Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model

Abstract

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4′,6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.