Blood vessels expressing CD31 and endomucin contribute to the formation of new hard tissue via osterix-expressing cells during the healing process of pulp revascularization in mice

Abstract

Background/purpose

Niche for osteogenesis forms near specialized blood vessels that highly express both CD31 and endomucin (Emcn). However, localization of the vessels remains unknown during wound healing process after pulp revascularization. We investigated the localization of both CD31 and Emcn-expressing vessels and osterix (Osx)-expressing cells after pulp revascularization.

Materials and methods

Five-week-old male C57BL/6J mice underwent pulp revascularization in the mesial root canal of the right first maxillary molar. The mice were sacrificed at 1 hour, 7 days, 14 days, and 21 days post-surgery, and immunofluorescence for CD31, Emcn, and Osx was performed. Additionally, calcein labeling was used to visualize newly formed mineralized tissue.

Results

At 1 hour post-surgery, blood clots were present, but no blood vessels or pulp tissues were observed. By day 7, the blood clots had disappeared, and CD31(+)/Emcn(+) blood vessels originating from the periapical tissue were found beneath the apical half of the root canal walls, alongside newly formed hard tissue and Osx(+) cells. By day 14, these blood vessels extended from the apex to the root canal orifice, and Osx(+) cells were observed nearby. Both CD31(+)/Emcn(+) vessels and Osx(+) cells, as well as mineralized tissue, significantly increased by day 14 (P<0.05), resulting in narrowing of the root canal.

Conclusion

The findings suggest that CD31(+)/Emcn(+) vessels play a key role in pulp revascularization, providing a supportive micro-environment for Osx(+) cells, which contribute to mineralized tissue formation and thickening of the root canal walls.

Introduction

For immature permanent teeth diagnosed with pulp necrosis or apical periodontitis due to caries or trauma, apexification has been performed traditionally as a treatment method for immature teeth, aiming for closure of the apical region with newly formed mineralized tissue.1 Nevertheless, due to the thinning of root canal walls and the large crown-to-root ratio caused by the cessation of root development, there is a risk of root fractures.2 To overcome these disadvantages, Iwaya et al. reported a novel therapy called pulp revascularization to treat necrotic immature teeth.3 Several years later, Banchs and Trope developed a protocol that forms a blood clot in the root canal after sterilization and over-instrumentation of the apex, which acts as a scaffold to attract stem/progenitor cells into the root canal, enabling tissue repair.4

Histological observations of pulp revascularization in human immature permanent teeth have reported the presence of fibrous connective tissue, including bone-like or cementum-like tissue, in the root canal.5–8 However, histological changes remain unclear. To investigate the detailed healing process involved in pulp revascularization, we established a mouse experimental model.9 The study demonstrated that capillaries and fibroblasts infiltrated into the root canal from the apical region, resulting in the formation of cementum-like hard tissue. Capillaries are known to play a significant role during wound healing, not only as a component of granulation tissue but also in the clearance of foreign bodies and the supply of oxygen and nutrients to tissues.10 Recently, attention has been drawn to the interactions between capillaries and progenitor cells, highlighting their role as niches for bone-forming cells. However, the localization and blood vessels proliferating in the root canal, and the existence of progenitor cells contributing to hard tissue formation remains unknown.

CD31 is a surface marker of vascular endothelial cells that is highly expressed at junctions between adjacent endothelial cells.11 CD31 functions as an adhesive stress-response protein that contributes to the maintenance of endothelial cell junctions, angiogenesis and the early recovery of vascular permeability barriers after inflammation. 12 Endomucin (Emcn), a mucin-like glycoprotein discovered in vascular endothelial cells, is known to be expressed in endothelial cells of capillaries and veins but not in arteries.13,14 Several morphologically and functionally distinct capillary subtypes have been reported. 15,16 During bone modeling in the growth plates of developing mouse long bones, Type H capillaries characterized by high expression levels of CD31 and Emcn, are abundant beneath the hypertrophic cartilage of the growth plate. Osterix (Osx)-expressing osteoprogenitor cells are observed near these capillaries, and the interaction between them promotes angiogenic-osteogenic coupling.15,17 In the dental pulp, it has been reported that capillaries expressing both CD31 and Emcn contribute to angiogenic-odontogenic coupling with odontoblasts during primary dentin formation.18 We have also demonstrated that CD31-expressing (CD31 (+)) and Emcn-expressing (Emcn (+)) capillaries exist in mouse dental pulp and contribute to dentin formation.19

Based on these findings, we hypothesized that CD31(+)/Emcn(+) capillaries invade the root canal from the apex during the healing process of pulp revascularization, acting as niches for mesenchymal stem/progenitor cells and contributing to the formation of newly developed mineralized tissue. Therefore, we investigated the localization of CD31(+)/Emcn(+) vessels and of Osx(+) cells after a pulp revascularization procedure to understand the relationship between the healing process histologically and their contribution using a mouse experimental model.

