The hDPSCs purchased from Lonza (PT-5025, Lonza Bioscience) were cultured in DPSC SingleQuot Growth Medium (DPSCGM) (PT-4516, Lonza Bioscience) in a humidified atmosphere with 5% CO₂ at 37 °C. For osteogenic differentiation, hDPSCs at passage 4 or 5 were seeded at a density of 2 × 10⁴ cells per well in 24 well collagen-coated plates. After 24 h, the media was changed with osteogenic media (Alfa-MEM with 1% penicillin-streptomycin, 5% fetal bovine serum, 50 μM L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone) in presence or absence of Thiamet-G (S7213, Selleckchem.com) with various concentrations. The culture media was changed every 2 or 3 days.

Human dental pulp stem cells (hDPSCs) viability was determined using the MTS assay. hDPSCs were seeded into 96-well plates at 5 × 10³ cells/well density in a serum-free medium and incubated in a humidified atmosphere with 5% CO₂ at 37 °C. The next day, cells were treated with vehicle (DMSO) or Thiamet-G (1 μM, 10 μM, 50 μM, & 100 μM) in DPSCGM and further incubated at 37 °C for 24 h and 48 h. After the indicated drug treatment period, 20 μL MTS (G3582, Promega) solution was put into each well and incubated at 37 °C for an hour. The absorbance was examined using a SpectraMax ABS Microplate Reader at a wavelength of 490 nm. Cell viability was calculated using Excel.

hDPSCs at passage 4 or 5 were cultured with vehicle (DMSO) or Thiamet-G (1 and 10 μM) in osteogenic differentiation media for 7 and 14 days. To detect the osteogenic differentiation of hDPSCs, alkaline phosphatase (ALP) activity was performed using an ALP activity assay kit (ab83369, Abcam), according to the manufacturer’s protocol. The absorbance was measured at 405 nm using a SpectraMax ABS Microplate Reader. The standard curve and ALP activity were calculated using Excel, following the manufacturer’s instructions.

hDPSCs at passage 4 were cultured in osteogenic media with vehicle (DMSO) or Thiamet-G (10 μM) for 7 days. Then, RNA was extracted using RNeasy® Micro Kit (74004; Qiagen) and transcribed to cDNA using Omniscript RT kit (205111; Qiagen), following the instructions in the manual. RT-qPCR was performed using the StepOnePlus RT-PCR system device. The 2^−ΔΔCT method was used to determine the relative alteration in gene expression, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The nucleotide sequences of the primers used in this study are listed in Supplementary Table 1. The obtained data was analyzed using Excel and GraphPad Prism 8.

All experiments were approved by Kyungpook National University School of Dentistry, Intramural Animal Use and Care Committee (KNU 2020-0107). For this study, 8-week-old male Institute of Cancer Research (ICR) mice were used for pulp cavity preparation. The adult mice were housed at 22 °C ± 2 °C temperature, 55% ± 5% humidity, and artificial illumination lit for 12 h with free access to food and water.

Eight-week-old male mice were anesthetized with an intraperitoneal injection of avertin (250 mg/kg; T4802-5G, Sigma Aldrich). The pulp cavity on the upper right first molar was prepared using a 0.6 mm round bur and refined with K-files to minimize heat generation under a dissecting microscope. After pulp cavity exposure, mice were randomly assigned to two groups, and either 100 μM Thiamet-G or 1% dimethyl sulfoxide (DMSO) with pluronic® F-127 (P2443-250G, Sigma Aldrich) was locally delivered into the exposed pulp using a Hamilton syringe. Following drug delivery, the exposed pulp cavity was covered with mineral trioxide aggregate (MTA) and light-cured composite resin. The mice were then housed for 3, 5, and 42 days for further evaluation. 5–10 mice were used for each group.

