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.


CONCLUSIONS
These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro.
Advanced Operative Dentistry and Endodontics, The Nippon Dental University, Graduate School of Life Dentistry at Niigata, Niigata City, Japan.
Rie
R
Yamada
Kayoko
K
Kitajima
Kyoko
K
Arai
Masaru
M
Igarashi
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D062109:Dermatologic Surgical Procedures / Q000379:methods; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology / Q000637:transplantation; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology / Q000637:transplantation; D005456:Fluorescent Dyes; D053538:Keratin-18 / Q000032:analysis; D053539:Keratin-19 / Q000032:analysis; D053533:Keratin-8 / Q000032:analysis; D007633:Keratins / Q000032:analysis*; D007797:Laminin / Q000032:analysis; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009930:Organic Chemicals; D010513:Periodontal Ligament / Q000166:cytology* / Q000378:metabolism; D011498:Protein Precursors / Q000032:analysis; D013552:Swine; D013997:Time Factors; D046509:Tissue Culture Techniques; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
D003470:Culture Media; D005456:Fluorescent Dyes; D053538:Keratin-18; D053539:Keratin-19; D053533:Keratin-8; C486595:Krt8 protein, mouse; D007797:Laminin; D009930:Organic Chemicals; C070080:PKH 26; D011498:Protein Precursors; C032253:involucrin; D007633:Keratins; D003094:Collagen
cytokeratin; engraft; epithelial cells; periodontal ligament; three-dimensional culture