Characteristics of Tissue-engineered Dentin Generated by Immortalized Rat Odontoblast Cells

Repair and regeneration of dentin pose an interesting challenge, as odontoblast cells are post mitotic cells. Therefore an immortalized odontoblast cell-line which has the required biochemical machinery to produce mineralized tissue in-vitro has been generated. Objective: In this study these transformed odontoblast cells were implanted into animal models to determine their potential to form dentin in vivo. Methods: About 5x10⁶ of T4-4 cells were incubated with a 0.5x0.5 cm² of Collagraft (Zimmer, Warsaw, IN) at 37^oC for 30 min, and then transplanted subcutaneously on the dorsal surface and intramuscularly (quadriceps muscles) in 15-week-old Sprague-Dawley rats. Collagraft with RPC-C2A cells was used as a control. Radiographs were closely examined for signs of mineralized tissue formation every 10 days. After 12 weeks the transplant was excised, fixed with 10% formalin, and embedded in paraffin, decalcified with buffered 10% EDTA (pH 7.4). Results: Histological and immunohistochemical evaluations demonstrated that the morphology of the transformed cells resembled odontoblast-like cells. Characteristic phenotypic changes associated with mineralized tissue formation were observed at the microenvironment of the implantation site. Immunohistochemical analysis confirmed the presence of dentin matrix proteins. Topographic mapping of the mineralized surface by atomic force microscopy (AFM) revealed that T4-4 cells on collagraft membrane were well organized with odontoblast process parallel to each other. Dentinal tubules were also observed to be surrounded by a mineralized matrix. The structural characteristics of the matrix were similar to native dentin. Conclusion: The data demonstrate that the immortalized cells have a characteristic odontoblast phenotype and have the unique capacity to form de-novo dentin in vivo. This work is supported by NIH-NIDCR grant DE 11657.