Odontogenic exosomes simulating the developmental microenvironment promote complete regeneration of pulp-dentin complex in vivo

Abstract

Introduction: Establishing an optimized regenerative microenvironment for pulp-dentin complex engineering has become increasingly critical. Recently, exosomes have emerged as favorable biomimetic nanotherapeutic tools to simulate the developmental microenvironment and facilitate tissue regeneration.

Objectives: This study aimed to elucidate the multifaceted roles of exosomes from human dental pulp stem cells (DPSCs) that initiated odontogenic differentiation while sustaining mesenchymal stem cell (MSC) characteristics in odontogenesis, angiogenesis, and neurogenesis during pulp-dentin complex regeneration.

Methods: Differential centrifugation was performed to isolate exosomes from normal DPSCs (DPSC-Exos) and DPSCs that initially triggered odontogenic differentiation (DPSC-Od-Exos). The impact of these exosomes on the biological behavior of DPSCs and human umbilical vein endothelial cells (HUVECs) was examined in vitro through CCK-8 assay and Transwell migration assay, as well as assays dedicated to assessing odontogenic, angiogenic, and neurogenic capabilities. In vivo, Matrigel plugs and human tooth root fragments incorporating either DPSC-Exos or DPSC-Od-Exos were subcutaneously transplanted into mouse models. Subsequent histological, immunohistochemical, and immunofluorescent analyses were conducted to determine the regenerative outcomes.

Results: DPSC-Exos and DPSC-Od-Exos revealed no remarkable difference in their characteristics. In vitro analyses indicated that DPSC-Od-Exos significantly facilitated the proliferation, migration, and multilineage differentiation of DPSCs compared with DPSC-Exos. Furthermore, DPSC-Od-Exos elicited a more pronounced effect on the tubular structure formation of HUVECs. Consistently, Matrigel plug assays confirmed that DPSC-Od-Exos exhibited superior performance in promoting endothelial differentiation of DPSCs and stimulating angiogenesis in HUVECs. Notably, DPSC-Od-Exos contributed to complete pulp-dentin complex regeneration in human tooth root fragments, characterized by enriched neurovascular structures and a continuous layer of odontoblast-like cells, which extended cytoplasmic projections into the newly formed dentinal tubules.

Conclusion: By simulating the developmental microenvironment, multifunctional DPSC-Od-Exos demonstrated promising potential for reconstructing dentin-like tissue, vascular networks, and neural architectures, thereby enhancing our understanding of the therapeutic implications of DPSC-Od-Exos in regenerative endodontic treatment.

Keywords: Angiogenesis; Dental pulp stem cells; Exosomes; Neurogenesis; Odontogenesis.

Graphical abstract

Fig. 1

Human DPSCs exhibited MSC attributes and secreted typical exosomes. (A, B) DPSCs were characterized as typical MSCs. (A) P4 DPSCs adherent to the plastic surface displayed a spindle-like shape. In addition, DPSCs promoted the accumulation of orange-red calcified nodules, the formation of bead-like lipid droplets, and the production of light-blue extracellular matrix under proper induction. Scale bars: 200 μm and 20 μm (high magnification). (B) Flow cytometry signified that as opposed to the negatively expressed CD34, CD73, CD90, and CD105 were highly expressed in DPSCs. (C, D) When subjected to OIM, DPSCs initiated odonto/osteogenic differentiation in 3 days, although they still maintained MSC stemness. (C) Compared with SOX2 gene expression without a marked difference at 3 days, the expression of ALP was substantially augmented. (D) Coincidently, ALP staining and activity evaluation confirmed that DPSCs initially triggered odonto/osteogenic differentiation in 3 days. (E-G) Regular DPSC-Exos and DPSC-Od-Exos obtained from DPSCs that underwent initial odontogenic differentiation for 3 days met the requirements of exosomes. (E) As detected by TEM, DPSC-Od-Exos and DPSC-Exos had similar morphological features and exhibited saucer-like microstructures. Scale bar: 100 nm. (F) It was denoted by NTA that DPSC-Od-Exos and DPSC-Exos, ranging from 107.2 nm to 151.1 nm, had an average diameter of around 130 nm. (G) The WB assessment showed that DPSC-Od-Exos and DPSC-Exos were highly enriched in transmembrane protein CD63 and cytoplasmic protein TSG101, and no difference was notably distinguished, conforming the characteristics of exosomes. Noted: Different letters above the bars indicated statistically significant difference at p < 0.05.

