Exosome-like vesicles derived from Hertwig's epithelial root sheath cells promote the regeneration of dentin-pulp tissue

Abstract

Background: The formation of dentin-pulp involves complex epithelial-mesenchymal interactions between Hertwig's epithelial root sheath cells (HERS) and dental papilla cells (DPCs). Earlier studies have identified some of the regulatory molecules participating in the crosstalk between HERS and DPCs and the formation of dentin-pulp. In the present study we focused on the role of HERS-secreted exosomes in DPCs and the formation of dentin-pulp. Specifically, we hypothesized that exosome-like vesicles (ELVs) might mediate the function of HERS and trigger lineage-specific differentiation of dental mesenchymal cells. To test our hypothesis, we evaluated the potential of ELVs derived from a HERS cell line (ELVs-H1) in inducing in vitro and in vivo differentiation of DPCs. Methods: ELVs-H1 were characterized using transmission electron microscopy and dynamic light scattering. The proliferation, migration, and odontoblast differentiation of DPCs after treatment with ELVs-H1, was detected by CCK8, transwell, ALP, and mineralization assays, respectively. Real time PCR and western blotting were used to detect gene and protein expression. For in vivo studies, DPC cells were mixed with collagen gel combined with or without ELVs and transplanted into the renal capsule of rats or subcutaneously into nude mice. HE staining and immunostaining were used to verify the regeneration of dentin-pulp and expression of odontoblast differentiation markers. Results: ELVs-H1 promoted the migration and proliferation of DPCs and also induced odontogenic differentiation and activation of Wnt/β-catenin signaling. ELVs-H1 also contributed to tube formation and neural differentiation in vitro. In addition, ELVs-H1 attached to the collagen gel, and were slowly released and endocytosed by DPCs, enhancing cell survival. ELVs-H1 together with DPCs triggered regeneration of dental pulp-dentin like tissue comprised of hard (reparative dentin-like tissue) and soft (blood vessels and neurons) tissue, in an in vivo tooth root slice model. Conclusion: Our data highlighted the potential of ELVs-H1 as biomimetic tools in providing a microenvironment for specific differentiation of dental mesenchymal stem cells. From a developmental perspective, these vesicles might be considered as novel mediators facilitating the epithelial-mesenchymal crosstalk. Their instructive potency might be exploited for the regeneration of dental pulp-dentin tissues.

Keywords: Hertwig's epithelial root sheath cell; epithelial-mesenchymal interaction; exosome-like vesicle; odontogenic differentiation; pulp-dentin regeneration..

Figure 1

Exosomal vesicles mediate interaction of HERS and DPCs cells (A) Schematic diagram showing transwell coculture of HERS-H1 and DPCs cells. (B) HERS-H1 cells were labeled by DiO (B i), DPCs cells were stained with phallotoxins, and nuclei were stained with DAPI (B ii). DPCs cells cocultured with HERS-H1 cells endocytosed exosomal vesicles (white arrow) released from HERS-H1 cells (B iii), whereas DPCs cells cocultured with GW4869-pretreated HERS-H1 cells did not show the endocytosed exosomal vesicles (B iv). (C) HERS-H1 cells upregulated the expression of DSPP and DMP1 in DPCs cells, which was attenuated by pretreatment with GW4869. (D) TEM analysis of ELVs. (E) DLS showed the particle size distribution of ELVs-H1. (F) Western blot analysis of the surface markers of ELVs. (G) DPCs cells were incubated with DiO-labeled ELVs (green) for 2, 24, and 48 h, respectively. Nuclei of DPCs cells were stained with DAPI (blue). (TEM: transmission electron microscope; DLS: dynamic light scattering). Scale bars are shown. *p < 0.05 vs. Con; #p < 0.05 vs HERS-H1.

Figure 2

HERS-H1 cells-derived ELVs enhanced proliferation and migration of DPCs cells. (A) The proliferation of DPCs measured by the CCK8 assay. (B) Migrated cells per field of view from 4 different experiments. (C) Representative images of the capacity for cell migration shown by the transwell test. Scale bars represent 100 μm. *p < 0.05 vs. Con.

Figure 3

HERS-H1 cells-derived ELVs enhanced odontogenic differentiation of DPC cells in vitro and in vivo. (A) Western blot analysis showing the significantly upregulated expression of odontoblastic markers (DSPP, DMP1, ALP, and RUNX2) in DPC cells after treatment with various concentrations of ELVs for 3 d. (B) ALP activity assay showing that treatment with ELVs increased the alkaline phosphatase activity of DPC cells with or without OM induction. (C) Representative images of Alizarin Red S staining showing that treatment with ELVs increased the odontogenic differentiation of DPC cells. (D) H&E staining showing that mineralized tissue was generated in the DPCs+ELVs group (black arrow). Immunohistochemical staining showing the upregulated expression of odontoblastic markers (OCN, DSPP, and DMP1) in the DPCs+ELVs group (white arrow). (OM: osteogenic medium; DSPP: dentin sialophosphoprotein; DMP1: dentin matrix protein 1; ALP: alkaline phosphatase; RUNX2: runt related transcription factor 2). Scale bars represent 50 μm. *p < 0.05 vs. Con.

