Materials and Methods: The rat dental pulp tissue (Wistar, male, 5wk) was obtained sterilely from incisors and cultured. The tissue was subcultured for 2 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, 14wk). SOFTEX was taken to identify the mineralization of transplants. After 6 weeks, the transplants with surrounding tissues were removed and embedded in paraffin, and the occurance of collagen and osteocalcin was examined by routine light microscopy and CLSM. The fine structure was further studied by the conventional TEM.
Results: In the monolayer cultures, alkaline phosphatase (ALP) activity was increased by adding β-glycerophosphate. Mineralizing nodules with the DSPP mRNA expression were observed in the cultures. In the transplants, formation of hard tissue was demonstrated. Type-I and -III collagen, osteocalcin and DSP were observed in the mineralizing matrix. In the experimental group, we observed an abundant invasion of capillary network in the transplants containing cultured cells and alginate scaffold. In contrast to the control (alginate implantation) group, certain mineralizing dentin-like tissue, which was presumptively formed by the cultured cells, was evident in the experimental group.
Conclusions: The expression of DSPP mRNA and formation of X-ray opaque tissue containing collagen indicate the formation of dentin-like structure by the transplantation of subcultured cultured cells.