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, 12 (3), 410-418

Regeneration of Neural Networks in Immature Teeth With Non-Vital Pulp Following a Novel Regenerative Procedure

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Regeneration of Neural Networks in Immature Teeth With Non-Vital Pulp Following a Novel Regenerative Procedure

Maha M F Mounir et al. Int J Stem Cells.

Abstract

Background and objectives: Recombinant amelogenin protein (RAP) was reported to induce soft-tissue regeneration in canine infected endodontically treated permanent teeth with open apices. To characterize identities of the cells found in the RAP regenerated tissues compared to authentic pulp by identifying: 1) stem cells by their expression of Sox2; 2) nerve fibers by distribution of the axonal marker peripherin; 3) axons by their expression of calcitonin gene-related peptide (CGRP); 4) the presence of astrocytes expressing glial fibrillary acidic proteins (GFAP).

Methods: A total of 240 open-apex root canals in dogs were used. After establishment of oral contamination to the pulp, the canals were cleaned, irrigated, and 120 canals filled with RAP, and the other 120 with calcium hydroxide.

Results: After 1, 3, and 6 months, teeth were recovered for immune-detection of protein markers associated with native pulp tissues. Regenerated pulp and apical papilla of RAP group revealed an abundance of stem cells showing intense immunoreactivity to Sox2 antibody, immunoreactivity of peripherin mainly in the A-fibers of the odontoblast layer and immunoreactivity to CGRP fibers in the central pulp region indicative of C-fibres. GFAP immunoreactivity was observed near the odontoblastic, cell-rich regions and throughout the regenerated pulp.

Conclusions: RAP induces pulp regeneration following regenerative endodontic procedures with cells identity by gene expression demonstrating a distribution pattern similar to the authentic pulp innervation. A- and C-fibers, as well as GFAP specific to astrocytic differentiation, are recognized. The origin of the regenerated neural networks may be derived from the Sox2 identified stem cells within the apical papilla.

