Bone or Tooth dentin: The TGF-β signaling is the key

Abstract

To investigate the cell linkage between tooth dentin and bones, we studied TGF-β roles during postnatal dentin development using TGF-β receptor 2 (Tgfβr2) cKO models and cell lineage tracing approaches. Micro-CT showed that the early Tgfβr2 cKO exhibit short roots and thin root dentin (n = 4; p<0.01), a switch from multilayer pre-odontoblasts/odontoblasts to a single-layer of bone-like cells with a significant loss of ~85% of dentinal tubules (n = 4; p<0.01), and a matrix shift from dentin to bone. Mechanistic studies revealed a statistically significant decrease in odontogenic markers, and a sharp increase in bone markers. The late Tgfβr2 cKO teeth displayed losses of odontoblast polarity, a significant reduction in crown dentin volume, and the onset of massive bone-like structures in the crown pulp with high expression levels of bone markers and low levels of dentin markers. We thus concluded that bones and tooth dentin are in the same evolutionary linkage in which TGF-β signaling defines the odontogenic fate of dental mesenchymal cells and odontoblasts. This finding also raises the possibility of switching the pulp odontogenic to the osteogenic feature of pulp cells via a local manipulation of gene programs in future treatment of tooth fractures.

Keywords: 3.2 kb Col1-CreERT2; Cell lineage tracing; Gli1-CreERT2; cell fate switch; evolution; tooth development.

Figure 1

Lineage tracing of Gli1^Lin tdTomato⁺ cells in the root pulp, and development of molar root defects in Gli1^Lin cKO including, impaired mineralization, changes of Od cell morphologies, and the loss of dentinal tubules. a. Lineage tracing of Gli1^Lin cells in root formation. Gli1^CreERT2/+, R26R^tdTomato/+ mice were administrated one dose of tamoxifen at postnatal day 5 (P5) and followed by 3 chasing timepoints: 1-, 9-, and 23-days post injection, separately. b. One-time injection of tamoxifen (administered at P5 and harvested at P28) induced severe tooth root defects in Gli1^Lin cKO (right panels) by representative Micro-computed tomography (μCT) analyses; and c. quantitation of changes of root length, dentin thickness, and mineral density (n = 4-6; **P< 0.01; ***P< 0.001; ****P< 0.0001); d. Representative Calcein (green color) and alizarin red (red color) double injections (5 day apart) revealed drastic reduction in mineral apposition rate in the Gli1^Lin cKO root (lower panel) and crown (upper panel). Quantitation data showed that these changes were statistically significant (n = 4-6; **P< 0.01); e. Representative Masson's Trichrome staining images showed expanded root canals and thin dentin plus a replacement of postulated polarized odontoblasts by a single layer of non-polarized in the Gli1^Lin cKO root (right panels); f. Acid-etched SEM images displayed a lack of dentin tubules in the Gli1^Lin cKO root (lower right) and a moderate reduction in the Gli1^Lin cKO crown dentin tubules (upper right); g. Statistic data analyses showed that those changes were significant (n=4; ***P< 0.001; ****P< 0.0001). Od, odontoblast.

Figure 2

Defected dentin matrices, a replacement of polarized odontoblasts (Ods) by a flat layer of osteoblast (Ob)-like cells, and molecular changes occurred in the Gli1^Lin cKO roots. a. Representative polarized light images displayed a disorganized dentin fiber with no sign of dentin tubules; b. H&E images showing a flat layer of Ob-like cells in the Gli1^Lin cKO roots; and (c-g) Immunofluorescence staining images showed the following changes in the Gli1^Lin cKO root: a sharp decrease in OSX with no sign of polarized cells (c); a great reduction of NESTIN in pre-dentin and a lack of dentin tubules (d); disperse DSPP distribution (e) and COL1 staining (f) in the Gli1^Lin cKO roots; and a drastic increase in DMP1 in the Gli1^Lin cKO root dentin (g).

Figure 3

The RNAscope data analyses revealed a switched mRNA expression profile characterized with increased osteogenic markers and decreased odontogenic markers in the Gli1^Lin cKO root compared to the control. a) A drastic decrease of Osx mRNA levels in the Gli1^Lin cKO root (right panel); b) A great decrease or lack of Dspp expression in Gli1^Lin cKO Ob-like cells; and c) an increase in Dmp1 mRNAs in the Gli1^Lin cKO Ob-like cells; and d) Statistical data analyses revealed the above changes were statistically significant (n = 4~7; **P< 0.01; ***P< 0.001). Ob, osteoblast.

Figure 4

Lineage tracing of 3.2 Col1^Lin tdTomato⁺ cells and tooth crown defects in 3.2 Col1^Lin cKO. a. Lineage tracing of 3.2 Col1^Lin cells in crown (dominant) and root. 3.2-kb Col1^CreERT2/+, R26R^tdTomato/+ mice were administrated 1 dose of tamoxifen at postnatal day 5 (P5) and followed by 3 chasing timepoints: 1, 9, and 23 days, separately. b. One-time injection of tamoxifen (@P5 and harvested at P28) induced remarkable tooth crown phenotypes in 3.2 Col1^Lin cKO (right panels) were revealed by representative μCT analyses, in which red open arrows pointed ectopic ossification in the crown pulp; c. Masson's Trichrome staining showed thin crown dentin and non-polarized Ob-like cells plus ectopic ossification in the pulp chamber; d. Polarized light microscope images revealed irregular fiber organization in the ectopic ossification tissue; and e. SEM images displayed short/thin dentin tubules in the 3.2 Col1^Lin cKO crown (also see Figure S7 for abnormal changes of root dentin tubules and quantitation data of changes of the tubule numbers). Od, odontoblast; Pre-od, pre-odontoblasts; Ob, osteoblast.

Figure 5

Removal of Tgfβr2 caused ectopic ossification in the crown pulp with molecular changes in the 3.2 Col1^Lin cKO crown. a-c. Representative immunofluorescent images showed a great decrease of OSX in the 3.2 Col1^Lin cKO crown cells plus a high expression level in the Ob-like cells along the ossification cite (a); sharp increase of DMP1 in the ossification site (b) and reduced DSPP expression.; d-e. The RNAscope images revealed a decrease of Dspp expression in Ods of 3.2 Col1^Lin cKO but an ectopic expression in the Ob-like and Ocy-like cells (d), and an increase in Dmp1 mRNAs in the Ods and Ob-like cells of 3.2 Col1^Lin cKO (e). Ob, osteoblast; Ocy, osteocyte; Od, odontoblast.

Figure 6

Changes of β-catenin in the Gli1^Lin cKO pulp and the overall working hypothesis. a. Representative images showed sharp increases of β-catenin in the Gli1^Lin cKO pulp/early odontoblasts (Ods) at both protein (right upper panels) and mRNA (right lower panel); and b. We propose that TGF-β signaling plays a vital role during odontogenesis by controlling the fate of pulp cells toward the odontogenic pathway, in which Osterix (OSX) positively regulates expressions of NESTIN and DSPP during Od polarization and dentin tubule/matrix formation (see text for details).