Abstract
Tissue engineering using living cells is emerging as an alternative to tissue or organ transplantation. The purpose of the present study was to fabricate cell sheets of a thickness sufficient for transplantation, and shortening of the fabrication periods. Human alveolar bone periosteal cells (HABPCs) were seeded at a density of 3.6×104 cells/ml on to the 24 wells of temperature-responsive cell culture dishes at 1, 7,and 11 days. Fourteen days after the first seeding, the HABPC sheets were harvested from 15 of the temperature-responsive culture dishes and stacked one over the other to fabricate HABPC multilayered sheets. The HABPC multilayered sheets were cultured for 1, 3, or 5 days and examined by histological and immunohistochemical analyses. Alkaline phosphatase staining and immunohistochemical staining for anti human type I collagen, osteopontin, osteocalcin, and runt-related transcription factor 2 showed HABPC differentiation occurring from the outer layers of the HABPC multilayered sheets. The results suggested an increase in new bone formation from 3 to 5 days. Mineralized-like tissues were observed in the HABPC multilayered sheets with von Kossa staining at three days. In this study, we were able to shorten the fabrication period of the HABPC multilayered sheets by using a higher cell density for the initial cell seeding into the temperature-responsive cell culture dishes. In addition, we have reported a method for the preparation of HABPC multilayered sheets of sufficient thickness for transplantation. Nihon Shishubyo Gakkai Kaishi (J Jpn Soc Periodontol) 55(1):24-36, 2013.
