Three-Dimensional Organotypic Systems for Modelling and Understanding Molecular Regulation of Oral Dentogingival Tissues

Abstract

Three-dimensional organotypic models benefit from the ability to mimic physiological cell-cell or cell-matrix interactions and therefore offer superior models for studying pathological or physiological conditions compared to 2D cultures. Organotypic models consisting of keratinocytes supported by fibroblasts embedded in collagen matrices have been utilised for the study of oral conditions. However, the provision of a suitable model for investigating the pathogenesis of periodontitis has been more challenging. Part of the complexity relates to the different regional epithelial specificities and connective tissue phenotypes. Recently, it was confirmed, using 3D organotypic models, that distinct fibroblast populations were implicated in the provision of specific inductive and directive influences on the overlying epithelia. This paper presents the organotypic model of the dentogingival junction (DGJ) constructed to demonstrate the differential fibroblast influences on the maintenance of regionally specific epithelial phenotypes. Therefore, the review aims are (1) to provide the biological basis underlying 3D organotypic cultures and (2) to comprehensively detail the experimental protocol for the construction of the organotypic cultures and the unique setup for the DGJ model. The latter is the first organotypic culture model used for the reconstruction of the DGJ and is recommended as a useful tool for future periodontal research.

Keywords: dentogingival; periodontitis; periodontium; three dimensional organotypic.

Figure 1

Diagram showing the differences in epithelial growth between an epithelium recombined with SCT versus DCT. When the epithelium is recombined with SCT, the epithelium will continue to increase in thickness; therefore, SCT supports epithelial growth (A). Conversely, when the epithelium is recombined with DCT, it fails to grow and atrophies over time (B). SCT = superficial connective tissue, DCT = deeper connective tissue, E = epithelium.

Figure 2

Haematoxylin and eosin section of a 3D organotypic construct, consisting of H400 epithelium cultured over collagen matrix enriched with PDL fibroblasts (connective tissue equivalent). The fibroblasts have been stained pink in this section. Image used with permission [19].

Figure 3

Haematoxylin and eosin sections of human gingival fibroblasts (HGFs) and human periodontal ligament fibroblasts (HPDLFs) demonstrating the differences in epithelial growth following a 21-day culture. Image used with permission from Wiley [2].

Figure 4

H&E sections showing the reduction in epithelial growth of HGF construct following treatment with 500 ng/mL DKK1 over 8 days (A) compared to control which showed progressive epithelial growth (B). Following testing for a range of Wnt agonists and antagonists, SFRP4 was identified to be differentially expressed in HPDLFs (C) and this was reinforced by the proteomic expression of SFRP4 in the PDL tissues (D). H&E sections of constructs transfected with siRNA-SFRP4 (E,F). Treatment of HPDLF constructs with si-RNA-SFRP4 led to an increase in epithelial growth (E) whilst the same treatment on HGF constructs resulted in no epithelial changes compared to control, scrambled si-RNA (F). All images used with permission from Wiley [2]. H&E: haematoxylin and eosin; ** denotes p-value < 0.01.

Figure 5

Diagram showing the epithelial response to mucosal wounding. Following superficial wounding to the mucosa in the SCT, the epithelium predictably heals over (A). If a deep wound is created (B), the epithelium will migrate apically and halt upon contact with the DCT (B). This edge of epithelium (arrowed) acquires a new phenotype due to the stimulus from the DCT. SCT = superficial connective tissue; DCT = deep connective tissue; E = epithelium.

Figure 6

The edge concept. H&E section of the normal dentogingival tissues in health (A), the junctional epithelium is arrowed in red. Image used with permission from Wiley [2]. Schematic showing the spatial relationships of the different connective tissue components relative to the dentogingival epithelia (B). The gingival lamina propria, as an SCT, supports gingival epithelium, while the PDL, as a DCT is associated with the development of an atrophic epithelial phenotype, resembling the JE. The interface where the epithelium meets the DCT is known as the edge (arrowed). The anatomical relationship could be modelled in vitro, using organotypic cultures consisting of an SCT construct overlying a DCT collagen–fibroblast matrix (C). The larger DCT collagen bed enables the formation of an edge, where the epithelial cells would come in contact with the DCT (arrowed in C). Corresponding H&E section showing the abrupt termination of epithelial growth following contact with HPDLFs (arrowed in (D)). Image used with permission from Wiley [45] The control setup consisted of SCT construct supported by SCT; the arrows point to where the epithelial cells would contact the DCT (E). Corresponding histological section showing the continuous epithelial growth along the length of HGF construct, until the point where it ceases to migrate (arrowed) (F). Image used with permission from Wiley [45]. H&E: haematoxylin and eosin, SCT: superficial connective tissues, DCT: deep connective tissues.

Figure 7

Diagram showing the experimental sequence and timing in constructing the 3D organotypic culture system. The protocol begins with the formation of the fibroblast–collagen matrix, followed by the addition of epithelial cells at day 7, air lifting at day 14 and harvesting of the samples for processing at day 21.

Figure 8

Diagram showing the experimental sequence and timing in constructing in vitro model of the dentogingival junction (DGJ). The protocol begins with the setup of the donor construct, followed by the addition of the epithelial cells at day 7. The larger recipient construct is prepared also at day 7, followed by the transfer of the donor construct to the recipient collagen matrix at day 14. The specimen is harvested at day 21.