In Vivo Investigation of the Osteogenic Potential of Human Periodontal Ligament Cell Sheet Cultured on Amniotic Membrane

Statement of the problem: The ultimate aim of this study is to develop a novel culture sheet for bone regeneration using periodontal ligament (PDL) cells cultured on an amniotic membrane (AM). The 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 PDL cells sheet using the membrane as a substrate (ref. 1, 2). In addition, we have previously reported the possibility of PDL cells sheet on AM having osteogenic potential in vitro. In this study, we report new findings from an investigation of in vivo cell kinetics of PDL cells sheet on AM.

Materials and methods: AM, collected from the placenta during cesarean section, was used. The PDL cells were obtained from healthy erupted maxillary third molars removed for orthodontic reasons. The PDL cells were washed and primarily cultured. Cells after 3-4 passages were used as PDL cells. PDL cells were cultured on AM, with epithelial cells peeled off and removed, for about three weeks in bone differentiation-inducing medium. PDL cells cultured on AM were HE stained and immunostained with fluorescent antibodies. PDL cells 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 PDL and AM were approved by the Ethical Committee and the Animal Care Committee of Kyoto Prefectural University of Medicine. 

Results: The cell proliferation marker Ki-67, the mesenchymal cell marker vimentin, and the osteoblast marker osteocalcin were found to be expressed in the transplanted PDL cells sheet on AM. Vimentin and osteocalcin were also expressed after transplantation with the properties of cell sheets maintained even in an in vivo environment.

Conclusions: To our knowledge, no report has been published on the use of AM as a substrate to culture PDL cells. In addition, the kinetics of PDL cells sheet on AM in an in vivo environment remain unclear. Thus, in the present study, we transplanted PDL cells sheet on AM into experimental animals to examine cell migration and proliferation. The transplanted PDL cells sheet on AM showed cells positive for the mesenchymal cell marker vimentin and the osteoblast marker osteocalcin. This indicates that even after implantation, the properties of the PDL cells are retained. In addition, since proteins known to be expressed by osteoblasts during differentiation were also found, the transplanted PDL cells sheet may also have the osteogenic potential. Although further investigation of the transplantation period and cell kinetics is necessary, the results from the present study suggest that the PDL cells sheet on AM has potential as a culture sheet for bone regeneration. 

References:

1. Amemiya T, Adachi K, et al. Immunohistochemical study of human periodontal ligament-derived cells cultured on amniotic membrane. Jpn J Conserv Dent 53: 214–221, 2010.
2. Adachi K, Amemiya T, et al. Human periodontal ligament cell sheets cultured on amniotic membrane substrate. Oral Dis, in press.