Assessing bone regeneration potential of 3D scaffold-free cell pellets from periodontal ligament and bone marrow stem cells.

BACKGROUND: Bone defects pose a significant clinical challenge in clinical treatment, where stem cell-based tissue engineering strategies have emerged as a promising approach for bone regeneration. Notably, accumulating evidence suggests that scaffold-free three-dimensional cell pellets demonstrate therapeutic potential through direct implantation into bone defects area. METHODS: Two types of stem cells were isolated from bone and tooth, then cultured separately. Surface markers (CD34, CD45, CD90, CD105, CD146, STRO-1) were analyzed by flow cytometry. Bone marrow-derived and dental-derived CPs were cultured in ascorbic acid-supplemented growth medium. Histological morphology from CS to CP was examined through H&E staining. Cell pellets' biological properties were assessed in vitro via immunofluorescence, qPCR, and Western blot, and bone regeneration was evaluated using rat calvarial defect models. RESULTS: We demonstrated comparable morphology and immunophenotype between BMSCs and PDLSCs. H&E staining and TUNEL assays revealed tightly organized histological structures and low apoptosis rates in 5-day cultured cell pellets. Immunofluorescence analysis showed no significant differences in COL-1 or early osteogenic marker ALP between PDLSC-CP and BMSC-CP; however, BMSC-CP exhibited higher osteoblast-related BSP expression, whereas PDLSC-CP displayed elevated bone remodeling-associated OPN levels. Consistent trends were observed in qPCR and Western blot analyses. In rat calvarial defect models, both CP types induced significant bone formation, with BMSC-CP demonstrating enhanced osteogenic capacity compared to PDLSC-CP. CONCLUSION: Both BMSC-CPs and PDLSC-CPs demonstrate osteogenic potential in scaffold-free 3D environments, whereas standardized controls and mechanistic investigations are required to establish their distinct therapeutic advantages.