Materials and methods

Animals

All animal experiments were performed according to the Guidelines for the Treatment of Animals at the Tokyo Dental College (Approval No. 242301). C57BL/6 mice (5 weeks old) were purchased from Sankyo Labo Service Corporation (Tokyo, Japan).

Experimental procedure of regenerative endodontics

All procedures are shown schematically in Fig. 1A. The procedures were performed according to the method described by Komada et al.9 A total of forty-eight male mice were used in this study. Mice were anesthetized intraperitoneally using a mixed anesthetic of medetomidine hydrochloride (0.3 mg/kg, Nippon Zenyaku Kogyo, Koriyama, Japan), midazolam (4 mg/kg, Astellas Pharma, Tokyo, Japan) and butorphanol tartrate (5 mg/kg, Meiji Seika Pharma, Tokyo, Japan). After right first maxillary molar (M1) was isolated using a polytetrafluoroethylene (PTFE) membrane (Trusco Nakayama Corp., Tokyo, Japan), the mesial occlusal enamel of M1 was removed using a round-type diamond point (0.5 mm in diameter, Hinatawada Seimitsu MFG. Co., Ltd., Tokyo, Japan) with a portable micro-motor system (Viva Mate G5, Nakanishi Inc., Kanuma, Japan). An endodontic access opening into the pulp chamber was then made using a round-type tungsten carbide bur (0.4 mm in diameter, Komet, Lemgo, Germany) and the coronal pulp was removed. The dental pulp of the mesial root canal was mechanically removed with a #8 Kfile (MANI, Utsunomiya, Japan) and was chemically cleaned with 10% sodium hypochlorite solution (Neo Cleaner; Neo Dental Chemical Products Co., Tokyo, Japan) and 3% ethylenediaminetetraacetic acid solution (Smear Clean; Nishika, Shimonoseki, Japan). After removing the pulp, a #8 Kfile was inserted via the mesial root canal and bleeding from the outside of the apical foramen was initiated. Hemostasis was accomplished using a sterile article point to form a blood clot. The upper part of the blood clot (just below the root canal orifice) was covered with mineral trioxide aggregate (MTA) cement (Bio MTA cement, Morita, Tokyo, Japan). The primer was applied by active rubbing (Clearfill® Universal Bond Quick 2, Kuraray Noritake Dental Inc, Tokyo, Japan) and was air dried until the liquid surface stopped moving in the cavity. After light curing, the cavity was sealed with resin composite (Clearfill® Majesty® ES flow, Kuraray Noritake Dental Inc).

Figure 1.

Schematic diagram of the experimental protocol (A) Schematic diagram depicting the series of experimental procedures for pulp revascularization (B) Experimental design to analyze histological and immunofluorescent histological changes during the healing process (C) Region of interest (ROI).

Tissue preparation

Mice were sacrificed at 1 h and at days 7, 14 and 21 after the surgery (Fig. 1B). After deep anesthesia, perfusion fixation with 4% (w/v) paraformaldehyde solution (pH 7.4) (FUJIFILM Wako Pure Chemical Corp., Tokyo, Japan) was performed transcardially at each time point, and the dissected upper jaws were immersed in the same fixative for 24 h. After decalcification with Morse’s solution (10% sodium citrate and 22.5% formic acid; FUJIFILM Wako Pure Chemical Corp.) at 4 °C for 24 h, the samples were gradually cryoprotected in 10%, 20% and 30% (w/v) sucrose solution, embedded into a gel (SCEM, SECTION-LAB Co., Ltd., Yokohama, Japan) and quickly frozen in cold isopentane (FUJIFILM Wako Pure Chemical Corp.). Sagittal cryosections (12-μm-thick) were cut in accordance with Kawamoto’s film method using an adhesive film (Cryofilm Type IIIC (16UF), SECTION-LAB Co., Ltd.) and a tungsten carbide microtome (Leica, Wetzlar, Germany) at −20 °C for immunofluorescence.20

