Novel ready-to-use regenerative medicine with cryopreserved 3D nanostructured fiber scaffold and dental pulp stem cells for bone regeneration

This study aims to evaluate the feasibility of cryopreserved three-dimensional nanostructured fiber scaffolds supplemented with human dental pulp stem cells (hDPSCs), and to demonstrate their potential as ready-to-use constructs for bone regeneration. Nanofiber-based polyglycolic acid (nPGA) with different surface densities were used to evaluate the effect of cryopreservation on the mechanical properties of the nPGA matrix. hDPSCs were seeded onto nPGA matrix to prepare hDPSCs-nPGA complex, which were cryopreserved for 1 or 4 weeks. After thawing, cell proliferation, cell surface antigen expression, and osteogenic differentiation capacity of hDPSCs within the cryopreserved complex were assessed. The microstructure of the hDPSCs-nPGA complex was examined using scanning electron microscopy (SEM). The strength retention ratio of nPGA with lower surface densities was maintained after cryopreservation. hDPSCs exhibited time-dependent proliferation on nPGA matrices. SEM confirmed that hDPSCs adhered and spread along nPGA fibers in three dimensions regardless of freezing duration. hDPSCs, after cryopreservation as an hDPSCs-nPGA complex expressed specific surface antigens. Alkaline phosphatase (ALP) and Alizarin red staining showed positive results after osteogenic induction. ALP expression on hDPSCs-nPGA complex was significantly up-regulated ( P < 0.05). The phenotype, proliferative capacity, and osteogenic differentiation potential of hDPSCs were maintained after cryopreservation as hDPSCs-nPGA complexes. These findings demonstrate that cryopreserving hDPSCs within a cell-integrated scaffold is a novel and effective strategy to maintain their stemness and differentiation potential. This innovative preservation approach enables long-term storage in a cell-associated matrix form and may provide ready-to-use, cell-based therapeutic constructs for bone regeneration.