Exosomes derived from mesenchymal stem cells (MSCs) have demonstrated regenerative potential for cell-free bone tissue engineering, nevertheless, certain challenges, including the confined therapeutic potency of exosomes and ineffective delivery method, are still persisted. Here, we confirmed that hypoxic precondition could induce enhanced secretion of exosomes from stem cells from human exfoliated deciduous teeth (SHEDs) via comprehensive proteomics analysis, and the corresponding hypoxic exosomes (H-Exo) exhibited superior potential in promoting cellular angiogenesis and osteogenesis via the significant up-regulation in focal adhesion, VEGF signaling pathway, and thyroid hormone synthesis. Then, we developed a platform technology enabling the effective delivery of hypoxic exosomes with sustained release kinetics to irregular-shaped bone defects via injection. This platform is based on a simple adsorbing technique, where exosomes are adsorbed onto the surface of injectable porous poly(lactide-co-glycolide) (PLGA) microspheres with bioinspired polydopamine (PDA) coating (PMS-PDA microspheres). The PMS-PDA microspheres could effectively adsorb exosomes, show sustained release of H-Exo for 21 days with high bioactivity, and induce vascularized bone regeneration in 5-mm rat calvarial defect. These findings indicate that the hypoxic precondition and PMS-PDA porous microsphere-based exosome delivery are efficient in inducing tissue regeneration, hence facilitating the clinical translation of exosome-based therapy.

Recent studies have shown that secretion of bioactive factors from mesenchymal stem cells (MSCs) plays a primary role in MSC-mediated therapy; especially for bone marrow-derived MSCs (BMSCs). MSCs from dental apical papilla (SCAPs) are involved in root development and may play a critical role in the formation of dentin and pulp. Bioactive factors secreted from SCAPs actively contribute to their environment; however, the SCAPs secretome remains unclear. To address this and gain a deeper understanding of the relevance of SCAPs secretions in a clinical setting, we used isobaric chemical tags and high-performance liquid chromatography with tandem mass spectrometry to profile the secretome of human SCAPs and then compared it to that of BMSCs. A total of 2,046 proteins were detected from the conditioned medium of SCAPs, with a false discovery rate of less than 1.0%. Included were chemokines along with angiogenic, immunomodulatory, antiapoptotic, and neuroprotective factors and extracellular matrix (ECM) proteins. The secreted levels of 151 proteins were found to differ by at least twofold when BMSCs and SCAPs were compared. Relative to BMSCs, SCAPs exhibited increased secretion of proteins that are involved in metabolic processes and transcription and lower levels of those associated with biological adhesion, developmental processes, and immune function. In addition, SCAPs secreted significantly larger amounts of chemokines and neurotrophins than BMSCs, whereas BMSCs secreted more ECM proteins and proangiogenic factors. These results may provide important clues regarding the molecular mechanisms associated with tissue regeneration and how they differ between cell sources.

Dental pulp necrosis are serious pathologic entities that causes tooth nutrition deficiency and abnormal root development, while regeneration of functional pulp tissue is of paramount importance to regain tooth vitality. However, existing clinical treatments, which focus on replacing the necrotic pulp tissue with inactive filling materials, fail to restore pulp vitality and functions, thus resulting in a devitalized and weakened tooth. Currently, dental pulp regeneration via stem cell-based therapy for pulpless teeth has raised enormous attention to restore the functional pulp. Here, a novel design of injectable simvastatin (SIM) functionalized gelatin methacrylate (GelMA) cryogel microspheres (SMS) loaded with stem cells from human exfoliated deciduous teeth (SHEDs) was established to refine SHEDs biological behaviors and promote in vivo vascularized pulp-like tissue regeneration. In this system, SIM encapsulated poly (lactide-co-glycolide) (PLGA) nanoparticles were incorporated into GelMA cryogel microspheres via cryogelation and O₁/W/O₂ emulsion method. SMS with sustained release of SIM promoted SHEDs adhesion, proliferation and exhibited cell protection properties during the injection process. Furthermore, SMS enhanced SHEDs odontogenic differentiation and angiogenic potential, and SHEDs loaded SMS (SHEDs/SMS) are beneficial for human umbilical vein endothelial cells (HUVECs) migration and angiogenesis, demonstrating their potential for use in promoting vascularized tissue regeneration. SHEDs/SMS complexes were injected into cleaned human tooth root segments for subcutaneous implantation in nude mice. Our results demonstrated that SHEDs/SMS could induce vessel-rich pulp-like tissue regeneration in vivo and that such an injectable nano-in-micro multistage system for the controlled delivery of bioactive reagents would be suitable for clinical application in endodontic regenerative dentistry.

The dental pulp plays a crucial role in the long-term maintenance of tooth function. The progress of endodontic treatment and pulp tissue regeneration engineering has made pulp-regeneration therapy promising in clinical practice. However, the mechanisms of pulp regeneration and the role of dental stem cells in development and regeneration have not been fully elucidated. Bridging the gaps between clinical operation and basic research is urgently needed. With the application of single-cell sequencing technology in dental research, the landscapes of human dental pulp cells have begun being outlined. However, the specific cellular heterogeneity of dental pulp cells, especially that of dental stem cells, at different spatial and temporal levels, is still unclear. In this study, we used single-cell RNA sequencing analysis of pulp samples at four different developmental stages and combined the findings with immunohistochemical staining to explore the development of dental pulp and stem cells. The results revealed temporal changes in the proportion of pulp cells during development. For example, mononuclear phagocytes accounted for a higher proportion in early samples. Odontoblasts identified by DMP1 had a higher expression of ion channel-related and neurodevelopment-related genes. Subpopulations were identified in fibroblasts, odontoblasts, and mesenchymal stem cells. We identified a subclass of odontoblasts that expresses DGKI and RRBP1 present in early developmental samples. A population of earlier mesenchymal stem cells expressed the SEPTIN gene, which may have greater proliferative and differentiation potential. Furthermore, dental pulp stem cells can differentiate into two directions: mineralization and myogenesis. In summary, the specific cellular heterogeneity of dental pulp cells was revealed at different spatial and temporal levels. These findings may shed light on the mechanism of tooth development. The gene expression profile of developing pulp cells may help to select cells for regenerative engineering and improve the success of dental pulp regeneration.