Cryopreserved dentin matrix as a scaffold material for dentin-pulp tissue regeneration

Cryopreservation has been identified as an efficient approach to preserve tissue engineered products for a long term. Our prior studies have suggested that the treated dentin matrix (TDM) could be an ideal bioactive scaffold for dental tissue regeneration. In this study, we hypothesize that the cryopreservation could effectively maintain the survival and viability of dentinogenesis-related proteins of TDM and the cryopreserved dentin matrix (CDM) would provide the suitable biological scaffold and inductive microenvironment for the regeneration of dentin-pulp like tissue. CDM-3 and CDM-6 were prepared by cryopreserving TDM in liquid nitrogen (−196 °C) with cryoprotectant for 3 months and 6 months, respectively. Various biological characteristics of CDM, including mechanical properties, cell proliferation, and odontogenesis ability, were investigated. To further evaluate the inductive capacity of CDM, human dental follicle cells were encapsulated within CDM, and implanted the scaffold into a mouse model for 8 weeks, and the grafts were harvested and assessed histologically. The CDM showed superior mechanical properties than TDM. Compared to TDM, CDM can release more dentinogenesis-related proteins due to the larger pore diameter. Cell proliferation with the addition of CDM extract liquid was similar to that of TDM in the first five days. Human dental follicle cells, under the effect of CDM extract liquid, highly expressed bone sialoprotein, collagen-1, alkaline phosphatase, indicating that CDM, regarded as the inductive microenvironment, plays an important role in odontogenesis. Most importantly, in vivo, CDM could induce dental follicle cells to regenerate new dentin-pulp like tissues, such as dentinal tubules, predentin, collagen fibers, nerves, and blood vessels which were positive for dentin sialophosphoprotein, dental matrix protein-1, Tubulin, and collagen-1. In conclusion, CDM is an ideal biological scaffold material for human dentin-pulp like tissue regeneration. These findings indicated that TDM could be preserved as the tissue engineering scaffold that is readily available for patient treatments. Furthermore, the success of cryopreservation of TDM may also provide an insight into preserving other bioactive scaffold materials of tissue engineering.