A Correlation between Wnt/Beta-catenin Signaling and the Rate of Dentin Secretion

Abstract

Introduction: Odontoblasts produce dentin throughout life and in response to trauma. The purpose of this study was to identify the roles of endogenous Wnt signaling in regulating the rate of dentin accumulation.

Methods: Histology, immunohistochemistry, vital dye labeling, and histomorphometric assays were used to quantify the rate of dentin accumulation as a function of age. Two strains of Wnt reporter mice were used to identify and follow the distribution and number of Wnt-responsive odontoblasts as a function of age. To show a causal relationship between dentin secretion and Wnt signaling, dentin accumulation was monitored in a strain of mice in which Wnt signaling was aberrantly elevated.

Results: Dentin deposition occurs throughout life, but the rate of accumulation slows with age. This decline in dentin secretion correlates with a decrease in endogenous Wnt signaling. In a genetically modified strain of mice, instead of tubular dentin, aberrantly elevated Wnt signaling resulted in accumulation of reparative dentin or osteodentin secreted from predontoblasts.

Conclusions: Wnt signaling regulates dentin secretion by odontoblasts, and the formation of reparative or osteodentin is the direct consequence of elevated Wnt signaling. These preclinical data have therapeutic implications for the development of a biologically based pulp capping medicant.

Keywords: Aging; Wnt signal; odontoblasts; pulp.

Figure 1.. Dentin deposition rate declines with age.

Representative Movat’s pentachrome stained maxillary molar sections showing the dentin thickness and pulp volume in (A) 1M, (B) 3M to (C) 6M groups. In Movat’s pentachrome, dentin and alveolar bone stain green to yellow, and PDL and dental pulp stain red. (D) Quantification of the pulp area and dentin area over the total pulp + dentin area from 1M, 3M, 6M and 12M mice (N=3 for each group). Vital dye labeling showing the new dentin formation in mice around (E) 1M and (F) 6M old in a 10-day interval. Calcein (green) was first injected, Alizarin red (red) was injected 10 days later, and mice were harvest 2 days after the last injection. Blue dotted lines indicate the crown. Abbreviations: d: dentin; p: pulp; ab: alveolar bone; g: gingiva; scale bars: 100 μm. Data are expressed as mean ± standard deviation; *p < 0.05; ***p < 0.0001.

Figure 2.. The decline in dentin deposition is paralleled by a decline in the population of Wnt-responsive odontoblasts.

Xgal^+ve, Wnt-responsive cells in the maxillary first molars at the age of (A) 1M, (B) 3M and (C) 6M. (D) Quantification of Xgal^+ve pixels in the pulp by age. Microscopy images showing Lef1 expression in odontoblasts in (E) 1M, (F) 4M and (G) 6M groups. (H) Quantification of Lef1^+ve pixels in the pulp by age. (I) GFP^+ve, Wnt-responsive cells in the maxillary first molar dental pulp of 1M-old mice, 5 days after tamoxifen treatment. GFP^+ve, Wnt-responsive cells were examined with (J) 1-month tracing and (K) 3-month tracing. (L) Quantification of GFP^+ve, Wnt-responsive cells in the pulp area. Co-Immunostaining of apoptosis marker Caspase 3 (Red) with Wnt-responsive cells (Green) in odontoblasts of (M) 1M, (N) 4M and (O) 6M mice. Immunostaining for the proliferation marker PCNA in odontoblasts in (P) 1M, (Q) 4M and (R) 6M mice. (S) Quantification of PCNA^+ve pixels in the pulp by age. Pentachrome staining showing the odontoblasts in the maxillary first molar in (T) 1M, (U) 4M and (V) 6M mice. Double head arrows indicate the odontoblast layer. Abbreviations: d, dentin; p, pulp; scale bars: 25 μm. Data are expressed as mean ± standard deviation; *p < 0.05; **p < 0.01; *** p < 0.0001.

Figure 3.. Wnt signaling regulates the rate of dentin secretion.

(A) Immunohistochemistry identifies DMP1 expression (brown) in a maxillary first molar. (B) Immunohistochemistry confirmed the GFP expression (purple) in a maxillary first molar in DMP1^GFP mice. Immunohistochemistry for β-catenin (brown) in odontoblasts in (C) control and (D) daβcat^Ot mutant mice (42-d-old). The sections were counterstained with fast green. Representative micro-CT sagittal-sectional reconstructed images of incisor from (E) control and (F) daβcat^Ot mutant mice (42-d-old). Representative micro-CT sagittal-sectional reconstructed images of the maxillary first molar from (G) control and (H) daβcat^Ot mutant mice (42-d-old). Pentachrome staining of the maxillary first molar from (I) control and (J) daβcat^Ot mutant mice (42-d-old). Dentin stains green to yellow, with the more mature dentin staining yellow. Abbreviations: d, dentin; p, pulp. Scale bars: 25 μm (A-D), and 200 μm (E-J).

Figure 4.. Accelerating deposition converts a tubular dentin into osteodentin.

Representative Pentachrome staining of a maxillary first molar in (A) control and (B) daβcat^Ot mutant mice. Representative cross-section of Aniline Blue staining of the maxillary first molar in (C) control and (D) daβcat^Ot mutant mice. Immunostaining of Osterix of the maxillary first molar in (E) control and (F) daβcat^Ot mutant mice. Immunostaining of Nestin in (G) 1M, (H) 4M and (I) 6M control mice. (J) Immunostaining of Nestin in daβcat^Ot mutant mice. Abbreviations: d, dentin, p, pulp. Scale bar: 25 μm.