83In the dental field, many researchers have isolated the dental epithelial and mesen-
chymal tissues from embryonic tooth germs of experimental animal and dissociated
such tissues with surgical operation by using stereomicroscopic guidance and enzy-
matic treatments to obtain single stem cells. It has been reported that bioengineering
cell aggregates reconstructing epithelial and mesenchymal tooth germ cells by using
the cellular centrifugation have the potential for tooth formation after in vivo trans-
plantation (Hu et al. 2006 ; Yamamoto et al. 2003 ). Even when bioengineered cell
aggregate were mixed with epithelial and mesenchymal stem cells isolated from
tooth germ, the correct tooth structure could be generated by the self-reorganisation
through the cell rearrangement of epithelial and mesenchymal cells (Song et al.
2006 ). This technique is an innovative approach to replicate tooth organogenesis;
however, the further improvements have been required in terms of the generating
frequency of bioengineered tooth and the correct tissue formation.
5.4.3 Three-Dimensional Cell-Manipulation Method:
The ‘Organ Germ Method’
To achieve precise replication of the developmental processes in organogenesis, an
in vitro three-dimensional novel cell-manipulation method designated as a bioengi-
neered organ germ method has been recently established (Nakao et al. 2007 ). We
demonstrated the possibility of developing a bioengineered tooth germ using com-
pletely dissociated single epithelial and mesenchymal cells from tooth germs at
ED14.5 mice. The most important breakthrough using our method is the achieve-
ment of three-dimensional cell compartmentalisation of epithelial and mesenchy-
mal cells at a high cell density in a collagen gel. This bioengineered tooth germ can
achieve initial tooth development with the appropriate cell-to-cell compaction
between epithelial and mesenchymal cells in vitro organ culture. Bioengineering
tooth germ reproduced by our method successfully replicates the multicellular
assembly, including ameloblasts, odontoblasts, pulp cells and dental follicle cells,
underlying the epithelial-mesenchymal interactions as well as natural tooth devel-
opment. The bioengineered tooth germ generates a structurally correct tooth after
transplantation in an organ culture in vitro as well as following placement into a
subrenal capsule in vivo (Nakao et al. 2007 ) (Fig. 5.4a). Tooth morphology is
defined by not only the tooth length and crown size as macromorphological feature
but also the cusp numbers/position as micromorphological feature. These morpho-
logical properties are regulated by specific gene expression at the boundary surface
of immature oral epithelium and neural crest-derived mesenchyme in the embryonic
jaw. Our bioengineered tooth germ could be reconstructed by adjusting various cell-
to- cell contact length between the epithelial and mesenchymal cell layer, and
thereby the crown width and cusp number of occurred bioengineered tooth were
dependent on the contact length of epithelial and mesenchymal cell layer in bioen-
gineered tooth germ (Ishida et al. 2011 ).
5 Functional Tooth Regeneration