Objective
Periodontal ligament (PDL) cells and their substrates play key roles in periodontal regeneration. However, there has been no report on the use of amniotic membrane (AM) as a substrate for culturing PDL cells. In the current study, we conducted an analysis of PDL cells cultivated on AM to determine the distribution of factors responsible for maintaining the characteristics of PDL.

Materials and Methods
Amniotic membrane was obtained from women undergoing cesarean sections, whereas PDL tissue was obtained from human maxillary third molars. The harvested PDL cells were maintained in explant culture for three or four passages, following which they were cultured on AM.

Results
After 3 weeks of culture, the PDL cells had grown well on AM. Immunofluorescence showed that these cells were capable of proliferating and potentially maintaining their PDL-like properties. In addition, strong cell–cell adhesion structures, namely desmosomes and tight junctions, were shown to be present between cells. Electron microscopy images showed that the cultured PDL cells had differentiated and proliferated on AM with lateral conjugation and adhesion to AM.

Conclusion
We conclude that AM may represent a suitable substrate for culturing PDL cells and that PDL cells cultured on AM show sheet formation.