Cryopreservation of human dental roots using vitrification for autologous human tooth tissue banking

This study aims to preliminarily evaluate the feasibility of autologous transplantation of tooth tissues cryopreserved with vitrification, by investigating the influence of cryopreservation with vitrification on human dental root, regarding the morphology, microhardness, cell apoptosis, proliferation and differentiation. Freshly extracted human permanent premolars were collected with crown removed. Dental roots were cryopreserved using a commercial vitrification medium (Kitazatousa). After six-month storage in liquid nitrogen, cryopreserved roots were thawed, and then evaluated using histological and immunohistochemical methods. Microhardness of dentine was measured with a Vickers indenter. Cells in periodontal ligament and dental pulp tissues were isolated and characterized. The proliferation, immunophenotype, apoptosis and differentiation ability of cells isolated from cryopreserved roots were evaluated. The data was analyzed using one-way analysis of variance (ANOVA) and Student's t-test. The gross and histological morphology of dental roots was not significantly changed after vitrification and thawing. A few tiny cracks were found in 3 of all 10 cryopreserved samples. No obvious changes were found in microstructure of dentine under SEM observation. Dental pulp cells and periodontal ligament cells were successfully isolated from tissues of cryopreserved human dental roots. There were also no significant differences of those periodontal ligament cells in the two groups regarding morphology, immunophenotype, viability, proliferation and apoptosis. The osteogenic and adipogenic differentiation capability of periodontal ligament cells was maintained by cryopreservation with vitrification. In the conditions of this study, cryopreservation with vitrification preserves cell survival, hardness and structural integrity of dental roots. Vitrification can be a potential way to preserve tooth tissue for future auto-transplantation and autologous cell therapy.