Translation Approach for Dentine Regeneration Using GSK-3 Antagonists

The canonical Wnt/β-catenin signaling pathway is crucial for reparative dentinogenesis following tooth damage, and the modulation of this pathway affects the rate and extent of reparative dentine formation in damaged mice molars by triggering the natural process of dentinogenesis. Pharmacological stimulation of Wnt/β-catenin signaling activity by small-molecule GSK-3 inhibitor drugs following pulp exposure in mouse molars results in reparative dentinogenesis. The creation of similar but larger lesions in rat molars shows that the adenosine triphosphate (ATP)–competitive GSK-3 inhibitor, CHIR99021 (CHIR), and the ATP noncompetitive inhibitor, Tideglusib (TG), can equally enhance reparative dentine formation to fully repair an area of dentine damage up to 10 times larger, mimicking the size of small lesions in humans. To assess the chemical composition of this newly formed dentine and to compare its structure with surrounding native dentine and alveolar bone, Raman microspectroscopy analysis is used. We show that the newly formed dentine comprises equal carbonate to phosphate ratios and mineral to matrix ratios to that of native dentine, both being significantly different from bone. For an effective dentine repair, the activity of the drugs needs to be restricted to the region of damage. To investigate the range of drug-induced Wnt-activity within the dental pulp, RNA of short-term induced (24-h) molars is extracted from separated roots and crowns, and quantitative Axin2 expression is assayed. We show that the activation of Wnt/β-catenin signaling is highly restricted to pulp cells in the immediate location of the damage in the coronal pulp tissue with no drug action detected in the root pulp. These results provide further evidence that this simple method of enhancement of natural reparative dentinogenesis has the potential to be translated into a clinical direct capping approach.


Primary rat cell culture from pulp explants
Primary pulp cell cultures from explants of maxillary and mandibular first molars of 5-day-old, Wistar rats were established for preliminary drug testing assays (cell viability assessment, dose and time dependent relative Axin2 gene expression).
A standard medium (α MEM, UltraGlutamine, Lonza) enriched with 15% FBS (Gibco) and 1% Antibiotic / Antimycotic Solution (ABAM, Sigma) was prepared. Ten 5-day-old Wistar rats were sacrificed by cervical dislocation and the non-erupted first molars were extracted immediately under aseptic conditions using 20G needles and microscopes. Teeth were washed twice in ice cold PBS (Sigma) and the pulp tissue was removed from the developing teeth using fine straight tip tweezers and 27G needles. The pulp explants were placed in a 24-well culture dish (one pulp per well) and kept on ice. After initial attachment the explants were covered with 400µl medium and incubated (37°C, 5% CO2/95% O2, 100% humidity). Medium was replaced on day 2 (500µl), thereafter 2-3 times a week. Cells were passaged at 80% confluence with trypsin (TrypLE Express, Gibco) and expanded (Appendix Fig. 2A).

Cell viability assessment
Primary pulp cells cultured from 5 day-old rats were plated in 96 well plates at 10'000 cells/well and incubated (37°C, 5% CO2/95% O2, 100% humidity) using standard culture medium. After 24h of incubation, the medium was replaced with 100µl conditioned (drug + media) or control medium (medium alone) for another 24h. The following concentrations of CHIR and TG were tested: 1mM, 100µM, 50µM, 20µM, 10µM, 1µM, 100nM and 10nM. Stock concentrations were diluted in DMSO in order to equalize the final amount of DMSO per well. Concentrations ranging from 10nM to 20µM comprised 0.5µl DMSO + 99.5µl medium (0.5% v/v), 50µM and 3 100µM comprised 2.5µl DMSO + 97.5µl medium (2.5% v/v) and 1mM comprised 25µl DMSO + 75µl medium (25% v/v). DMSO only was tested at the above-mentioned concentrations. A control plate was set up with the same conditioned / control medium without cells in order to account for chemical interference of test compounds with the MTS assay (CellTiter 96 AQuous, Promega). After the 24-hour incubation period, representative images (Appendix Fig. 2, B-J) were taken and 20µl of MTS tetrazolium compound were added to each well using multichannel pipettes. Plates were incubated for another 2.5h and the absorbance of the produced formazan was measured at 490nm using a 96-well plate reader (ClarioStar, BMG Labtech). Background absorbance obtained from control plates was subtracted individually and data was normalized.
The experiment was performed in duplicates and repeated independently.
For RNA extraction, cells (3 wells per group) were harvested using 350µL RLT buffer enriched with 1% β-mercaptoethanol (Rneasy Mini Kit, Quiagen) and stored at -80°C. RNA was extracted using the Rneasy Mini Kit (Quiagen) according to the manufacturer's instructions. RNA quantification was measured by NanoDrop spectrophotometer (Thermo Fisher) and the absorbance ratios of 260/280 and 260/230 were recorded for quality control. Revers transcription and real time qPCR analysis was performed with Axin2 as readout. All experiments were repeated independently in triplicates.