Monitoring Notch Signaling-Associated Activation of Stem Cell Niches within Injured Dental Pulp

Abstract

Dental pulp stem/progenitor cells guarantee tooth homeostasis, repair and regeneration throughout life. The decision between renewal and differentiation of these cells is influenced by physical and molecular interactions with stromal cells and extracellular matrix molecules forming the specialized microenvironment of dental pulp stem cell niches. Here we study the activation of putative pulp niches after tooth injury through the upregulation of Notch signaling pathway. Notch1, Notch2, and Notch3 molecules were used as markers of dental pulp stem/progenitor cells. Upon dental injury, Notch1 and Notch3 are detected in cells related to vascular structures suggesting a role of these proteins in the activation of specific pulpal perivascular niches. In contrast, a population of Notch2-positive cells that are actively proliferative is observed in the apical part of the pulp. Kinetics of these cells is followed up with a lipophilic DiI labeling, showing that apical pulp cells migrate toward the injury site where dynamic regenerative/repair events occur. The knowledge of the activation and regulation of dental pulp stem/progenitor cells within their niches in pathologic conditions may be helpful for the realization of innovative dental treatments in the near future.

Keywords: Notch signaling; Notch2; cell proliferation; dental pulp; niches; regeneration; stem cells; tooth injury.

Figure 1

Distribution of Notch proteins in the injured dental pulp. (A) Schematic illustration showing dental pulp injury after cavity preparation. Arrowheads indicate the areas of Notch activation within the pulp. (B) Notch1 (N1) staining is visible in cells lining blood vessels within the central part of the pulp. (C) Notch3 (N3) labeling is detected in perivascular cells of the central part of the pulp. (D) Notch2 (N2) staining is mainly found in apical pulp cells at the tooth apex. (E,F) Whole mount immunostaining showing Notch2 protein distribution at the apical part of an injured pulp. Scale bars: 50 μm (B–D), 200 μm (E,F).

Figure 2

Proliferation of dental pulp cells correlates with Notch2 protein expression in the apex of injured molar teeth. BrdU in red color, Notch2 (N2) staining in green color. (A) Proliferating dental pulp cells in intact teeth. (B) Proliferating pulp cells in injured teeth. Note that cell proliferation increases upon injury. (C–E) Confocal microscope analysis showing co-expression of BrdU (C) and Notch2 (D) in the apical part of the pulp after merging of the pictures (E). Scale bars: 50 μm (A–E).

Figure 3

Migration of DiI-labeled cells from the apical part of the pulp toward the injury site. Bright-field pictures (D,G,J), dark-field pictures (B,E,H,K), merged pictures (C,F,I,L). Red arrows and arrowheads indicate the areas of DiI (red color) injection. Light blue arrowheads indicate the final location of migrating DiI-positive cells. Black and white arrows indicate injured teeth. (A) Schematic illustration showing dental pulp injury after cavity preparation. (B,C) Dental pulps immediately after injury and DiI injection (T0). (D–F) Dental pulps 4 days after cavity preparation and DiI injection (T4). (G–I) Intact dental pulps immediately after DiI injection (T0). (J–L) Intact dental pulps 4 days after DiI injection. Abbreviations: d, dentin; p, pulp; T0, time zero days; T4, time 4 days. Scale bars: 200 μm (B–L).

Figure 4

Migration of DiI-labeled cells from the apical part of the pulp toward the injury site. (A–C) DiI labeled cells at the apical part of the pulp (T0). Dark-field pictures. DiI labels cells in red color while DAPI stains in blue color the nuclei of pulp cells in cryosections. (D–L) Migration of DiI-labeled cells from the root apex toward the injury site - whole pulp view. Bright-field pictures (D,G,J), dark-field pictures (E,H,K), merged pictures (F,I,L). (D–I) Dental pulps immediately after injury and DiI injection (T0). (J–L) Dental pulps 1 day after cavity preparation and DiI injection (T1). Black and white arrows indicate the area of the tooth injury. DiI-positive pulp cells are seen in red color. Light blue arrowheads indicate the final location of migrating. (M–R) Migration of DiI-labeled cells from the apical part of the pulp toward the injury site—cryostat sections. DiI-labeled cells in red color. Bright-field pictures (M,P), dark-field pictures (N,Q), merged pictures (O,R). Black and white arrows indicate injured teeth. (M–N) Intact dental pulps 1 day after DiI injection (T1). Note that in DiI-positive cells remain in the apical part of the pulp. (P–R) Dental pulps 1day after cavity preparation and DiI injection (T1). Light blue arrowheads indicate the final location of migrating DiI-positive cells. Abbreviations: p, pulp; T0, time zero days; T1, time 1 day. Scale bars: 100 μm (A,B,D–R), 20 μm (C).

Figure 5

Schematic representation of the suggested relationship between Notch signaling, dental pulp stem cell niches, and their activation upon tooth injury. (A) Potential stem/progenitor cells within the dental pulp at the crown level. Cells related to perivascular niches and odontoblast progenitors (i.e., sub-odontoblastic cells) will express a specific Notch receptor after dental injury at the crown level. (B) A model summarizing the cellular events after dental injury. Cavity preparation activates stem cell niches located in different parts of the dental pulp (light blue asterisks). Stem/progenitor cells start to migrate toward the injury site (blue arrows) and once on place might differentiate into odontoblast-like cells and participate to the formation of reparative dentin. Abbreviations: d, dentin; DPSC, dental pulp stem cells; HSC, hematopoietic stem cells; o, odontoblasts; p, pulp.