Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments

Objective This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three‐dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Methods Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH‐26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air–liquid interface for 7 days. Three‐dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three‐dimensional culture tissues at 8, 14 and 21 days (in vitro model). Results Grafted three‐dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal‐layer‐like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo‐epidermal junction in vivo at 7 and 14 days, but not in vitro. Conclusion These results suggest that differentiation of three‐dimensional culture tissues differs in vivo and in vitro.