To evaluate the inhibitory effects of resveratrol on inflammatory response in rat dental pulp and its influence on the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. Thirty-six rats were divided into control, pulpitis and resveratrol groups (n=12) using a random number table. The dental pulp was cut open and added lipopolysaccharide to establish the pulpitis model, and the dental pulp of resveratrol group was covered with resveratrol paste. The pathological changes in dental pulp tissues were observed by hematoxylin-eosin staining. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in dental pulp tissues were detected by enzyme-linked immunosorbent assay. The mRNA and protein levels of TLR4 and NF-κB in dental pulp tissues were detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. The disorder and cellular inflammatory response grades of dental pulp tissues in resveratrol group were lower than those in pulpitis group (P<0.05), but the two groups had similar grade of hard tissue formation (P>0.05). The levels of TNF-α and IL-6, and the mRNA and protein levels of TLR4 and NF-κB in dental pulp tissues were all higher in pulpitis and resveratrol groups than those in control group (P<0.05). The levels of TNF-α and IL-6, and the mRNA and protein levels of TLR4 and NF-κB in dental pulp tissues were all lower in resveratrol group than those in pulpitis group (P<0.05). Resveratrol inhibits the inflammatory response in dental pulp tissues probably by suppressing the TLR4/NF-κB signaling pathway, thereby protecting dental pulp cells.

We aimed to evaluate the regulatory effects of miR-148a-3p on stem cell osteogenic differentiation and enamel development by targeting runt-related transcription factor 2 (RUNX2) and E-cadherin, respectively. TargetScan software was utilized to predict the binding sites between miR-148a-3p and osteogenic marker gene RUNX2 or E-cadherin. The changes in miR-148a-3p expression during osteogenic differentiation of epidermal stem cells were detected. After transfection with miR-148a-3p mimics or miR-148a-3p inhibitor, epidermal stem cells were induced towards osteogenic differentiation, and the changes in RUNX2 expression were measured. The changes in miR-148a-3p expression during enamel development regulated by epidermal stem cells were determined. After liposome-mediated transfection with miR-148a-3p mimics or miR-148a-3p inhibitor, epidermal stem cells were induced towards ameloblast development. Cell proliferation and apoptosis abilities were tested using methyl thiazolyl tetrazolium assay and flow cytometry, respectively. The expression of miR-148a-3p was detected by RT-PCR. The protein expressions of Wnt1, β-catenin, RUNX2 and E-cadherin were measured by Western blotting. Epidermal stem cells differentiated into osteoblasts through osteogenic induction culture. On 5, 12, 15 and 30 d, epidermal stem cells gradually differentiated into osteoblasts through epithelial aggregation and depression, mesenchymal aggregation, and dentin and enamel secretion. After transfection, compared with negative control (NC) group, the cell viability of miR-148-3p group significantly decreased (P<0.05), and the apoptosis rate increased (P<0.01). The viability of miR-148-3p inhibitor group significantly increased (P<0.05), while the apoptosis rate reduced (P<0.01). The dual-luciferase reporter assay showed that miR-148a-3p targeted Wnt1. Compared with NC group, the expression of miR-148a-3p significantly rose in miR-148a-3p group (P<0.001), and the protein expression levels of Wnt1, β-catenin, RUNX2 and E-cadherin significantly decreased. Compared with NC group, the expression of miR-148a-3p in miR-148a-3p inhibitor group significantly decreased (P<0.05), and the protein expression levels of Wnt1, β-catenin, RUNX2 and E-cadherin significantly increased. MiR-148a-3p is highly expressed in and regulates osteogenic differentiation and enamel development through targeting RUNX2 and E-cadherin respectively via the Wnt1/β-catenin pathway, which plays an important role in cell differentiation, stem cell proliferation and enamel development.

The aim was to investigate changes between autophagy and apoptosis in alveolar bone mesenchymal stem cells (BMSCs) under orthodontic pressure and the effect on osteogenesis in BMSCs. Twenty-four beagle canine teeth were used as experimental models. The control group received no treatment, while in the experimental groups, the canines were moved distally with 150 g of orthodontic force for 7 days, 14 days and 28 days. Bone remodeling was detected by H&E staining, while autophagy and apoptosis in the alveolar bone were analyzed by immunohistochemistry. 3-MA and Z-VAD-FMK were used to modulate autophagy and apoptosis. Transmission electron microscopy and western blotting were used to analyze autophagy and apoptosis in BMSCs, while their osteogenesis was analyzed by ALP staining, alizarin red staining, and osteogenic gene analysis. Compared to the control group, we observed significant distal movement of the canines in each of the experimental groups, the distance moved as the loading time was extended. In the experimental groups, the alveolar bone on the pressure side was clearly absorbed. On the pressure side, teeth were positive for LC3 after loading for 7 days and 14 days, whereas teeth were positive for caspase-3 after loading for 14 days and 28 days. The number of autophagosomes and the conversion of LC3-II/LC3-I gradually decreased, nevertheless, the number of apoptotic bodies and the expression of caspase-3 increased gradually. ALP activity, mineralized nodule formation, and osteogenic gene expression in BMSCs gradually decreased. Finally, the down-regulation of autophagy inhibited osteogenesis in BMSCs, whereas the restriction of apoptosis led to adverse consequences. Collectively, these data indicate that autophagy of the alveolar bone and BMSCs under orthodontic pressure gradually decreased as the loading time was extended, in contrast, the extent of apoptosis gradually increased; the osteogenic differentiation of BMSCs was positively correlated with autophagy but negatively correlated with apoptosis.