Mice were euthanized by cervical dislocation after 3, 5, and 42 days following local drug delivery; maxillae were separated, fixed in 4% paraformaldehyde (PFA), decalcified with 0.5% ethylenediaminetetraacetic acid (EDTA), dehydrated in graded ethanol (EtOH), cleared in xylene, and embedded in paraffin. Frontal wax sections were then prepared with a thickness of 7 μm using a microtome. At first, histomorphological alterations were analyzed by performing HE staining and MTC staining, as described previously (Aryal et al., 2021). For immunohistochemistry, anti-NESTIN (AB11306, Abcam), anti-TGF-β (ab92486, Abcam), anti-OPN (sc-73631, Santa Cruz Biotechnology), anti-MPO (bs-4943R, Bioss), anti-TNF-α (ab9739, Abcam), anti-NF-κB (bs-50467R, Bioss), anti-O-GlcNAc (RL2) (MA1-072, Invitrogen), and anti-RUNX2 (ab192256, Abcam) primary antibodies were used with goat anti-Mouse IgG H&L (HRP) (ab6789, Abcam) or goat F(ab')2 anti-Rabbit IgG F(ab')2 (HRP) (ab6112, Abcam) secondary antibodies. The color reaction was visualized using a diaminobenzidine tetrahydrochloride (DAB) reagent kit (C09-12, Origene). The experiment was conducted using at least three biological replicates. Then, the images of the immune-stained sections were arranged using Photoshop.

After 6 weeks of Thiamet-G or DMSO treatment, mice were sacrificed by cervical dislocation, fixed in 4% PFA and maxillae were analyzed using micro-CT imaging (Skyscan1272; Bruker). Three-dimensional reconstructions were prepared using NRecon software to quantify the hard tissue formed in the region of interest as described previously (n = 3) (Aryal et al., 2021). p < 0.05 was considered statistically significant.

All histological and immunostaining images were captured using a DM2500 microscope (Leica) equipped with a digital CCD camera (DF310 FX, Leica). Data are presented as mean ± SD, with at least three independent biological replicates performed for each experiment. Normality and homogeneity of variances were assessed prior to statistical analysis. Two-group comparisons were performed using the unpaired two-tailed Student’s t-test, while multiple group comparisons were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. Statistical analyses were conducted using Microsoft Excel and GraphPad Prism 9 (GraphPad Software, USA). A p-value <0.05 was considered statistically significant.

To understand the potential role of Thiamet-G in dental pulp stem cell expansion and differentiation, we used in vitro cell cultivation using hDPSCs (Figure 1). An MTS assay was conducted to assess cytotoxicity and cell viability of the hDPSCs after treatment with various concentrations of Thiamet-G (1 μM, 10 μM, 50 μM, and 100 μM) (Figure 1A). The MTS assay results indicated that Thiamet-G exhibited no toxicity in hDPSCs and promoted dose-dependent cellular proliferation (Figure 1B). We then evaluated the ALP activity of hDPSCs treated with Thiamet-G (1 μM and 10 μM) for 7 and 14 days under osteogenic differentiation conditions (Figure 1C). After 7 days, the ALP activity was almost similar across all groups. However, after 14 days, the ALP activity significantly increased in Thiamet-G-treated groups in a dose-dependent manner, with 10 μM showing the highest activity (Figure 1D). Based on these, 10 μM was selected for further in vitro experiments. Then, we examined the global O-GlcNAc level using Western blot. Western blot data showed significantly increased global O-GlcNAc protein levels in Thiamet-G-treated groups compared to the control groups (Supplementary Figure S1). Thereafter, we examined the expression levels of reparative dentin formation markers, Alp, bone morphogenetic protein-2 (Bmp2), bone sialoprotein (Bsp), dentin sialophosphoprotein (Dspp), glycogen synthase kinase-3 beta (Gsk3β), osteocalcin (Ocn), osteopontin (Opn), and Runx2, after 7 days of Thiamet-G treatment with induced osteogenic differentiation, using RT-qPCR (Figure 1E). Our results showed significantly elevated expression patterns of all reparative dentin markers following Thiamet-G treatment. However, the mRNA expression of Ocn showed no significant changes compared to the control (Figure 1E).

