in vivo tooth radicular cyst biopsies, showing a cystic cavity lined by an epithelial layer, surrounded by collagen and fibroblasts. Additionally, the cavity area increased while the limiting epithelial area decreased. The highest epithelial area-to-total area ratio was observed in day 1 spheroids, while the lowest was found on day 7 (p < 0.05).

Conclusion

The incorporation of fibroblasts improved the in vitro cystogenesis model, since it did not interfere with the model’s development and more closely mimicked the in vivo microenvironment.