Fig. 2

DPSC-Od-Exos were dramatically beneficial for the odontogenic differentiation of DPSCs in vitro. (A) As confirmed by the uptake assay, DPSC-Od-Exos and DPSC-Exos positively stained by PKH26 could be effectively internalized by DPSCs, thus leading to extensive distribution in the cytoplasm. Scale bar: 50 μm. (B) The application of DPSC-Od-Exos and DPSC-Exos had similar efficacy and triggered a significantly enhanced proliferation of DPSCs. (C) Compared with DPSC-Exos, DPSC-Od-Exos could time-dependently promote the migratory capacity of DPSCs, as suggested by the remarkably increased number of migrated cells. Scale bar: 100 μm. (D) It was elucidated by RT-qPCR that, in contrast with DPSC-Exos, DPSC-Od-Exos could elicit an outstanding enhancement in the expression of odonto/osteogenic-correlated genes, such as DSPP, DMP-1, and OCN. (E) In line with gene expression, DPSC-Od-Exos were more potent than DPSC-Exos in facilitating ALP staining and activity. Scale bar: 100 μm. (F) By performing ARS staining, we further illustrated that DPSC-Od-Exos notably fostered the deposition of orange-red mineralized nodules, therefore contributing to a higher OD value in semi-quantification analysis. Scale bar: 100 μm. Noted: Different letters above the bars indicated statistically significant difference at p < 0.05.

Fig. 3

DPSC-Od-Exos were capable of inducing endothelial and neurogenic differentiation of DPSCs in vitro. (A-C) DPSC-Od-Exos exerted a notable promoting effect on DPSC endothelial cell differentiation. (A) In contrast with DPSC-Exos, DPSC-Od-Exos enormously enhanced the expression level of angiogenesis-related genes, including ANG II, VEGF, and PDGFA. (B) Consistently, DPSC-Od-Exos were more suitable inducers in upregulating the immunostaining intensity of VEGF compared with DPSC-Exos. Scale bar: 20 μm. (C) The tube formation assay indicated that as opposed to blank control that presented disorganized networks, both DPSC-Od-Exos and DPSC-Exos were dedicated to forming regular tubular-like structures. However, in sharp contrast with DPSC-Exos, DPSC-Od-Exos contributed to producing capillary-like networks in an orderly manner, thus drastically enhancing the total length, total branching length, and the number of junctions, nodes, branches, and meshes. Scale bars: 500 μm and 200 μm (high magnification). (D, E) DPSC-Od-Exos possessed substantial benefits in stimulating the neurogenic differentiation of DPSCs. (D) As depicted by RT-qPCR, the expression of neurogenic-associated markers GDNF and Nestin was massively strengthened by DPSC-Od-Exos. (E) In accordance with this, DPSC-Od-Exos had obvious superiority over DPSC-Exos in enhancing the immunostaining of Nestin. Scale bar: 50 μm. Noted: Different letters indicated statistically significant difference (p < 0.05).

Fig. 4

DPSC-Od-Exos exhibited more robust promoting potential in the angiogenesis of HUVECs in vitro. (A) The uptake assay verified that DPSC-Od-Exos and DPSC-Exos could be extensively taken up by HUVECs, resulting in a wide distribution of red fluorescence in the HUVEC cytoplasm. Scale bar: 50 μm. (B) As demonstrated by the CCK-8 assay, DPSC-Od-Exos and DPSC-Exos at 50 μg/mL time-dependently potentiated the proliferative property of HUVECs. (C) Concentrated on the migratory capability of HUVECs, we discovered that DPSC-Od-Exos could be more effective than DPSC-Exos in elevating the number of migrated cells in a time-dependent manner. Scale bar: 100 μm. (D) According to RT-qPCR, compared with DPSC-Exos, DPSC-Od-Exos considerably promoted the expression level of angiogenic genes, including ANG II, VEGF, PDGFA, and MMP-9. (E) The immunostaining against VEGF delineated that the expression intensity was substantially facilitated after the employment of DPSC-Od-Exos. Scale bar: 20 μm. (F) It was clarified through tube formation assay that although DPSC-Od-Exos and DPSC-Exos were able to enhance the production of regularly organized tubular structures, DPSC-Od-Exos exhibited better performance than DPSC-Exos, remarkably increasing the number of junctions, nodes, branches, and meshes as well as the total length and total branching length. Scale bars: 500 μm and 200 μm (high magnification). Noted: Different letters indicated statistically significant difference between groups (p < 0.05).

