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Bioengineered Teeth from Cultured Rat Tooth Bud Cells

¹ University Federal of São Paulo, Department of Otorhinolaryngology and Human Communication Disorders, São Paolo, Brazil;
² Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, and Department of Oral and Developmental Biology, Harvard Medical School, Boston, MA 02115, USA; and
³ Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA;

View larger version (106K): [in a new window]   Figure 1. Cultured 4-dpn rat tooth bud cells. After 6 days in culture, tooth bud cells appeared heterogeneous, containing both rounded epithelial and fibrotic mesenchymal cell types (A,B). Cytokeratin-14-positive positive-control oral epithelial cells and dental epithelial cells in mixed cell culture appeared brightly fluorescent (arrows, C and D, respectively). Dental cells seeded onto PGA and PLGA scaffolds (E and F, respectively).

View larger version (118K): [in a new window]   Figure 2. Analysis of 12-week implants. Light micrographs of PGA (A) and PLGA (B) scaffold implants harvested after 12 wks of growth in the omentum. Radiographic analysis of the same PGA (A') and PLGA (B') implants revealed distinctly mineralized tissues.

View larger version (112K): [in a new window]   Figure 3. Histological analysis of 12-week implants. H&E-stained control and experimental tissues. Positive control intact 4-dpn tooth bud implants exhibited well-formed dentin, enamel, and pulp tissues (A,A'). Dental-cell-seeded PGA (B,B') and PLGA (C,C') scaffold tooth tissues also generated dentin, enamel, and pulp tissues. Infiltrating lymphocytes were occasionally observed in dental implants (C,C', arrows). Goldner’s stain of positive control intact tooth bud implants revealed blue-stained dentin, red-stained immature enamel, and gray-stained mature enamel (D,D'). PGA- and PLGA-bioengineered teeth both exhibited blue-stained dentin, while PGA generally produced mature, gray-stained enamel at 12 wks, and PLGA generated both red- and gray-stained mature enamel (E,E',F,F'). Abbreviations: d, dentin; e, enamel; em, enamel matrix; pe, pre-enamel; pu, pulp.

View larger version (120K): [in a new window]   Figure 4. Immunohistochemical analysis of amelogenin expression. Control tooth bud implants exhibited amelogenin expression in forming enamel (A), while control antibody appeared negative (A'). PGA- (B,B') and PLGA- (C,C') bioengineered teeth also exhibited positive amelogenin expression (arrows).