Molecular Analyses of Bioengineered Tooth Structures

We previously reported the successful bioengineering of small but complete tooth crowns, by seeding single cell suspensions of dissociated pig tooth buds onto biodegradable polymer scaffolds, and growing them in the omenta of rats for 20-30 weeks (Young et al., 2002). Bioengineered tooth structures contained odontoblasts and dentin, dental pulp, Hertwig’s epithelial root sheath, and fully formed enamel organs with dental enamel. Objective: To perform molecular and cellular analyses of developing tooth structures. Methods: Biodegradable scaffolds were seeded with dissociated porcine third molar cells, implanted into the omentum of athymic Rowett rats, and allowed to develop over 2, 4, 8, 11, 18, 21 and 25 weeks. Tissues were excised, fixed, decalcified, embedded in paraffin, sectioned and stained with hematoxylin/eosin, and examined microscopically. Results: At 4 weeks, multiple small (10-40 cells) circular clusters of epithelial cells surrounded by pulp-like mesenchymal tissues were observed. By 8 weeks, these small foci appeared to have formed larger circular structures typically surrounded by dentin and odontoblasts, and lined with columnar ameloblast-like cells adjacent to stratum intermedium and stellate reticulum tissues. By 11 and 18 weeks, tooth structures closely resembling natural cap-stage tooth buds had formed, consisting of a layer of epithelial cells adjacent to condensed mesenchymal cells. By 18 to 25 weeks many mature tooth structures had formed which contained enamel, dentin and pulp as previously reported. Conclusions: Bioengineered tooth structures appear to originate from small tooth bud-like clusters of cells, that appear to recapitulate normal embryonic tooth development. Molecular analyses of wnt, bmp, amelogenin and dentin sialoprotein expression in developing bioengineered tooth structures are currently being performed using in situ hybridization, immunohistochemistry and laser-capture RT-PCR. Supported by a Harvard School of Dental Medicine Seed grant and the Center for the Integration of Medicine and Innovative Technology (CIMIT).