Spontaneously formed mesenchymal stem cell spheroids enhance alveolar bone regeneration and suppress inflammation following tooth autotransplantation

Objectives: This study aimed to evaluate the effects of spontaneously formed mesenchymal stromal cells (MSCs) spheroids on alveolar bone regeneration and inflammatory responses following tooth autotransplantation in a murine model. Methods: Spontaneous spheroids were generated from mouse calvaria-derived MC3T3-E1 cells using a low-adhesion culture system. Extracted molars were transplanted with spheroids embedded in a collagen-based scaffold. Monolayer cells, scaffold, and tooth-only groups served as controls. Bone regeneration was assessed by micro-CT and histological analyses at 1, 2, and 4 weeks post-transplantation. Gene expression related to osteogenesis and inflammation was analyzed by quantitative PCR. Results: In vitro analyses demonstrated that MC3T3-E1 spheroids exhibited significantly increased alkaline phosphatase activity, mineralization, and expression of pluripotency and osteogenesis related genes compared with monolayer cells. In vivo, the spheroid group showed significantly enhanced new bone formation, with higher bone volume fraction (BV/TV) throughout the observation period (p < 0.05). Expression of osteogenic markers, including osteocalcin (OCN) and collagen type I, was significantly upregulated, whereas inflammatory cytokines IL-6 and TNF-α were consistently suppressed. Histological evaluation revealed organized collagen fiber alignment suggestive of periodontal ligament-like tissue regeneration. Conclusions: MSC-derived spontaneous spheroids promote alveolar bone regeneration and suppress inflammatory responses following tooth autotransplantation. This spheroid-based strategy may improve regenerative outcomes in tooth autotransplantation.