Human Tooth Development and Dental Stem Cell Yield: Radiographic Approach

Our long-term objective is to generate biologically based, bioengineered replacement tooth substitutes exhibiting the physical properties and functions of natural teeth. We previously demonstrated the successful generation of bioengineered tooth crowns from harvested pig and rat dental stem cells (DSCs). To facilitate characterizations of human DSCs, we desired to develop methods to correlate human tooth developmental stage with anticipated DSC number. Objective: To develop a novel radiographic developmental classification system for human third molar teeth, for correlation with anticipated DSC yield. Methods: Human teeth were obtained from patients scheduled for diagnostic third molar extraction using approved protocols (CONEP-13083). Harvested human tooth pulp cells were plated at low density (2.5x105 cells/mL, 5 mL total) into 75cm2 (T75) culture flasks, grown in 5% CO2 at 37°C, and analyzed for CFU and cell proliferation assays at 14 and 21 days. Confluent human tooth cell cultures were harvested, split into equal portions, statically seeded onto PGA/PLGA scaffolds, implanted into immuno-suppressed rats, and grown for 20 weeks. Results: CFU and cell proliferation assays revealed distinct differences in hDSC populations isolated from developmentally staged teeth. The greatest cell yields and CFU capability were obtained from Stage 1 and 2 dental pulp. Stage 1 maintained DSC colonies after 21 days in culture. The lowest cell yields were obtained from Stage 3, 4 and 5 teeth. Conclusion: Our novel radiographic tooth development classification method for estimating DSC yields will be useful in assessing the utility of harvested teeth for future dental tissue engineering applications. These studies were supported by the University Federal São Paulo, São Paulo Brazil, CNPq,FAPESP 07/51227-4, Tufts University, Boston, MA, The Center for the Integration of Medicine and Innovative Technology (CIMIT), and NIH/NIDCR grants TW007665-02 and DE016132 (PCY).