Odontogenic exosomes simulating the developmental microenvironment promote complete regeneration of pulp-dentin complex in vivo

DPSC-Od-Exos from DPSCs undergoing initial odontogenic differentiation facilitated complete regeneration of pulp-dentin complex by enhancing odontogenesis, angiogenesis, and neurogenesis, shedding light on their promising prospects in advancing regenerative endodontic therapy. • Compared with normal exosomes derived from dental pulp stem cells (DPSC-Exos), exosomes secreted by DPSCs undergoing initial odontogenic differentiation (DPSC-Od-Exos) elicited a more robust enhancement on the proliferation and migration as well as odontogenic, endothelial, and neurogenic differentiation of DPSCs. • DPSC-Od-Exos were superior to DPSC-Exos in prompting the proliferative, migratory, and angiogenic potential of human umbilical vein endothelial cells (HUVECs). • As reveled by the Matrigel plug assay, DPSC-Od-Exos performed better than DPSC-Exos in ameliorating the formation of vascular tube-like structures of DPSCs and HUVECs. • After subcutaneous transplantation with DPSCs, DPSC-Od-Exos contributed to the regeneration of complete pulp-dentin complex in tooth root fragments, which encompassed enriched neurovascular structures and a continuous layer of odontoblast-like cells extending their cytoplasmic projections into the newly formed dentinal tubules. Establishing an optimized regenerative microenvironment for pulp-dentin complex engineering has become increasingly critical. Recently, exosomes have emerged as favorable biomimetic nanotherapeutic tools to simulate the developmental microenvironment and facilitate tissue regeneration. This study aimed to elucidate the multifaceted roles of exosomes from human dental pulp stem cells (DPSCs) that initiated odontogenic differentiation while sustaining mesenchymal stem cell (MSC) characteristics in odontogenesis, angiogenesis, and neurogenesis during pulp-dentin complex regeneration. Differential centrifugation was performed to isolate exosomes from normal DPSCs (DPSC-Exos) and DPSCs that initially triggered odontogenic differentiation (DPSC-Od-Exos). The impact of these exosomes on the biological behavior of DPSCs and human umbilical vein endothelial cells (HUVECs) was examined in vitro through CCK-8 assay and Transwell migration assay, as well as assays dedicated to assessing odontogenic, angiogenic, and neurogenic capabilities. In vivo, Matrigel plugs and human tooth root fragments incorporating either DPSC-Exos or DPSC-Od-Exos were subcutaneously transplanted into mouse models. Subsequent histological, immunohistochemical, and immunofluorescent analyses were conducted to determine the regenerative outcomes. DPSC-Exos and DPSC-Od-Exos revealed no remarkable difference in their characteristics. In vitro analyses indicated that DPSC-Od-Exos significantly facilitated the proliferation, migration, and multilineage differentiation of DPSCs compared with DPSC-Exos. Furthermore, DPSC-Od-Exos elicited a more pronounced effect on the tubular structure formation of HUVECs. Consistently, Matrigel plug assays confirmed that DPSC-Od-Exos exhibited superior performance in promoting endothelial differentiation of DPSCs and stimulating angiogenesis in HUVECs. Notably, DPSC-Od-Exos contributed to complete pulp-dentin complex regeneration in human tooth root fragments, characterized by enriched neurovascular structures and a continuous layer of odontoblast-like cells, which extended cytoplasmic projections into the newly formed dentinal tubules. By simulating the developmental microenvironment, multifunctional DPSC-Od-Exos demonstrated promising potential for reconstructing dentin-like tissue, vascular networks, and neural architectures, thereby enhancing our understanding of the therapeutic implications of DPSC-Od-Exos in regenerative endodontic treatment.