Rete Ridge Formation in Three-dimensional Engineered Gingiva

Objectives: Rete ridges provide an increase in surface area of epidermal-dermal/epithelial-connective tissue junction, allowing better tissue integrity and possible regenerative potential. The objective of this study was to generate rete ridges in 3D-engineered human gingiva.
Methods: Primary human gingival fibroblasts (HGF) and keratinocytes (HGK) were isolated from gingiva obtained following gingivectomy/crown lengthening surgeries (7 patients). HGF+HGK were cultured onto electrospun collagen scaffolds (ES-collagen) to fabricate engineered tissues. Before HGK inoculation, the tissues in test group were lasered with a fractional CO₂ laser at 25% density to ablate small wells into the “connective tissue layer” for keratinocytes to proliferate into rete-like pattern (Provisional Patent Application No. 62/779,276). At day 4, engineered tissues were strained to 10% in static strain devices. Tissues within the strain devices were cultured for another 6 days prior to histological analysis. The control groups had same culturing conditions except that one set of tissues received laser treatment without straining (laser-control) and a second set of tissues received neither laser nor strain manipulation (control). Each experiment was repeated 3 times.
Results: HGF+HGK were stratified into two distinct layers on the ES-collagen. The test group showed rete ridges-like structure similar to natural rete ridges. In addition, samples in the test group had distinctive increase in epithelial thickness and surface area of epithelial-connective tissue junction compared to control group. The laser-control group did not show a well-defined rete structure.
Conclusions: Based on this pilot study, the fractional CO₂ laser in combination with the strain device technique can be used to generate rete ridges structure in 3D-engineered gingiva. Additionally, creating rete ridges by this technique resulted in an increase in epithelial thickness. Additional studies are necessary to determine possible higher regenerative potential of 3D-engineered gingiva modified by rete ridges.