Regenerative endodontics has been described as a paradigm shift in dentistry, despite its current limitation to immature teeth and reparative rather than regenerative outcomes. Cell-free treatments are favored because of regulatory issues. However, the recruitment of host-derived stem cells to the desired site remains challenging. We investigated whether dental pulp-derived exosomes, which are extracellular vesicles that contain proteins, lipids, RNA, and DNA and thus mirror their parental cells, may be used for this purpose. The use of exosomes may present appreciable advantages over the direct use of transplanted stem cells due to a higher safety profile, easier isolation, preservation, and handling. Here we harvested exosomes from a cultured third-molar pulp cell and assessed them by transmission electron microscopy and Western blotting. Human mesenchymal stem cells (MSCs) were exposed to these exosomes to assess exosome uptake, cell migration, and proliferation. In addition, a fibrin gel (i.e., a diluted fibrin sealant), was assessed as a delivery system for the exosomes. Our results show that exosomes attracted MSCs, and the fibrin gel enhanced their effect. Moreover, exosomes improved the proliferation of MSCs. Therefore, we propose that pulp-derived exosomes in combination with a fibrin gel could be a powerful combination for clinical translation towards improved cell-free regenerative endodontics and thus represent a new way to fill dental hard tissues.

Introduction: In carious teeth, transforming growth factor beta 1 (TGF-b1) is released from the dentin matrix and possibly activated in an acidic environment. Conversely, EDTA solutions with a neutral to slightly alkaline pH are used in clinics to promote cell homing in regenerative endodontic procedures. We hypothesized that citric acid (CA) might be more beneficial. Methods: TGF-b1 release from human dentin disks conditioned with either 10% CA (pH = 2) or 17% EDTA (pH = 8) and the behavior of human stem cells toward such pretreated dentin were studied. The protein concentration in conditioning solutions after 10 minutes of dentin exposure was determined using a pHindependent slot blot technique. Results: There was a 5-fold higher concentration of the target protein in CA (382 AE 30 ng/disk) compared with EDTA (66 AE 3 ng/ disk, P < .005). Using confocal laser scanning microscopy on immunofluorescent-labeled disks, we identified a high density of TGF-b1 in peritubular dentin after CA treatment. A migration assay showed that CA conditioning attracted significantly more stem cells toward the dentin after 24 hours compared with EDTA (P < .05) or phosphate-buffered saline (P < .005). To investigate whether the cell response to these dentin surfaces could be affected by different pretreatments, we cultured stem cells on conditioned dentin disks and found that CA had a significantly (P < .05) better effect than EDTA on cell attachment and cell survival. Conclusions: CA conditioning could be useful and may have significant benefits over current treatments.

Regenerative endodontic procedures are an alternative to conventional root-canal treatment and apexification. There are two different tissue engineering approaches that are currently followed, both aiming at the colonisation of the cleaned pulp space by pluripotent cells and subsequent pulp regeneration. Firstly, the transplantation of mesenchymal stem cells (MSCs), and secondly a cell-free strategy that relies on bioactive molecules to trigger the recruitment of the patient’s own cells. The first approach is hampered by costs and regulatory issues. Despite great initial enthusiasm with a clinically used cell-free approach that relies on induced bleeding into the pulp space, results have been revealed to be rather unpredictable, and mere repair rather than regeneration of the pulp-dentin complex is what is typically achieved. Moreover, the extent of further root development is variable, and the concept is limited to immature teeth. This article discusses a third possible way of regenerative endodontics that involves the application of MSC-derived exosomes. These are extracellular vesicles that contain proteins, lipids, and nucleic acids, reflecting the secretome of MSCs. Based on the first in vitro and in vivo studies, exosomes appear to be a potent tool to improve pulp regeneration. This narrative review aims to investigate the therapeutic use of human MSCs or dental pulp-derived exosomes in regenerative endodontics. Furthermore, the focus of this review is on targeting important questions that should be investigated in future in-vivo and clinical studies, such as the choice of scaffold material for exosome delivery into the pulp space.