Fig. 5

DPSC-Od-Exos performed better than DPSC-Exos in enhancing angiogenesis when incubated with DPSCs or HUVEVs in Matrigel plugs. (A-E) The combined application of DPSC-Od-Exos and DPSCs remarkably contributed to the formation of abundant capillary-like structures. (A) In visual observation, a more apparent reddish-brown appearance could be identified following the introduction of DPSC-Od-Exos. (B, C) As revealed by immunofluorescence staining against VEGF, DPSC-Od-Exos were more effective than DPSC-Exos in promoting the formation of positively stained tubular networks. Scale bar: 20 μm. (D, E) In accordance with the above findings, DPSC-Od-Exos elicited a better angiogenic performance in facilitating the expression of CD31, suggesting that DPSC-Od-Exos were capable of inducing DPSC endothelial cell differentiation. Scale bar: 50 μm. (F-J) Compared with DPSC-Exos, DPSC-Od-Exos considerably enhanced the angiogenic potential of HUVECs. (F) By visual inspection, DPSC-Od-Exos showed notable advantages in stimulating microvessel formation for HUVECs, thus giving rise to red coloration. (G, H) Under a confocal microscope, DPSC-Od-Exos were superior to DPSC-Exos in increasing the number of VEGF-positive vascular lumens. Scale bar: 20 μm. (I, J) It was evidenced through immunohistochemical assessment that the immunoactivity of CD31 to newly formed capillary tube-like networks was markedly promoted by DPSC-Od-Exos when compared with DPSC-Exos (I), which was also confirmed by quantification analysis (J). Scale bar: 50 μm. Noted: Different letters indicated statistically significant difference (p < 0.05).

Fig. 6

DPSC-Od-Exos promoted complete regeneration of pulp-dentin complex by enhancing DPSC odontogenesis, angiogenesis, and neurogenesis. (A) As demonstrated by HE staining, the sole DPSC transplantation led to the formation of disorganized soft tissue in the root canal. Notably, following the introduction of DPSC-Od-Exos and DPSC-Exos, pulp-like tissue with higher cell density and more regular structure could be observed. In contrast with the DPSC-Exo-treated group, DPSC-Od-Exos contributed to the formation of vascular-enriched pulp tissue and continuous tubular dentin layer with the alignment of odontoblast-like cells. Scale bar: 50 μm. The dashed line: the interface between original dentin and regenerated dentin; D: original dentin; rD: regenerated dentin; rP: regenerated pulp; black star: tubular dentin; blue arrowhead: odontoblast-like cells; yellow arrow: blood vessels. (B) According to immunofluorescence staining and quantification analysis, compared with DPSC-Exos, DPSC-Od-Exos displayed more robust potential to promote the deposition of DSPP-positive tubular dentin, the reconstitution of CD31-positive vascular networks, and the sprouting of NF200-positive sensory nerve fibers, indicating their promising versatile roles in inducing functional pulp-dentin complex regeneration. Scale bar: 20 μm. The dashed line: the interface between original dentin and regenerated dentin. Noted: Different letters above the bars indicated statistically significant difference at p < 0.05.

Fig. 7

Schematic illustration of the multifaceted roles of DPSC-Od-Exos in pulp-dentin complex regeneration. DPSC-Od-Exos were obtained from DPSCs that initially triggered odontogenic differentiation via ultracentrifugation. After encapsulation in Matrigel with DPSCs, DPSC-Od-Exos were injected into the pulp cavity of human tooth root fragments and then subcutaneously implanted into nude mice. As revealed by histological evaluation, DPSC-Od-Exos contributed to the regeneration of complete pulp-dentin complex, which encompassed enriched neurovascular structures and a continuous layer of odontoblast-like cells extending their cytoplasmic projections into the newly formed dentinal tubules.