Objectives <p>This study aimed to investigate the regenerative potential of a novel three-dimensional, scaffold-free human periodontal ligament stem cell-derived cell pellet (PDLSC-CP) for bone tissue regeneration and alveolar ridge preservation. The study evaluated the efficacy of PDLSC-CP in vitro and in vivo, comparing it with traditional cell sheets (PDLSC-CS).</p> Methods <p>PDLSCs were isolated from extracted premolars and characterized for their mesenchymal stem cell properties, including fibroblast-like morphology, colony-forming ability, multilineage differentiation potential, and specific surface marker expression. PDLSC-CP and PDLSC-CS were constructed and compared through histomorphological, immunofluorescence, real-time PCR, and western blot analyses. In vivo evaluations were performed using rat calvarial defect models and canine alveolar ridge preservation models, with micro-CT and histological analyses to assess bone regeneration and ridge preservation.</p> Results <p>PDLSC-CP demonstrated superior histomorphological characteristics, including higher cell density, richer extracellular matrix (ECM), and enhanced osteogenic potential compared to PDLSC-CS. In vitro analyses revealed significantly elevated expression of ECM proteins (fibronectin, collagen type I) and osteogenic markers in PDLSC-CP. In vivo evaluations confirmed its remarkable capacity for bone regeneration in rat calvarial defects and alveolar ridge preservation in canine models. Micro-CT and histological analyses showed that PDLSC-CP promoted extensive and dense new bone formation, with improved bone volume fraction, trabecular thickness, and mineralization.</p> Conclusions <p>The findings highlight the therapeutic potential of PDLSC-CP as a promising tool for bone tissue engineering and regenerative medicine. Its ability to enhance bone regeneration and alveolar ridge preservation underscores its clinical relevance.</p> Graphical Abstract <p></p>