Cell Sheet Engineering for Periodontal Regeneration - New Trends in Tissue Engineering and Regenerative Medicine - page 8

3.1. Small animal studies

Human PDL (hPDL) cell sheets were successfully created using temperature responsive
dishes, and the characteristics of hPDL cell sheets were investigated (Hasegawa et al., 2005).
In this study, explant culture methods were utilized for the primary culture of hPDL cells.
The hPDL cell sheets cultured with ascorbic acid were recovered from the culture dishes as a
contiguous sheet accompanied by abundant extracellular matrix components, including
type I collagen, integrin β1 and fibronectin. Then, hPDL cell sheets were transplanted as cell
pellets into a mesial dehiscence model in athymic rats. Four weeks after surgery, newly
formed immature fibers with obliquely anchored dentin surfaces were observed in all the
experimental sites, whereas no such findings were observed in any control sites (Figure
2).These results suggest that this procedure based upon the principles of cell sheet engineer‐
ing can be applied to periodontal regeneration.

A B

Figure 2. PDL regeneration at 4 weeks postsurgery.

A: Nontransplanted control site. B: hPDL transplanted experimental site. Regeneration of
periodontal ligament-like structure was observed only in the experimental site. Azan stain‐
ing. Modified and reprint from Hasegawa et al., 2005.

Next, the optimal culture condition was examined. Because the osteoinductive medium,
which contains 50 µg/ml of ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexame‐
thasone, enhanced both osteoblastic/cementoblastic and the periodontal differentiation of
PDL cells in vitro , we compared hPDL cell sheets cultured in the absence and presence of
these osteoinductive supplements in a xenogeneic transplantation model (Flores et al.,
2008a). Three layered hPDL cell sheets were constructed with fibrin gel and transplanted
with a human dentin block into the back of a subcutaneous athymic rat. The constructs were
excised for histological investigation 6 weeks after the transplantation. The three-layered
hPDL cell sheets-dentin block constructs induced a new cementum-like hard tissue on the
surface of the dentin in more than 60% of the samples. Collagen fibers were inserted perpen‐