Human Dental Pulp Cell Culture and Transplantation using Alginate Scaffold

Objective: We research stem cells in human dental pulp for the regenerative medicine. The present study examines the transplantation of cultured cells using alginate as a scaffold. Methods: The human dental pulp tissue was obtained sterilely from extracted tooth and cultured. The tissue was subcultured for 13 passages. We added Β-glycerophosphate to examine the cell differentiation, and examined alkaline phosphatase activity in the mineralizing tissue. For studying the differentiation of odontoblasts, the expression of dentin sialophosphoprotein (DSPP) mRNA was examined by the reverse transcriptase polymerase chain reaction (RT-PCR). We mixed the subcultured cells with 1.5% alginate solution to form cell clusters, which were transplanted subcutaneously into the dorsum of nude mice (KSN/Slc -nu/nu,male,7wk). SOFTEX was taken to identify mineralization in the transplants. After 6 weeks, the tissue was removed, embedded in paraffin and the collagen was examined. The fine structure was studied by the conventional TEM. Results: 1) Monolayer cultures: Fibroblast-like cells were observed in the 13th passage. Alkaline phosphatase activity was increased by adding Β-glycerophosphate. Mineralized nodules were observed in the cultures. The expression of DSPP mRNA was observed. 2) Transplantation: Formation of hard tissue was observed in the transplants. Type-I and -III collagen were observed in the mineralizing tissue. Conclusions: 1) The 13th passage cells differentiated odontoblast-like cells. 2) The expression of DSPP mRNA and formation of X-ray opaque tissue containing collagen fibers indicated the formation of dentin-like structure by the transplanted subcultured cells.