Immunohistochemical Study of Periosteal-Derived Cell Sheet Cultured on Amniotic Membrane Aiming at Periodontal Tissue Regeneration

Statement of the problem: The amniotic membrane (AM) is a thin membrane that covers the outermost layer of the placenta, and is composed of parenchyma of a constant thickness. It can mostly be aseptically collected from the placenta, and is easily obtained without any ethical or technical problem because it is usually discarded after delivery. It has been used for various surgeries, attracting attention for its usefulness and effectiveness as a culture substrate as well as graft material. We have focused on such usefulness of AM, and prepared various cultured cell sheets (oral mucosal epithelial, periodontal ligament-derived, and pulp-derived cell sheets) using the membrane as a substrate (ref. 1, 2). Some sheets have been clinically applied, demonstrating their effectiveness in novel regenerative therapies. In the present study, these cell culture systems were applied to the culture of periosteum-derived cells (PDCs). In several recent studies, PDCs were cultured in vitro in a sheet form and transplanted for regenerating periodontal tissue. We developed the use of a suitable substrate for culturing PDCs. PDCs were grown in vitro and cultured in a sheet form on AM. In addition, the cell kinetics of the cultured sheet were investigated in vivo.

Materials and methods: AM, collected from the placenta during cesarean section, was used. The periosteal tissue on the alveolar bone was collected during mucoperiosteal flap during surgeries, such as wisdom tooth extraction. The periosteal tissue was washed and primarily cultured. Cells after 3-4 passages were used as PDCs. PDCs were cultured on AM, with epithelial cells peeled off and removed, for about three weeks in bone differentiation-inducing medium. PDCs cultured on AM were HE stained and immunostained with fluorescent antibodies. PDCs cultured on AM were transplanted under the renal capsule of a BALB/c nude mouse, and removed four weeks later. The present study and the use of periosteal tissue and AM were approved by the Ethical Committee (C-1038) and the Animal Care Committee (M25-268) of Kyoto Prefectural University of Medicine.

Results: PDCs proliferated on AM, forming a layered structure, and showed the expressions of cell proliferation (Ki-67), mesenchymal cell (vimentin), and osteoblast (bone Gla protein) markers on immunostaining images. In addition, desmosomal protein (desmoplakin), tight junction protein (ZO-1), basement membrane protein (laminin 5), and basement membrane collagen (collagen VII) were expressed, forming a single cell sheet. Vimentin and bone Gla protein were also expressed after transplantation with the properties of cell sheets maintained even in an in vivoenvironment.

Conclusions: Important factors for regenerating periodontal tissue defects include growth factors, cells, and substrates. To our knowledge, no report has been published on the use of AM as a substrate to culture PDCs. In addition, the kinetics of PDCs on AM in an in vivo environment remain unclear. Thus, in the present study, we transplanted PDCs cultured on AM into experimental animals to examine cell migration and proliferation. As a result, PDCs form a single cell sheet on AM, suggesting that AM is a suitable culture substrate. Some periosteal cells can differentiate into osteoblasts or osteocytes. Bone Gla protein of osteoblasts is also expressed on the cell sheet, suggesting that the properties were maintained even in an in vivoenvironment. Thus, PDCs cultured on AM may form a cell sheet capable of regenerating periodontal tissues. Long-term follow-up studies after transplantation should be conducted.

References:

1. Amemiya T, Nakamura T, et al. Immunohistochemical study of oral epithelial sheets cultured on amniotic membrane for oral mucosal reconstruction. Biomed Mater Eng. 20: 37-45, 2010.
2. Adachi K, Amemiya T, et al. Human periodontal ligament cell sheets cultured on amniotic membrane substrate. Oral Dis, in press.