Axin2-expressing cells differentiate into reparative odontoblasts via autocrine Wnt/β-catenin signaling in response to tooth damage

Abstract

In non-growing teeth, such as mouse and human molars, primary odontoblasts are long-lived post-mitotic cells that secrete dentine throughout the life of the tooth. New odontoblast-like cells are only produced in response to a damage or trauma. Little is known about the molecular events that initiate mesenchymal stem cells to proliferate and differentiate into odontoblast-like cells in response to dentine damage. The reparative and regenerative capacity of multiple mammalian tissues depends on the activation of Wnt/β-catenin signaling pathway. In this study, we investigated the molecular role of Wnt/β-catenin signaling pathway in reparative dentinogenesis using an in vivo mouse tooth damage model. We found that Axin2 is rapidly upregulated in response to tooth damage and that these Axin2-expressing cells differentiate into new odontoblast-like cells that secrete reparative dentine. In addition, the Axin2-expressing cells produce a source of Wnt that acts in an autocrine manner to modulate reparative dentinogenesis.

Figure 1

Time course of reparative dentinogenesis in molar tooth damage model. Masson’s Trichrome staining of a superior first molar 1 day post-damage (A) and in situ hybridisation analysis of Dspp expression in a superior first molar 1 day post-damage (B). Masson’s Trichrome staining of a superior first molar 5 days post-damage (C) and in situ hybridisation analysis of Dspp expression in a superior first molar 5 days post-damage (D). Masson’s Trichrome staining of a superior first molar 14 days post-damage (E) and in situ hybridisation analysis of Dspp expression in a superior first molar 14 days post-damage (F). All damage was performed in CD-1 mice and representative sagittal sections are shown from four independent experiments. Scale bars are equivalent to 100 μm and an arrow indicates the site of damage.

Figure 2

Pulp cells proliferate in response to damage. Immunohistochemical staining of proliferating cells with a PCNA antibody in an undamaged molar at low (A) and high magnification (A’). Immunohistochemical staining of proliferating cells with a PCNA antibody in a damaged superior first molar 3 days post-damage at low (B) and high magnification (B’). All damages were performed in CD-1 mice and representative sagittal sections are from four independent experiments. Scale bars are equivalent to 100 μm, an arrow indicates a pulp exposure. Quantification of the PCNA positive cells (C) was performed on three high-powered fields on at least four specimens per group, *p = <0.05.

Figure 3

Wnt/β-catenin signaling is activated in proliferating cells in response to tooth damage. Immunohistochemical staining of TCF/Lef (Wnt reporter) cells with a GFP antibody in an undamaged (A) and damaged superior first molar 3 days post-damage (B) from TCF/Lef:H2B-GFP reporter mice. Real-time qPCR analysis of Axin2 gene expression in dental pulp tissue extracted from undamaged and damaged maxillary first molars (C), n = 4, *p = <0.05. Immunohistochemical staining of TCF/Lef:H2B-GFP cells with a GFP antibody (D), proliferating cells with a PCNA antibody (E) and merged image (F) in a damaged superior first molar 3 days post-damage from TCF/Lef:H2B-GFP reporter mice. Representative sagittal sections are shown from four independent experiment’s. Scale bars are equivalent to 100 μm. The dentine-pulp interface is outlined by a white dashed line drawn from the light field image. An arrow indicates pulp exposure and arrow heads indicate examples of double stained cells.

Figure 4

Odontoblast-like cells are descendants of Wnt active cells. Immunohistochemical staining of Axin2-expressing cells with GFP in a damaged superior first molar 3 days post-damage (A) and 14 days post-damage at low (B) and high magnification (B’). Immunofluorescent staining of Axin2-expressing cells with GFP (C), in situ hybridisation analysis of dspp expression (D) and merged image (E) of damaged superior first molar from Axin2^{CreERT2; Rosa26-mT-mG flox/+} mice 5 days post-damage. Representative sagittal sections are shown from four independent experiment’s. Scale bars are equivalent to 100 μm. The dentine-pulp interface is outlined by a white dashed line drawn from the light field image. An arrow indicates pulp exposure; arrow heads indicate examples of double stained cells and asterisk indicates the formation of a dentine bridge.

Figure 5

Inhibition of Wnt signaling in Axin2-expressing cells impairs reparative dentinogenesis. Real-time qPCR analysis of Wls gene expression in dental pulp tissue extracted from WT and Wls^fl/fl mice teeth 5 days post-damage (A) Masson’s Trichrome staining of a damaged superior first molar from WT mice (B), Wls^fl/fl mice (C) and Axin2^CreERT2;Wls^fl/fl mice 14 days post-damage (D). Representative sagittal sections are shown, scale bars are equivalent to 100 μm, an arrow indicates the site of damage and an asterisk indicates a dentine bridge. Quantification of PCNA positive cells in damaged superior first molar from WT and Axin2^CreERT2; Wls^fl/fl mice post-damage (E), n = 4, *p = <0.05.

Figure 6

Wnt/β-catenin signaling modulates reparative dentinogenesis. Pulp cells rapidly proliferate in response to tooth damage shown by PCNA staining, with a significant peak in proliferation occurring 3 days post-damage and returning to baseline 14 days post-damage (Fig. 2). New odontoblast-like cells are detected by DSPP expression as early as 5 days post-damage and a dentine bridge is seen 14 days post-damage (Fig. 1). Our data shows that pulp exposure first triggers proliferation, followed by odontoblast differentiation and secretion of reparative dentine to form a dentine bridge. Wnt reporter mice (TCF/Lef:H2B-GFP) demonstrated proliferating cells are Wnt responsive 3 days post-damage (Fig. 3). Real-time qPCR analysis of Axin2 expression demonstrated that Axin2 is significantly elevated 3 days post-damage, indicating that the Wnt responsive cells are Axin2 positive ((Fig. 3C), Supplementary Figure 1). Lineage tracing of Axin2 cells in Axin2^{CreERT2, Rosa26 mTmG fl/+} mice demonstrated that these cells undergo a proliferative expansion and differentiate into odontoblast-like cells indicated by their co-expression of Dspp 5 days post-damage, characteristic odontoblast morphology and close association with the dentine bridge 14 days post-damage (Fig. 4). Loss of Wnt signaling in Wls^fl/fl mice demonstrated that damaged teeth no longer repair as a dentine bridge is absent 14 days post-damage compared to WT (Fig. 5B). Moreover, specifically deleting Wls in Axin2-expressing cells in Axin2^CreERT2; Wls^fl/fl mice severely impaired dentine bridge formation 14 days post-damage compared to WT (Fig. 5D). This suggests that Axin2 cells are producing their own source of Wnt to modulate their fate in an autocrine manner. Additionally, Wnt signaling is important for damage induced proliferation as the number of proliferating cells are significantly reduced in Axin2^CreERT2; Wls^fl/fl compared to WT at 3 and 5 days post-injury (Fig. 5E).