Wnt/β-catenin Promotes Cementum Apposition in Periodontal Regeneration

Abstract

Regeneration of periodontal tissue, particularly the cementum-periodontal ligament (PDL)-bone complex, has long been challenging because the differentiation kinetics of cells and the molecular pathways contributing to the regeneration process are largely unknown. We aimed to evaluate the cell behavior and molecular pathways that contribute to periodontal tissue regeneration in vivo. We analyzed the process of periodontal tissue regeneration through subrenal capsule transplantation of immediately extracted molars in mice. We showed that the regenerated periodontal tissue in the subrenal capsule was morphologically comparable to the intact periodontal tissue, with increased cellular cementum thickness in the apical region. Cell tracing analysis revealed that the cells comprising the regenerated periodontal tissue were derived from transplanted teeth and were indispensable for periodontal tissue regeneration, whereas recipient mouse-derived cells partly contributed to angiogenesis. Bioinformatics analysis based on the gene expression profile in the transplanted teeth indicated that Wnt/β-catenin signaling is involved in periodontal tissue regeneration, which was further confirmed through β-catenin immunohistochemistry. Moreover, the constitutive activation of β-catenin in the cells of transplanted teeth was found to promote accelerated cellular cementum apposition, while the conditional knockout of β-catenin in the cells of transplanted teeth suppressed cellular cementum apposition. Notably, the manipulation of Wnt/β-catenin signaling did not interfere with the bone-PDL-cementum complex, while endogenous osteoclast activity was affected in bone. Our results demonstrated the essential roles of endogenous PDL cells in periodontal tissue regeneration and that Wnt/β-catenin signaling is involved in this process, particularly cellular cementum apposition. Hence, controlling this pathway could promote cementum regeneration, which is a critical process for the regeneration of the cementum-PDL-bone complex. This study provides novel insights into cell behavior and signaling pathways that will advance practical periodontal tissue regeneration.

Keywords: Wnt signaling pathway; bone; cell tracing; periodontal ligament; periodontal tissue regeneration; signaling pathway; subrenal capsule.

Figure 1.

Transplantation of extracted teeth into the subrenal capsule of mice. (A) Schematic illustration of the immediate autologous transplantation of the extracted upper first molar into the subrenal capsule. (B) Experimental schedule. Samples were harvested at 1, 2, and 4 wk after transplantation. (C) Low-magnification histological image of the transplanted tooth after 4 wk and a control tooth without transplantation (8 wk). (D) High-magnification histological images of the transplanted teeth at the distal side of the interradicular septum (rectangle in Fig. 1C). (E) Staining for tartrate-resistant acid phosphatase (TRAP)/alkaline phosphate (ALP) activity in the transplanted teeth at the distal side of the interradicular septum. Immunohistochemistry using an anti-Dmp1 antibody (F) and anti-periostin antibody (G). (H) Picrosirius red (PSR) staining samples analyzed under bright field and polarized light. Bar: 200 µm (C); 50 µm (all other images).

Figure 2.

Fate of transplanted tooth–derived cells during periodontal tissue regeneration. (A) Schematic illustration of extracted tooth allogenic transplantation for cell tracing. The extracted molars of green fluorescent protein (GFP)–expressing mice were transplanted into the subrenal capsule of the wild-type (WT) littermates. (B) Schematic experimental schedule. Samples were harvested 4 wk after transplantation. (C) Detection of GFP-positive transplanted tooth–derived cells in the regenerated periodontal tissue at the subrenal capsule. (D) Ratio of GFP-positive cells in the periodontal ligament (PDL), bone, and cementum in regenerated periodontal tissue; n = 3, No statistical significance was detected between groups. (E, F) Immunostaining using anti-GFP and anti-endomucin antibodies at the regenerated PDL. Transplantation of extracted teeth of GFP-expressing mice into the subrenal capsule of WT mice (E) and extracted teeth of WT mice into the subrenal capsule of GFP-expressing mice (F). GFP-negative, endomucin-positive cells (white arrows) and GFP-positive, endomucin-positive cells (yellow arrows). GFP, green fluorescent protein. Bar: 200 µm (C, left), 50 µm (all other images).

Figure 3.

Effects of decellularization on periodontal tissue regeneration. (A) Schematic illustration of decellularized tooth transplantation. (B) Schematic experimental schedule. Samples were harvested 4 wk after transplantation. (C) Histology of decellularized tooth and untreated control tooth. (D) Histology of a decellularized tooth 4 wk after transplantation. (E) Immunohistochemistry to detect DMP1-positive cells at the cementum surface of the transplanted tooth 4 wk after transplantation. (F) Alkaline phosphatase (ALP) staining at the cementum surface of the transplanted tooth 4 wk after transplantation. Immunohistochemistry to detect osterix (Osx)–expressing cells (G) and periostin-positive area (H) at the cementum surface of transplanted teeth 4 wk after transplantation. Bar: 200 µm (D, left), 50 µm (all other images).

Figure 4.

Effects of constitutively active β-catenin on periodontal tissue regeneration. (A) Pathway enrichment analysis of differentially expressed genes between 2 wk after transplantation and Cont groups. Wnt/β-catenin signaling was suggested to be a variable pathway. (B) Immunohistochemistry staining of β-catenin during periodontal tissue regeneration at the cementum surface of the transplanted teeth. β-catenin–positive cells localized to the bone (white arrows) and cementum surfaces (yellow arrows). (C) Schematic illustration of allogenic tooth transplantation from mice expressing constitutively active (CA-βcat) into the subrenal capsule of wild-type (WT) littermates. (D) Experimental schedule. The constitutive activation of β-catenin was established via tamoxifen injection for 2 consecutive days into the CA-βcat–expressing mice before tooth transplantation. (E) Histology of the transplanted teeth of CA-βcat–expressing mice at the distal side of the interradicular septum (lower panels correspond to the rectangles in upper panels). (F) Quantification of the DMP1-positive cementum area at the distal side of the interradicular septum; n = 5, *P < 0.05. (G) Tartrate-resistant acid phosphatase (TRAP)/alkaline phosphate (ALP) activity staining of transplanted teeth at the distal side of the interradicular septum. (H) Quantification of the TRAP-positive multinuclear cell per regenerated bone area (N.Oc/BA) at the distal side of the interradicular septum; n = 3, *P < 0.05. (I) Immunohistochemistry staining for periostin. *Periostin-positive periodontal ligament (PDL) space. Bar: 200 µm (E, upper panel), 50 µm (all other images).

Figure 5.

Effects of conditional knockout of β-catenin on periodontal tissue regeneration. (A) Schematic illustration of allogenic tooth transplantation from conditional β-catenin–knockout (cKO-βcat) mice into the subrenal capsule of wild-type (WT) littermates. (B) Experimental schedule. The β-catenin knockout was established via tamoxifen injection for 2 consecutive days into the cKO-βcat mice before tooth transplantation. (C) Histology of the transplanted teeth of cKO-βcat mice at the distal side of the interradicular septum (lower panels correspond to the rectangles in upper panels). (D) Quantification of the DMP1-positive cementum area at the distal side of the interradicular septum; n = 5, *P < 0.05. (E) Tartrate-resistant acid phosphatase (TRAP)/alkaline phosphate (ALP) activity staining of transplanted teeth at the distal side of the interradicular septum. (F) Quantification of the TRAP-positive multinuclear cell per regenerated bone area (N.Oc/BA) at the distal side of the interradicular septum; n = 3, *P < 0.05. (G) Immunohistochemistry staining for periostin. *Periostin-positive periodontal ligament (PDL) space. Bar: 200 µm (C upper panel), 50 µm (all other images).