Keywords: Amelogenin; Apical periodontitis; Calcitonin gene-related peptide; Dental pulp necrosis; Glial fibrillary acidic protein; Peripherin; Regenerative endodontics; Sox2; Stem cell homing.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Premolar teeth at selected time intervals following treatment with recombinant amelogenin and calcium hydroxide. (A) Apical and mid root area of a premolar tooth 1-month post treatment with r-amelogenin showing regenerated cellular tissue inside canal consistent with pulp tissue (P), and apical papilla (AP) and dentin (D). H&E stain. Original magnification, ×100. (B) Apical and mid root area of a premolar tooth 1-month post treatment with r-amelogenin showing cells in the regenerated pulp tissue (P), apical papilla (AP) and cellular dentin (CD) Trichrome stain. Original magnification, ×100. (C) Apical area of premolar tooth 1-month post treatment with r-amelogenin showing regenerated regenerated pulp tissue (P) in the root canal, dentin (D) and apical papilla (AP). H&E stain. Original magnification, ×100. (D) Apical and mid-root area of premolar tooth 1-month post treatment with r-amelogenin showing intense to moderate immunoreactivity to the peripherin (PER) protein. Intense immunoreactivity is observed at the odontoblast layer and odontoblastic zone in the form of parallel conical arrays. Moderate to intense immunoreactivity in the central root canal and pulp camber. Strong immunoreactivity is seen in the apical papilla (AP). Dentin (D). Peripherin antibody. Original magnification, ×100. (E) Apical area of premolar tooth 1-month post treatment with r-amelogenin showing intense to moderate immunoreactivity to the CGRP antigen in the cells of the apical papilla (AP) running axially in the central area of root canal (red lines). Dentin (D). CGRP antibody. Original magnification, ×100. (F) Apical area of premolar tooth 1-month post treatment with r-amelogenin showing intense immunoreactivity to the GFAP antibody in the odontoblastic region (arrow) and mild to moderate immunoreactivity throughout the regenerated pulp tissue. Mild immunoreactivity is seen in the cells of the apical papilla (AP). Dentin (D). GFAP antibody. Original magnification, ×40. (G) Apical area of premolar tooth 3-month post treatment with r-amelogenin showing intense immunoreactivity to peripherin antibody in the regenerated pulp tissue (P) that lies at the pulp periphery near the odontoblast area (red lines). Dentin (D), periodontal ligament PDL (black arrow). Peripherin antibody. Original magnification, ×40. (H) Apical area of premolar tooth 3-month post treatment with r-amelogenin showing moderate to intense immunoreactivity to CGRP antibody in the regenerated pulp tissue (P) that lies at root canal apex and in mid root in the central pulp region (red lines). Apical papilla (AP) shows moderate immunoreactivity. Dentin (D). CGRP antibody. Original magnification, ×100. (I) Apical area of premolar tooth 3-month post treatment with r-amelogenin showing moderate to intense immunoreactivity to GFAP antibody in the odontoblastic region (red arrow) and mild to moderate immunoreactivity throughout the regenerated pulp tissue (P). Dentin (D), periodontal ligament PDL (black arrow). GFAP antibody. Original magnification, ×100. (J) Apical area of premolar tooth 6-month post treatment with r-amelogenin showing intense immunoreactivity to peripherin antibody in the regenerated pulp tissue (P) that lies at pulp periphery near the odontoblast area (red lines). Dentin (D), PDL (black arrow). Peripherin antibody. Original magnification, ×100. (K) Apical area of premolar tooth 6-month post treatment with r-amelogenin showing intense immunoreactivity to CGRP antibody in the regenerated pulp tissue (P) that lies at root canal apex and in mid root in the central pulp region (red lines). Dentin (D), PDL (black arrow). CGRP antibody. Original magnification, ×100. (L) Apical and mid area of premolar tooth 6-month post treatment with r-amelogenin showing moderate to intense immunoreactivity to GFAP antibody in the odontoblastic and cell-rich zone regions (red arrows) and mild to moderate immunoreactivity throughout the regenerated pulp tissue (P). Dentin (D), PDL (black arrow). GFAP antibody. Original magnification, ×100. (M) Apical area of premolar tooth 1-month post treatment with calcium hydroxide showing no immunoreactivity to Sox2 antibody in the empty root canals (RC). Dentin (D). Mineralized island can be seen in the empty RC (box). Sox2 antibody. Original magnification, ×100. (N) Apical and mid root area of a premolar tooth 3-month post treatment with calcium hydroxide showing empty root canals (RC). Odontoblast cell shows intense immunereactivity to peripherin antibody. Dentin (D). Granulation tissue (GT) shows mild immune reactivity to peripherin antibody. Peripherin antibody. Original magnification, ×100. (O) Apical area of premolar tooth 6-month post treatment with calcium hydroxide showing no immunoreactivity to CGRP antibody in the empty root canals (RC). Dentin (D). Granulation tissue shows no immunereactivity to CGRP antibody. CGRP antibody. Original magnification, ×100.
Fig. 1
Fig. 1