Calcein labeling was performed to visualize newly formed mineralized tissue. Mice were intraperitoneally administered calcein green (20 mg/kg body weight; FUJIFILM Wako Pure Chemical Corp.) one day before sacrifice. Their maxillae were removed and embedded in a gel (SCEM, SECTION-LAB Co., Ltd.) and then frozen in cold isopentane (FUJIFILM Wako Pure Chemical Corp.) without decalcification. Frozen undecalcified sections (12-μm-thick) were prepared with an adhesive film (Cryofilm Type IIIC (16UF), SECTION-LAB Co., Ltd.) according to a previously described method.20

Immunofluorescence

Cryosections were washed in TNB blocking buffer (0.1 M Tris–HCl, 0.15 M NaCl; FUJIFILM Wako Pure Chemical Corp.) and 0.5% blocking reagent (Thermo Fisher Scientific, FP1020, Waltham, MA, USA) and pretreated with 0.25% Triton X-100 (Thermo Fisher Scientific) for 10 min at room temperature. Sections of the decalcified samples were incubated with an Alexa Fluor 488-conjugated anti-CD31 goat polyclonal antibody (dilution 1:100, R&D Systems, Minneapolis, MN, USA), a rat monoclonal antibody against mouse Emcn (dilution 1:200, Santa Cruz Biotechnology Inc., Dallas, TX, USA), a rabbit polyclonal antibody against mouse Osx (dilution 1:800, Abcam, Cambridge, England) and an Armenian hamster monoclonal antibody against mouse CD31 (dilution 1:1000, Sigma–Aldrich Co., St. Louis, MO, USA) at 20 ± 5 °C for 8 h in the dark. After washing, the sections were incubated with secondary antibodies, an Alexa Fluor 555-conjugated donkey anti-rat IgG (dilution 1:1000, Thermo Fisher Scientific), an Alexa Fluor 647-conjugated goat anti-rabbit IgG (dilution 1:1000, Thermo Fisher Scientific) and an Alexa Fluor 647-conjugated goat anti-Armenian hamster IgG (dilution 1:1000, Thermo Fisher Scientific) for 2 h at 20 ± 5 °C. Nuclei were counterstained with Hoechst (dilution 1:1000, Thermo Fisher Scientific). The cryosections were also incubated with TNB blocking buffer which was not contained primary antibody as a negative control. Finally, the sections were mounted using 30% glycerol, covered with coverslips, and sealed with nail varnish. Fluorescent images were taken using a confocal laser-scanning microscope (LSM880 Airy NLO, Zeiss, Oberkochen, Germany).

Histological quantitative analysis

The region of interest (ROI) was defined as the area within the root canal bounded by a perpendicular line (red line) to the root axis (blue line) derived from the cement enamel junction (CEJ) and the root apex which was equal to the top of cementum (Fig. 1C). Quantitative analysis of newly formed mineralized tissue was performed using the area of calcification in ROI/area in ROI. In the density analysis of CD31 (+)/Emcn (+) vessels, we measured the CD31(+) and/or Emcn(+) areas in the entire dental pulp of the first molars using ImageJ (National Institutes of Health, Bethesda, MD, USA) according to the method described by Kambe et al.19 In brief, the merged images were split into green and red channels, and an optimal threshold (CD31: 0–115, Emcn: 0–160) that distinguished between a positive reaction and the background was set. Areas of green-colored (A) and red-colored (B) vessels were measured separately. Yellow-colored areas indicating merged colored (C) vessels were displayed after using an image calculator and were measured. The ratio of CD31 and Emcn double-expressing areas (C) was calculated as follows; Ratio of CD31(+)/Emcn(+) vessel area = C/(A + B − C) × 100.

Quantitative analysis of Osx(+) cells and Hoechst(+) cells was performed using Image J.21

Statistical analysis

All values are expressed as means ± standard deviation (SD). Analysis software (IBM SPSS 27.0J for Windows; SPSS Japan, Tokyo, Japan) was used for generating graphs and for statistical analysis. Significance differences were assessed using one-way analysis of variance (ANOVA) followed by Tukey honestly significant difference (HSD). Statistical significance was set at 0.05. Sample sizes were selected on the basis of previous experiments.9 The number ‘n’ indicates the number of individuals. Individual samples were sliced into 4–5 specimens.

