Background <p>Mesenchymal stem cell (MSC)-based tissue engineering is largely mediated by extracellular vesicles (EVs), particularly exosomes (Exos). The biological activity of Exos is strongly influenced by the isolation method, yet no standardized approach exists.</p> Methods <p>Dental pulp stem cell (DPSC)-derived Exos were isolated using ultracentrifugation (UC) or size exclusion chromatography (SEC). UC-Exos and SEC-Exos were characterized for morphology, size, yield, protein content, and proteomic profiles. Their effects on periodontal ligament stem cells (PDLSCs) were assessed, including proliferation, migration, and osteogenic differentiation. In vivo bone regeneration was evaluated in rat calvarial defects using Exos combined with bone-grafting material.</p> Results <p>SEC-Exos yielded exosomes with higher purity, greater particle counts, and lower protein contamination than UC-Exos. Proteomic profiling showed enrichment of osteogenic factors, including ENPP2 and afamin, in SEC-Exos. Functionally, UC-Exos promoted PDLSC proliferation and migration, while SEC-Exos more effectively enhanced osteogenic differentiation. Both Exos improved PDLSC functions dose-dependently and, when combined with bone grafts, consistently promoted calvarial bone regeneration. Notably, osteocalcin expression was higher in SEC-Exos–treated defects.</p> Conclusions <p>The isolation method critically determines the biological performance of DPSC-derived Exos. UC-Exos may be preferable for stimulating proliferation and migration, whereas SEC-Exos show advantages in osteogenic differentiation. Both approaches facilitated bone regeneration in vivo, suggesting that exosome isolation strategies should be tailored to experimental requirements and therapeutic objectives to optimize translational outcomes.</p> Significance Statement <p>The isolation method critically determines the biological performance of DPSC-derived exosomes. SEC-Exos, enriched in osteogenic factors such as ENPP2 and afamin, preferentially enhanced osteogenic differentiation, whereas UC-Exos promoted proliferation and migration. Both approaches supported consistent bone regeneration in vivo, underscoring that the optimal isolation strategy should be tailored to specific experimental and therapeutic objectives to advance translational applications.</p> Graphic Abstract <p>SEC isolation produced exosomes with higher yield, purity, and reduced protein contamination compared with UC. UC-Exos preferentially enhanced PDLSC proliferation and migration, whereas SEC-Exos more effectively promoted osteogenic differentiation. In rat calvarial defects, both Exos supported bone regeneration, with higher osteocalcin expression observed in the SEC-Exos group.</p>