Figure 4

HERS-H1 cells-derived ELVs enhanced tube formation and neurogenic differentiation in vitro. (A and B) In vitro tube formation of HUVECs and total number of nodes, meshes, and junctions of all tubing upregulated after treatment with ELVs (80 μg/mL). (C) Representative immunofluorescence images showing the increased expression of neurogenic differentiation markers (nestin and NF200) after treatment with ELVs (80 μg/mL) for 3 d. (NM: neurogenic medium). Scale bars are shown. *p < 0.05 vs. Con.

Figure 5

HERS-H1 cells-derived ELVs activated Wnt/β-Catenin signaling. (A) Immunoblots of exosomal (Hsp70, CD63) and cytoplasmatic cell (actin) markers; Wnt3a proteins in ELVs are presented in the panel. (B) Real time RT-PCR showing the upregulated expression of AXIN2 and TCF7 in DPC cells after treatment with ELVs (80 μg/mL). (C) Western blotting revealing the upregulated expression of β-catenin in DPC cells after treatment with ELVs (80 μg/mL), whereas the same marker was significantly downregulated in the ELVs+DKK1 and DKK1 groups. (D) Immunofluorescence staining of β-catenin in DPC cells after treatment with ELVs (80 μg/mL) alone or combined with DKK1. In the control, β-catenin mostly existed in the cytosol of DPC cells, even after addition of DKK1. ELVs induced the transference of β-catenin from the cytosol into the nucleus (white arrow). Accordingly, addition of DKK1 into ELVs could inhibit the transference of β-catenin from the cytosol into the nucleus. (E) Treatment with ELVs (80 μg/mL) upregulated the expression of DMP1 and DSPP, which was attenuated by treatment with DKK1. (DSPP: dentin sialophosphoprotein; DMP1: dentin matrix protein 1). Scale bars are shown. *p < 0.05 vs. Con; #p < 0.05 vs ELVs.

Figure 6

HERS-H1 cells-derived ELVs incorporated collagen gel enhanced cell survival. (A) ELVs within collagen gel were processed for SEM and confocal microscopy (white arrow). (B) The release efficiency of ELVs within the collagen gel was analyzed using the BCA method. (C) DPC cells endocytosed ELVs released from the collagen gel (white arrow). DPC cells were incubated onto collagen gel filled with DiO-labeled ELVs (green) for 2, 24, and 48 h, respectively. Cells were stained with phallotoxins (red) and nuclei were stained with DAPI (blue). (D) Live/dead staining of cultured DPC cells within the ELVs-containing collagen gel. Live cells are labeled with green, whereas red staining indicates dead cells. (E) Death rates of DPC cells within the collagen gel are shown. Scale bars are shown. *p < 0.05 vs. Con.

Figure 7

HERS-H1 cells-derived ELVs increased in vivo formation of pulp-dentin complex. (A) The implanted tooth root slice, with an internal diameter of 2 mm and a height of 3 mm. (B) Schematic of the preparation of in vivo transplants. DPC cells were resuspended with collagen gel mixed with ELVs, and then injected into TDM tubes. (C) HE staining showing odontoblast-like cells and regenerated dentin-like tissue (red arrows) at the interface between the dentin and pulp-like tissue. Immunofluorescence analysis showing the upregulated expression of odontogenic differentiation markers (DSPP and DMP1) in the Gel-ELVs+DPCs group, with positive staining (white arrows) represented by green stains. (DSPP: dentin sialophosphoprotein; DMP1: dentin matrix protein 1; TDM: treated dentin matrix; Rd: regenerated dentin-like tissue; Od: odontoblast-like cell; DP: dental pulp-like tissue). Scale bars are shown.

Figure 8

HERS-H1 cells-derived ELVs increased angiopoiesis in vivo. HE staining showing the newly-formed blood vessels (white arrows). Immunofluorescence showing the increased expression of angiogenic markers (CD31 and VEGF) and neurogenesis markers (MBP101 and NF200) in the Gel-ELVs+DPCs group (white arrows). (TDM: treated dentin matrix; BV: blood vessels; DP: dental pulp-like tissue). Scale bars are shown.