Results

Localization of CD31(+)/Emcn(+) vessels, Osx(+) cells and new hard tissue

At 1 h post-surgery, no CD31(+) or Emcn(+) blood vessels were observed in the root canal (Fig. 2A and B). CD31(+)/Emcn(+) blood vessels were seen in the bone marrow and in the periodontal ligament close to the alveolar bone (Fig. 2A and C), and calcein labeled the bone and cementum (Fig. 2D). Osx(+) cells were not observed within the root canal, while they were detected in cementoblasts of the apical region, in the periodontal ligament, and in osteoblasts on the alveolar bone surface (Fig. 2A, B and C). Osx(+) cells were localized near the CD31(+)/Emcn(+) vessels (Fig. 2A and C), and calcein labeled the bone and cementum (Fig. 2D).

Figure 2.

Confocal images showing the localization of CD31(+)/Emcn(+) blood vessels and Osx(+) cells at 1 h post-surgery (A) No CD31(+) (green) or Emcn(+) (red) blood vessels were observed within the root canal. CD31(+)/Emcn(+) blood vessels (yellow) were observed in the bone marrow and were localized closer to the alveolar bone in the periodontal ligament. Intracanal Osx(+) (magenta) cells were not observed (B) Higher magnification of the upper white boxed area in (A) (C) Higher magnification of the lower white boxed area in (A) (D) Confocal images illustrating calcein (green) and CD31(+) (cyan) and Emcn(+) (red) blood vessels (white). The calcein labeling beneath the dentin wall represents newly formed dentin generated by the day before sacrifice. Scale bars = 200 μm in A, D; scale bars = 20 μm in B, C. Abbreviation: AB, alveolar bone; DE, dentin; PDL, periodontal ligament; MTA, mineral trioxide aggregate; Emcn, endomucin; Osx, osterix.

By day 7, continuous CD31(+)/Emcn(+) blood vessels extending from the apex to the middle portion of the root canal were identified that were predominantly localized just beneath the root canal walls (Fig. 3A). In the apical half of the root canal, newly formed hard tissue was observed directly beneath the canal walls (Fig. 3B). At the orifice, CD31(+)/Emcn(+) vessels and Osx(+) cells were detected just beneath the MTA cement (Fig. 3C). However, the blood vessels were shorter, and their luminal structures were indistinct compared to those in the apical region (Fig. 3B). Osx(+) cells continuous with the apical cellular cementum were observed just beneath the root canal walls but were not localized near the central portion of the root canal (Fig. 3A). Osx(+) cells were observed in the vicinity of the CD31(+)/Emcn(+) vessels at the lower part of the root canal (Fig. 3B). At higher magnification, some Osx(+) cells were also identified beneath the MTA cement, with CD31(+)/Emcn(+) vessels present nearby (Fig. 3C). Regarding with newly formed hard tissue, calcein-labeling was observed directly beneath the canal walls in the apical half of the root canal (Fig. 3D and E). No calcein-labeled newly formed hard tissue was observed in the upper area (Fig. 3F). A small number of CD31(+) but Emcn negative (Emcn(−)) blood vessels were also present that were located closer to the center of the root canal rather than immediately beneath the canal walls (Fig. 3B and E). Emcn was uniformly expressed in the luminal structures, while CD31 had a reticular pattern across the lumens (Fig. 3B, C, E and F). Additionally, pseudopod-like structures (filopodia) extending from Emcn(+) cells were observed that were oriented toward the calcein-labeled newly formed hard tissue (Fig. 3E).

Figure 3.

Confocal images showing the localization of CD31(+)/Emcn(+) blood vessels and Osx(+) cells at 7 days post-surgery (A) Continuous CD31(+) (green) and Emcn(+) (red) blood vessels (yellow) extending from the apex to the middle portion of the root canal were identified that were predominantly localized just beneath the root canal walls (B) Higher magnification of the upper white boxed area in (A). CD31(+)/Emcn(+) blood vessels were detected just beneath the MTA cement (white arrowhead) (C) Higher magnification of the lower white boxed area in (A). Osx(+) cells were observed in the vicinity of the CD31(+)/Emcn(+) blood vessels (white arrowhead) (D) In the apical half of the root canal, newly formed mineralized tissue labeled with calcein was observed directly beneath the canal walls (E) Higher magnification of the upper white boxed area in (D). No calcein-labeled newly formed mineralized tissue was observed at the orifice (F) Higher magnification of the lower white boxed area in (D). Newly formed hard tissue labeled with calcein was observed directly beneath the canal walls. Scale bars = 200 μm in A, D; scale bars = 20 μm in B, C, E, F. Abbreviation: AB, alveolar bone; DE, dentin; PDL, periodontal ligament; MTA, mineral trioxide aggregate; Emcn, endomucin; Osx, osterix.

