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Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury.

ABSTRACT: The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1?; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2^CreERT2/+; R26R ^mTmG/+ reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC6055255 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury.

Zhao Y Y Yuan X X Liu B B Tulu U S US Helms J A JA

Journal of dental research 20180322 9

The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving ...[more]

PMID: 29566345

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Project description:Odontoblast synthesis of dentin proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentin in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentin, marked by biochemical and architectonic modifications including diminished tubularity. Scanning electron microscopy of the collagen framework in reactionary dentin revealed a radically modified yet highly organized meshwork as indicated by fractal and lacunarity analyses. Immuno-gold labelling demonstrated increased density and regular spatial distribution of dentin sialoprotein (DSP) in reactionary dentin. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. To further dissect the formation of this altered dentin matrix, the associated enzymatic machinery was investigated. Analysis of extracted dentin matrix indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentin. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed up-regulation of MMP-2. Parallel up-regulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane type 1- matrix metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of Toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth TLR-2 and the gene encoding the corresponding adaptor protein MyD88 were down-regulated whereas genes encoding TLR-4 and adaptor proteins TRAM and Mal/TIRAP were up-regulated. TLR-4 signalling mediated by binding of bacterial products has been linked to up-regulation of MMP-2. Further, increased expression of genes encoding components of the TGF-? signalling pathway, namely SMAD-2 and SMAD-4, may explain the increased synthesis of collagen by odontoblasts in caries. These findings indicate a radical adaptive response of odontoblasts to microbial invasion of dentin with resultant synthesis of modified mineralized matrix.

Project description:Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

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Dataset Information

Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury.

Publications

Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury.

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