Cultivated of Periodontal Ligament-derived Cells on Amniotic Membrane

Objectives: Recent development in tissue-engineering technology has led to the ex vivo cultivation of periodontal ligament (PDL) structures. Some studies have demonstrated that PDL-derived cells could regenerate periodontal tissues, and indicated that PDL cells and scaffolds have key roles in periodontal regeneration. However, there has been no report on the use of amniotic membrane (AM) as a scaffold for PDL-derived cell sheets. AM has high biocompatibility as well as anti-scarring and anti-infection properties. In the current study, we immunohistochemically evaluated PDL-derived cells cultured on AM. Methods: The protocol for this experiment was approved by the Human Studies Committee of Kyoto Prefectural University of Medicine (RBMR-R-21). AM was obtained from women undergoing caesarean section. PDL tissue was obtained from a healthy human maxillary third molar that had erupted. The PDL tissue was minced and then cultured in a culture medium as explants. After 3rd to 4th passages, the PDL-derived cells were first cultured on an AM carrier and then evaluated immunohistochemically for distribution of Ki-67, vimentin, desmoplakin and zonula occludens protein-1 (ZO-1) using an indirect fluorescent antibody method. Results: PDL-derived cells showed a monolayered structure after 2 weeks in culture on AM. Immunohistochemistry revealed that PDL-derived cells cultured on AM expressed Ki-67, vimentin, desmoplakin, ZO-1. Conclusion: These findings indicated that the cultivation of PDL-derived cells using AM enabled PDL-like differentiation. In addition, those cells showed desmosomes and tight junction-mediated cell-to-cell adhesion. We conclude that AM may represent a suitable scaffold for culturing PDL-derived cells, and that PDL-derived cells cultured on AM show sheet formation. This work was supported by Grant-in-Aid for Scientific Research (No. 22792000) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.