Objectives: Former research has led to the assessment that human dental pulp can serve as an alternative source for adult mesenchymal stem cells. To enable their use in clinical applications, cryopreservation of these cells is inevitable. However, little is known about the effects of cryopreservation on the proliferative kinetics and immunophenotype of human dental pulp stem cells (HDPSCs). In this study, the proliferative kinetics, as well as the expression of mesenchymal stem cell markers, before and after cryopreservation was compared. Furthermore, their multilineage differentiation capacity was evaluated.

Methods: HDPSCs were isolated from third molars using a mechanical and enzymatical digestion method. Cells were kept in culture, or were frozen and thawed. Proliferation rates were assembled to calculate doubling times and immunophenotyping was performed using the mesenchymal stem cell markers STRO-1, CD29, CD44, CD105, CD117 and CD146. Next, cells were cultured in conditioned media triggering adipogenic, osteogenic and chondrogenic differentiation of HDPSCs. Differentiation was then evaluated using antibodies against FABP, osteocalcin and aggrecan, respectively. Moreover, Oil red O, Alizarian Red S and Alcian blue stainings were used to assess multilineage differentiation of HDPSCs.

Results: Proliferative capacity of HDPSCs shortly after cryopreservation declined prior to re-establishment of normal proliferation rates. Furthermore, cells appeared to be positive for all used markers before and after cryopreservation. Immunological and histological staining demonstrated successful differentiation of HDPSCs into adipocytes, osteoblasts and chondrocytes.

Conclusion: The results of this study prove that HDPSCs do not show distinct expression patterns of stem cell markers before or after cryopreservation. Additionally, in the long term no differences in proliferative kinetics were visible between cultured or cryopreserved HDPSCs. This study demonstrated that cryopreservation can serve as an adequate manner to preserve HDPSCs for later clinical appliance. Moreover, the multilineage differentiation capacity of HDPSCs renders them a possible therapy in regenerative medicine.

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