2016 Volume 59 Issue 5 Pages 394-401
Purpose: An amniotic membrane (AM) with anti-inflammatory and anti-infective effects is attracting attention for its usefulness and effectiveness as a culture substrate and graft material, and is currently used in a number of surgical procedures. We previously prepared various cell sheets cultured with AM as the substrate, and demonstrated that some sheets may be effectively used in clinical settings for regenerative medicine. In recent studies, cultured cell sheets derived from the periosteum were transplanted in order to regenerate periodontal tissues. In the present study, periosteal-derived cells (PDCs) were cultured on AM to generate novel periosteal cell sheets, and the kinetics of the cell sheets were immunohistochemically investigated in vivo.
Methods: AM was removed from the placenta during cesarean section, and periosteal tissues on alveolar bones were collected upon the creation of a mucoperiosteal flap during oral surgery. Periosteal tissues were primarily cultured, followed by three to four passages to obtain PDCs. These cells were seeded on AM, and the epithelial cells that developed were scraped off and then cultured for three weeks. The resulting culture sheets were transplanted under the renal capsules of nude mice, and were removed after 4 weeks for H-E staining and immunostaining.
Results: PDCs showed a layered structure on AM. Immunostaining images revealed the expression of Ki-67 (a cell proliferation marker), vimentin (a mesenchymal cell marker), and osteocalcin (an osteoblast marker). Desmoplakin (a desmosome marker) and ZO-1 (a tight junction marker) were also expressed with cell sheet formation. Vimentin and osteocalcin were expressed even after transplantation, and the properties of the cell sheets were maintained.
Conclusion: PDCs proliferated on AM, forming single cell sheets, which demonstrates that AM is suitable as a culture substrate. The periosteum contains cells that have the ability to differentiate into osteoblasts and osteocytes. The cell sheets expressed osteoblast proteins, which were maintained even in an in vivo environment. Thus, PDC sheets may be prepared with AM. A long-term transplantation study will be conducted in the future.