By day 14, CD31(+)/Emcn(+) vessels were observed just beneath the root canal walls that extended from the apex to the orifice and exhibited multiple branching formations (Fig. 4A). Osx(+) cells were found along the root canal walls from the apex to the orifice, surrounding the entire root canal wall (Fig. 4A). Some Osx(+) cells were enclosed within the newly formed mineralized tissue (Fig. 4B and C). CD31(+)/Emcn(+) vessels were also observed near the Osx(+) cells (Fig. 4A, B and C). Calcein-labeled newly formed mineralized tissue was observed along the canal walls from the apical region to the orifice (Fig. 4D). Emcn(+) filopodia were detected near the calcein-labeled newly formed hard tissue (Fig. 4E and F). Blood vessels surrounded by newly formed mineralized tissue were also identified (Fig. 4E).

Figure 4.

Confocal images showing the localization of CD31(+)/Emcn(+) blood vessels and Osx(+) cells at 14 days post-surgery (A) CD31(+)/Emcn(+) blood vessels and Osx(+) cells were observed just beneath the root canal walls, extending from the apex to the orifice (B) Higher magnification of the upper white boxed area in (A). CD31(+)/Emcn(+) blood vessels surrounded by newly formed mineralized tissue were also identified (white arrowhead) (C) Higher magnification of the lower white boxed area in (A). Some Osx(+) cells were enclosed within the newly formed mineralized tissue (white arrowheads) (D) Calcein-labeled newly formed mineralized tissue was observed along the canal walls from the apical region to the orifice (E) Higher magnification of the upper white boxed area in (D). Emcn(+) filopodia were detected near the calcein-labeled newly formed hard tissue (F) Higher magnification of the lower white boxed area in (D). Scale bars = 200 μm in A, D; scale bars = 20 μm in B, C, E, F. Abbreviation: AB, alveolar bone; DE, dentin; PDL, periodontal ligament; MTA, mineral trioxide aggregate; Emcn, endomucin; Osx, osterix; NH, newly formed hard tissue.

By day 21, as seen on days 7 and 14, CD31(+)/Emcn(+) vessels were localized around the root canal walls (Fig. 5A). A small number of CD31(+) but Emcn(−) blood vessels was observed that were located closer to the center of the root canal rather than directly beneath the canal walls (Fig. 5A). Osx(+) cells were localized just beneath the root canal walls, surrounding the entire root canal wall (Fig. 5B and C). Narrowing of the root canal due to the calcein-labeled newly formed hard tissue was noted, with portions of the newly formed mineralized tissue appearing continuous between the inner and outer curvatures of the root canal (Fig. 5D). Emcn(+) filopodia were observed in the vicinity of the calcein-labeled newly formed hard tissue (Fig. 5E and F).

Figure 5.

Confocal images showing the localization of CD31(+)/Emcn(+) blood vessels and Osx(+) cells at 21 days post-surgery (A) CD31(+)/Emcn(+) blood vessels were localized around the root canal wall. Osx(+) cells were localized just beneath the root canal walls, surrounding the entire root canal wall (B) Higher magnification of the upper white boxed area in (A) (C) Higher magnification of the lower white boxed area in (A) (D) Narrowing of the root canal due to the calcein-labeled newly formed hard tissue was noted (E) Higher magnification of the upper white boxed area in (D). Emcn(+) filopodia were observed in the vicinity of the calcein-labeled newly hard tissue (F) Higher magnification of the lower white boxed area in (D). Scale bars = 200 μm in A, D; scale bars = 20 μm in B, C, E, F. AB, Abbreviation: alveolar bone; DE, dentin; PDL, periodontal ligament; MTA, mineral trioxide aggregate; Emcn, endomucin; Osx, osterix; NH, newly formed hard tissue.