After 42 days of Thiamet-G treatment, dentin-bridge formation in the pulp cavity was evaluated using micro-CT and MTC staining. Micro-CT analysis revealed a significantly increased percentage of hard tissue volume in the Thiamet-G-treated specimens compared to the control, indicating facilitated reparative dentin formation (Figures 2A-C). MTC staining further showed that the Thiamet-G-treated group exhibited dentin-bridge formation beneath the pulp-exposed area, with regular pulp cell arrangement and well-organized tertiary dentin (Figure 2E). In contrast, specimens treated with DMSO showed disordered arrangement (Figure 2D). These results suggest that Thiamet-G treatment promotes effective reparative dentin formation with sound pulp tissue.

As Thiamet-G treatment for 42 days promoted reparative dentin formation, we examined the early cellular and molecular responses of pulp cells against Thiamet-G treatment using histology and immunohistochemistry after 3 and 5 days of local drug delivery (Figure 3). Following 3 and 5 days of local drug delivery, histological alterations were examined using HE staining. Thiamet-G treatment produced time-dependent alterations in inflammatory cell presence. After 3 days of treatment, histological analysis showed a significant increase in leukocyte infiltration, whereas the number of hyperchromatic cells was reduced compared with controls (Figures 3A–B’; Supplementary Figure S2). In contrast, following 5 days of Thiamet-G exposure, both leukocyte numbers and hyperchromatic cell counts were significantly decreased (Figures 3C–D’; Supplementary Figure S2). These data indicate that Thiamet-G modulates inflammatory responses in a time-dependent manner, characterized by early immune cell accumulation followed by attenuation of inflammation with prolonged treatment. Thiamet-G-treated specimens exhibited a higher number of O-GlcNAc-positive cells at 3 days post-treatment compared to controls (Supplementary Figures S3A,B). Although the number decreased by day 5, it remained elevated relative to control levels particularly beneath injury area (Supplementary Figures S3C,D). After 3 and 5 days of Thiamet-G local delivery, we examined the role of Thiamet-G treatment in inflammation modulation by performing immunohistochemistry with its well-known markers MPO and TNF-α. At 3 days of Thiamet-G local delivery, decreased immunoreactions against MPO and TNF-α were observed compared to control (Figures 3E,F’,I–J’; Supplementary Figure S4). Similarly, Thiamet-G treatment for 5 days showed decreased localization of MPO and TNF-α compared to control specimens (Figures 3G,H’,K–L’; Supplementary Figure S4). Furthermore, localization of NF-κB, the marker for innate and adaptive immune responses, showed a decreased reaction after 3 and 5 days of Thiamet-G treatment compared to control (Figures 3M–P’; Supplementary Figure S4).

Further, we examine the localization patterns of NESTIN, TGF-β1, OPN, and RUNX2 in the in vivo experimental specimens as markers of reparative dentin formation. Immunohistochemistry using NESTIN antibody showed a stronger staining pattern, particularly around the injury area and reactionary dentin-forming region of Thiamet-G treated specimens for 3 and 5 days compared to the control (Figures 4A–D; Supplementary Figure S4). At 3 days of Thiamet-G treatment, the localization of TGF-β1, one of the markers of dental pulp tissue repair, was comparatively similar to that of control (Figures 4E,F; Supplementary Figure S4). However, Thiamet-G-treated specimens for 5 days showed more intense immunostaining against TGF-β1 than the control (Figures 4G,H; Supplementary Figure S4). Moreover, immunostaining against OPN, one of the markers for reparative dentin formation, also showed intense staining, particularly beneath the injury site in the specimens treated with Thiamet-G for 3 days and 5 days compared to the control (Figures 4I–L; Supplementary Figure S4). At 3 days of local delivery, more RUNX2-positive cells were observed beneath the injury area of Thiamet-G-treated specimens than in controls (Figures 4M,N; Supplementary Figure S4). Although the number of RUNX2-positive cells was less in Thiamet-G-treated specimens for 5 days compared to 3 days, it was still more than that of control specimens (Figures 4O,P; Supplementary Figure S4). Notably, a significant increase in RUNX2-positive cells was observed in the exposed pulp.