Exosomes Derived from Stem Cells from the Apical Papilla Promote Dentine-Pulp Complex Regeneration by Inducing Specific Dentinogenesis

Abstract

Regenerative endodontic procedures (REPs) are a new option for the treatment of dental pulp or periapical diseases in permanent teeth with open apices. Histologically, the new tissues formed in the root canal after REPs are mainly cementum- or bone-like mineralised tissues, but not the real dentine-pulp complex. Therefore, how to promote dentine-pulp complex regeneration and improve the clinical effects of REPs has become a prominent research topic. Stem cells from apical papilla (SCAP) are derived from the dental papilla that can differentiate into primary odontoblasts and dental pulp cells that produce root dentine and dental pulp. Exosomes are the key regulator for the paracrine activity of stem cells and can influence the function of recipient cells. In this study, SCAP-derived exosomes (SCAP-Exo) were introduced into the root fragment containing bone marrow mesenchymal stem cells (BMMSCs) and transplanted subcutaneously into immunodeficient mice. We observed that dental pulp-like tissues were present and the newly formed dentine was deposited onto the existing dentine in the root canal. Afterwards, the effects of SCAP-Exo on the dentinogenesis of BMMSCs were elucidated in vitro. We found that the gene and protein expression of dentine sialophosphoprotein and mineralised nodule formation in BMMSCs treated with SCAP-Exo were significantly increased. In summary, SCAP-Exo were endocytosed by BMMSCs and obviously improved their specific dentinogenesis. The use of exosomes derived from dental stem cells could comprise a potential therapeutic approach for dentine-pulp complex regeneration in REPs.

Figure 1

Identification of exosomes from stem cells of the apical papilla (SCAP-Exo). (a) Morphology of SCAP-Exo (yellow arrow) based on transmission electron microscopy. (b) Size distribution of particles in the pellet as measured by nanoparticle tracking analysis. (c) Western blot analysis showing that SCAP-Exo were positive for the exosomal-specific markers CD9 and Alix.

Figure 2

Exosomes from the stem cells of the apical papilla (SCAP-Exo) promoted the regeneration of the dentine-pulp complex in vivo. (a) Schematic diagram of the animal experiment: SCAP-Exo, bone marrow mesenchymal stem cells (BMMSCs), and gelatine sponges as scaffolds were inserted into the canal of the tooth fragments, while the control group was treated with the same preparation without SCAP-Exo. The tooth fragments were then implanted into the nude mice. (b) HE staining showed a newly continuous layer of dentine (dotted line), odontoblast-like cells with overt polarised morphology (yellow arrow), and enhanced vascular formation (red arrow) in the experimental group. (c, d) The thickness of the new dentine and the number of odontoblasts were higher in the SCAP-Exo group than that in the control group (^∗∗P < 0.01, ^∗∗∗P < 0.001, n = 10). Error bars indicate means ± SD.

Figure 3

Exosomes from the stem cells of the apical papilla (SCAP-Exo) were endocytosed by bone marrow mesenchymal stem cells (BMMSCs). PKH-26-labelled SCAP-Exo (red) was internalised into the cytoplasm of DAPI-labelled BMMSCs (blue). In the negative control group of BMMSCs without exosomes, only the nuclei of BMMSCs were stained with DAPI (blue).

Figure 4

Exosomes from stem cells of the apical papilla (SCAP-Exo) induced the odontogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). (a, b) CCK-8 and Ki-67 assays showed that SCAP-Exo had no significant effect on the proliferation of BMMSCs. Scale bars = 100 μm. (c) Alizarin red S staining showed that SCAP-Exo could increase mineralised nodule formation in a dose-dependent manner. Scale bars = 100 μm. (d) Real-time PCR analysis revealed that SCAP-Exo could augment DSPP mRNA expression levels in BMMSCs without exerting effects on Runx2 and ALP mRNA expression levels. (e) Western blot analysis indicated that the protein expression levels of ALP and Runx2 were not significantly changed, but that high doses (20 and 50 μg/mL) of SCAP-Exo significantly improved the protein expression levels of DSPP (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001, NS = not significant, n = 3). Error bars indicate means ± SD.