The proportion of CD31(+)/Emcn(+) area per total vessel area was significantly increased over time from 1.5% to 11.0% from 1 h to day 21. No statistically significant difference was observed between day 14 and day 21 (Fig. 6A). The proportion of Osx(+) cells among Hoechst(+) cells within the root canal significantly increased over time from 0% to 69.1% from 1 h to day 21 and statistically significant differences were observed in all groups (Fig. 6B).

Figure 6.

Comparison of specimens at 1 h, and at 7, 14 and 21 days post-surgery (A) Quantitative comparison of CD31(+)/Emcn(+) area/total vessel area. The data at 14 days and 21 days were significantly higher than at 1 h and at 7 days. However, there was no statistically significant difference between the two groups at 14 and 21 days (n = 5 per group) *P< 0.05. CD31(+)/Emcn(+) area was not none due to the influence of some background signals (B) Quantitative comparison of Osx(+) cells/Hoechst(+) cells. The proportion of Osx(+) cells to Hoechst(+) cells gradually increased from 0% to 69.1% (n = 8 per group) *P < 0.05. All data are expressed as means ± SD. Abbreviation: Emcn, endomucin; Osx, osterix.

Discussion

Previous studies on angiogenesis during the healing process of pulp revascularization have been limited. Our findings revealed that CD31(+)/Emcn(+) blood vessels originating from the apical region infiltrated and proliferated within the root canal following pulp revascularization. The formation of blood clots within the root canal plays a critical role, serving as a scaffold and containing various important growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), which signal endothelial progenitor cells and perivascular progenitor cells to promote angiogenesis.22 VEGF produced by perivascular cells is essential for angiogenesis.23 Sprouting angiogenesis originates from pre-existing capillary beds, with the filopodia of tip cells extending along VEGF concentration gradients. Tip cells express high levels of VEGF receptor 2 (VEGFR2).10,23 Emcn(+) filopodia form from capillaries or veins.24 Within the root canal, Emcn(+) filopodia are oriented toward newly formed mineralized tissue just beneath the root canal wall. This suggests that the extension of filopodia toward the root canal wall localizes CD31(+)/Emcn(+) capillaries just beneath the wall. During the primary dentinogenesis stage in murine pulp, CD31(+), Emcn(+), VEGFR1^high and VEGFR2^high capillaries produce transforming growth factor-β (TGFβ), which promotes odontoblast differentiation.18 Our findings indicate that there is a significant increase in Osx(+) cells along with the proliferation of CD31(+)/Emcn(+) vessels. This suggests that CD31(+)/Emcn(+) vessels in the root canal may produce growth factors that affect Osx(+) cells beneath the root canal wall.

Type H vessels (CD31^high, Emcn^high) are known to mediate angiogenic–osteogenic coupling with Osx(+) progenitor cells.15 The expression intensities of CD31 and Emcn in vessels in the root canal and apical tissue are similar. Unlike bone vasculature, there was no clear difference in immunofluorescence intensity between Type H and Type L vessels. CD31 within the root canal exhibited a reticular expression pattern along the vascular lumen, likely due to its high expression level at endothelial cell junctions.11 Morphologically, Type H vessels display columnar shapes without filopodia formation,25 whereas CD31(+)/Emcn(+) vessels in the dental pulp are characterized by extensive branching and filopodia formation, which is consistent with sprouting angiogenesis.18 Additionally, some CD31(+)/Emcn(+) vessels within the root canal may include lymphatic vessels.26 This suggests the presence of a distinct subtype or lymphatic vessels among the CD31(+)/Emcn(+) vasculature in the root canal, which differs from Type H vessels. CD31(+)/Emcn(−) vessels were observed throughout the healing process, predominantly near the root canal center rather than beneath the canal walls and they lacked filopodia. As Emcn is selectively expressed in capillaries and veins,13,14 CD31(+)/Emcn(−) vessels are likely arterioles. However, no transitional structures linking CD31(+)/Emcn(−) vessels to CD31(+)/Emcn(+) capillaries were identified, and the formation process of CD31(+)/Emcn(−) vessels remains unclear.