Premolar teeth at selected time intervals following treatment with recombinant amelogenin and calcium hydroxide. (A) Apical and mid root area of a premolar tooth 1-month post treatment with r-amelogenin showing regenerated cellular tissue inside canal consistent with pulp tissue (P), and apical papilla (AP) and dentin (D). H&E stain. Original magnification, ×100. (B) Apical and mid root area of a premolar tooth 1-month post treatment with r-amelogenin showing cells in the regenerated pulp tissue (P), apical papilla (AP) and cellular dentin (CD) Trichrome stain. Original magnification, ×100. (C) Apical area of premolar tooth 1-month post treatment with r-amelogenin showing regenerated regenerated pulp tissue (P) in the root canal, dentin (D) and apical papilla (AP). H&E stain. Original magnification, ×100. (D) Apical and mid-root area of premolar tooth 1-month post treatment with r-amelogenin showing intense to moderate immunoreactivity to the peripherin (PER) protein. Intense immunoreactivity is observed at the odontoblast layer and odontoblastic zone in the form of parallel conical arrays. Moderate to intense immunoreactivity in the central root canal and pulp camber. Strong immunoreactivity is seen in the apical papilla (AP). Dentin (D). Peripherin antibody. Original magnification, ×100. (E) Apical area of premolar tooth 1-month post treatment with r-amelogenin showing intense to moderate immunoreactivity to the CGRP antigen in the cells of the apical papilla (AP) running axially in the central area of root canal (red lines). Dentin (D). CGRP antibody. Original magnification, ×100. (F) Apical area of premolar tooth 1-month post treatment with r-amelogenin showing intense immunoreactivity to the GFAP antibody in the odontoblastic region (arrow) and mild to moderate immunoreactivity throughout the regenerated pulp tissue. Mild immunoreactivity is seen in the cells of the apical papilla (AP). Dentin (D). GFAP antibody. Original magnification, ×40. (G) Apical area of premolar tooth 3-month post treatment with r-amelogenin showing intense immunoreactivity to peripherin antibody in the regenerated pulp tissue (P) that lies at the pulp periphery near the odontoblast area (red lines). Dentin (D), periodontal ligament PDL (black arrow). Peripherin antibody. Original magnification, ×40. (H) Apical area of premolar tooth 3-month post treatment with r-amelogenin showing moderate to intense immunoreactivity to CGRP antibody in the regenerated pulp tissue (P) that lies at root canal apex and in mid root in the central pulp region (red lines). Apical papilla (AP) shows moderate immunoreactivity. Dentin (D). CGRP antibody. Original magnification, ×100. (I) Apical area of premolar tooth 3-month post treatment with r-amelogenin showing moderate to intense immunoreactivity to GFAP antibody in the odontoblastic region (red arrow) and mild to moderate immunoreactivity throughout the regenerated pulp tissue (P). Dentin (D), periodontal ligament PDL (black arrow). GFAP antibody. Original magnification, ×100. (J) Apical area of premolar tooth 6-month post treatment with r-amelogenin showing intense immunoreactivity to peripherin antibody in the regenerated pulp tissue (P) that lies at pulp periphery near the odontoblast area (red lines). Dentin (D), PDL (black arrow). Peripherin antibody. Original magnification, ×100. (K) Apical area of premolar tooth 6-month post treatment with r-amelogenin showing intense immunoreactivity to CGRP antibody in the regenerated pulp tissue (P) that lies at root canal apex and in mid root in the central pulp region (red lines). Dentin (D), PDL (black arrow). CGRP antibody. Original magnification, ×100. (L) Apical and mid area of premolar tooth 6-month post treatment with r-amelogenin showing moderate to intense immunoreactivity to GFAP antibody in the odontoblastic and cell-rich zone regions (red arrows) and mild to moderate immunoreactivity throughout the regenerated pulp tissue (P). Dentin (D), PDL (black arrow). GFAP antibody. Original magnification, ×100. (M) Apical area of premolar tooth 1-month post treatment with calcium hydroxide showing no immunoreactivity to Sox2 antibody in the empty root canals (RC). Dentin (D). Mineralized island can be seen in the empty RC (box). Sox2 antibody. Original magnification, ×100. (N) Apical and mid root area of a premolar tooth 3-month post treatment with calcium hydroxide showing empty root canals (RC). Odontoblast cell shows intense immunereactivity to peripherin antibody. Dentin (D). Granulation tissue (GT) shows mild immune reactivity to peripherin antibody. Peripherin antibody. Original magnification, ×100. (O) Apical area of premolar tooth 6-month post treatment with calcium hydroxide showing no immunoreactivity to CGRP antibody in the empty root canals (RC). Dentin (D). Granulation tissue shows no immunereactivity to CGRP antibody. CGRP antibody. Original magnification, ×100.
Fig. 2
Fig. 2
Premolar teeth at 1-month time interval following treatment with recombinant amelogenin. (A) Higher magnification of box in Fig. 1D showing positive peripherin Ag/Ab immunostaining (brown DAB chromogen) in 1~9 of 13 of the plotted areas, while 10~13 of the plotted areas show negative peripherin Ab/Ab reactivity (grey scale). Image analysis. Peripherin antibody. Original magnification, ×400. (B) Higher magnification of Fig. 1F showing positive GFAP Ag/Ab immunostaining (brown DAB chromogen) were detected in 1~20 out of 22 plotted areas. In contrast, 21~22 of the plotted areas showed negative GFAP Ab/Ab reactivity (grey scale). Image analysis. GFAP antibody. Original magnification, ×400.
Fig. 3
Fig. 3
Mean count for stem cells apical papilla SCAP+pulp stem cells. Quantitative image analysis immunohistochemical sections comparing between mean counts for SCAP+pulp stem cells (1000 pixel image scale/Mic. Mag. ×400).

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