Exosomes derived from stem cells from apical papilla promote craniofacial soft tissue regeneration by enhancing Cdc42-mediated vascularization

Background: Reconstruction of complex critical-size defects (CSD) in the craniofacial region is a major challenge, and soft tissue regeneration is crucial in determining the therapeutic outcomes of craniofacial CSD. Stem cells from apical papilla (SCAP) are neural crest-derived mesenchymal stem cells (MSCs) that are homologous to cells in craniofacial tissue and represent a promising source for craniofacial tissue regeneration. Exosomes, which contain compound bioactive compounds, are the key factors in stem cell paracrine action. However, the roles of exosomes derived from SCAP (SCAP-Exo) in tissue regeneration are not fully understood. Here, we explored the effects and underlying mechanisms of SCAP-Exo on CSD in maxillofacial soft tissue.

Methods: SCAP-Exo were isolated and identified by transmission electron microscopy and nanoparticle tracking analysis. The effects of SCAP-Exo on wound healing and vascularization were detected by measuring the wound area and performing histological and immunofluorescence analysis on the palatal gingival CSD of mice. Real-time live-cell imaging and functional assays were used to assess the effects of SCAP-Exo on the biological functions of endothelial cells (ECs). Furthermore, the molecular mechanisms of SCAP-Exo-mediated EC angiogenesis in vitro were tested by immunofluorescence staining, Western blot, and pull-down assays. Finally, in vivo experiments were carried out to verify whether SCAP-Exo could affect vascularization and wound healing through cell division cycle 42 (Cdc42).

Results: We found that SCAP-Exo promoted tissue regeneration of palatal gingival CSD by enhancing vascularization in the early phase in vivo and that SCAP-Exo improved the angiogenic capacity of ECs in vitro. Mechanistically, SCAP-Exo elevated cell migration by improving cytoskeletal reorganization of ECs via Cdc42 signalling. Furthermore, we revealed that SCAP-Exo transferred Cdc42 into the cytoplasm of ECs and that the Cdc42 protein could be reused directly by recipient ECs, which resulted in the activation of Cdc42-dependent filopodium formation and elevation in cell migration of ECs.

Conclusion: This study demonstrated that SCAP-Exo had a superior effect on angiogenesis and effectively promoted craniofacial soft tissue regeneration. These data provide a new option for SCAP-Exo to be used in a cell-free approach to optimize tissue regeneration in the clinic.