Following pulp revascularization, Osx(+) cells invaded and proliferated along the root canal walls. Some Osx(+) cells were embedded within the newly formed hard tissue, which suggests their involvement in mineralized tissue formation. During the process of root formation, Osx(+) cells differentiate into various types of cells that contribute to the root and periodontal tissues, including odontoblasts, cementoblasts, alveolar bone osteoblasts, and periodontal ligament cells. Even after the completion of root formation, those cells remain in the pulp and periodontal ligament as progenitor cells for odontoblasts and cementoblasts.27 Histological findings suggest that Osx(+) cells may originate from cementoblasts but they could also be supplied by the abundant stem/progenitor cells present in the apical region, and their true origin remains uncertain. During the formation of newly mineralized tissue, Osx(+) progenitor cells and odontoblasts produce Slit homolog 3 (SLIT3) and VEGF.15,17,18 Thus, these results suggest that Osx(+) cells in the root canal secrete VEGF and other growth factors that subsequently induce CD31(+)/Emcn(+) capillaries beneath the root canal walls. These interactions between CD31(+)/Emcn(+) vessels and Osx(+) cells likely contribute to the formation of new hard tissue during the healing process of pulp revascularization.

Newly formed hard tissue within the root canal exhibited a structure continuous with cellular cementum. The gradual thickening of the newly formed hard tissue beneath the root canal walls led to canal constriction. However, dentin-specific structures such as dentinal tubules were not observed, and there was no evidence of alveolar bone invasion or ectopic bone-like tissue formation (ankylosis) in the present and earlier studies.9 Our earlier study also demonstrated that immunoreactivity of nestin, odontoblast maeker, was not detected around newly formed hard tissue.28 Recent reports have suggested that the type of newly formed hard tissue after regenerative endodontic procedures depends on the residual pulp, and when the pulp is completely removed down to the root pulp, cementum-like hard tissue is formed.29 Our results indicate that the pulp tissue within the root canal was thoroughly removed during the procedure. Additionally, these findings suggest that the newly formed mineralized tissue is cementum-like in nature. The calcein labeling beneath the dentin wall at 1 h post-surgery represents newly formed dentin generated prior to the day of sacrifice. This suggests that the mechanical enlargement of the root canal was minimized. Moreover, mice are significantly smaller in size compared to other experimental animals, making it technically challenging to perform rubber dam isolation. Therefore, PTFE was used as a substitute for isolation. PTFE isolation effectively prevented saliva contamination in the operative field and the leakage of solutions outside the operative area, enabling a reliable adhesion protocol. In this mouse model, pulp revascularization was performed following the extirpation of healthy pulp tissue, without inflammation or infection in the pulp or apical tissue. Therefore, the healing outcome may vary depending on the extent of pulp vitality, inflammation and/or infection. Histological analyses using alternative models considering these conditions are necessary.

Efforts to regenerate pulp using stem cells from deciduous teeth for non-vital immature permanent teeth have been reported.30 However, ethical concerns surround the extirpation of healthy pulp from deciduous teeth. Additionally, the high-level processing required for cell culture presents challenges for application in dental clinics. Thus, there is a need to establish a simpler system for regenerative therapy. Our research has focused on tissue stem cells and growth factors to minimize invasiveness and simplify treatment. The apical region is rich in mesenchymal stem cells, which is likely to contribute significantly to pulp revascularization. To develop safer and more straightforward pulp regeneration therapy based on pulp revascularization, it is essential to elucidate the differentiation mechanisms of apically derived stem cells into pulp cells. Future research will employ lineage tracing and single-cell transcriptomic analyses to identify and study the differentiation pathways of apically derived stem cells that are involved in pulp revascularization.

In conclusion, our findings reveal that CD31(+)/Emcn(+) vessels in the apical area infiltrate and proliferate into the root canal along the dentin wall using blood clots as scaffolds during the healing process of pulp revascularization. This process occurs in coordination with Osx(+) cells and contributes to the formation of new hard tissue and the thickening of the root canal wall (Fig. 7). Accordingly, these results serve as a basis for understanding the healing process after pulp revascularization and might enable the therapeutic improvement of dental pulp regeneration in the future.

Figure 7.

Schematic diagram displaying the localization of Osx(+) cells and blood vessels within the root canal during the healing process of pulp revascularization. CD31(+)/Emcn(+) blood vessels invade and proliferate from outside the apical foramen into the root canal along the dentin wall. On the other hand, CD31(+)/Emcn(−) blood vessels are localized in the central part of the root canal. Osx(+) cells continuously invade and increase along the root canal wall in conjunction with cementoblasts at the apical region. Abbreviation: Emcn, endomucin; Osx, osterix.

Funding Statement

This work was supported by JPSP Grants-in-Aid for Scientific Research JP20K18544 (KM) and JP22K09971 (TM).