Apical periodontitis is a disease frequently observed, inducing discomfort. Nerves in periodontal ligament may be involved in the appearance of this symptom. Nerve endings of the periodontal ligament are reportedly stained with anti-calcitonin gene-related peptide (CGRP) antibody or anti-growth-associated protein 43 (GAP-43) antibody. Previous studies investigated the kinetics of structures positive for various antibodies on the formation of apical periodontitis, but no study has examined any GAP-43 immunoreactive structure. The purpose of this study was to experimentally induce apical periodontitis of rat molars and examine changes in a GAP-43 immunoreactive structure in the periodontal ligament. Using 10-week-old male Wistar rats, the mesial root of the lower right first molar was investigated. After general anesthesia, the mesial root canal pulp was exposed, and kept open for 2, 4, and 6 weeks. The mandible involving the molars was decalcified, and paraffin-embedded according to conventional methods to prepare sections 5 µm thick. Hematoxylin and eosin (H-E) staining and immunohistochemistry with anti-GAP-43 antibody were conducted. The morphology of an immunopositive structure was examined, and its expression per unit was statistically investigated. In the control group, a GAP-43 positive reaction was observed in the 1/3 area on the apical side. Morphologically, thin, short structures and thick branches were noted. In the experiment group, GAP-43 immunoreactive structures were only a few structures with branches. Furthermore, a GAP-43 positive reaction was localized in the apical area. We compared the percent GAP-43 immunopositive area per unit between the control and experiment groups. There was no significant difference at any point. This study showed that the morphology and distribution of GAP-43 immunoreactive structures existing in the periodontal ligament changed in the presence of apical periodontitis, suggesting that these changes may be related to the discomfort associated with apical periodontitis.

Epithelial projections (EP), which form as Hertwig’s epithelial root sheath (HERS) extends, play an important role in the formation of multi-rooted teeth. However, three-dimensional (3D) observations of the process in which EP form the furcation area have not yet been conducted. To investigate the formation of the furcation area by EP, we used a 3D reconstruction model of the furcation area in rats, which form subpulpal lobes in the same manner as humans, and examined the extent of cell division around the epithelium. Wistar rats between postnatal days 3-18 (PN3-18) (n=3/age) were fixed by the perfusion of 4% paraformaldehyde solution, and the maxillae were dissected out and decalcified. Tissues were embedded in paraffin by the conventional protocol, and 5-µm-thick serial sections and some sections for immunohistochemistry using an anti-proliferating cell nuclear antigen antibody of the frontal plane were prepared. All serial sections were stained by hematoxylin and eosin, and the structures of HERS, EP, dentin projections, and subpulpal lobes were overlayed after delineation every 5 sections from the images obtained of the maxillary second molar for the 3D reconstruction using ITK-snap software. Reconstructed images showed that a portion of HERS in the cervical region had extended into the dental papilla on PN3 to form EP. Four EP formed and extended with age at different speeds. On PN11, EP fused with one another in the center of the dental papilla and started to form subpulpal lobes. During this process, apical elongation in the apical direction of EP was not observed. Significant cell proliferation was detected in the dental papilla around the epithelium of the cervical loop. Collectively, these results indicate that the subpulpal lobes form immediately after the fusion of EP, and HERS extend in the apical direction following the fusion of EP.

Although TGF-β is known to be related to tumor proliferation, invasion and epithelial-mesenchymal transition (EMT), its effects on oral carcinomas remain unclear. This study aimed to elucidate the effects of TGF-β on oral squamous cell carcinoma (OSCC). In the present in vitro study, three human OSCC cell lines; SAS (tongue cancer), Ca9-22 (mandibular gingival cancer) and HSC-3 (cervical lymph nodes metastasis of tongue cancer), were treated with TGF-β1 or TGF-β signaling inhibitors, and assayed for proliferation and invasion. Expressions of EMT-related markers; E-Cadherin, N-Cadherin, Snail and Slug, as well as matrix metalloproteinases (MMPs) were evaluated by immunofluorescence staining and RT-qPCR. TGF-β1 treatment induced morphological change of SAS to spindle shape, significant proliferation of SAS and Ca9-22 (p<0.05), and significant increase in invasion capacity of all three cell lines (p<0.05). Inhibition of TGF-β signaling significantly suppressed proliferation of all three cell lines (p<0.05). By treatment with TGF-β1, reduced expression of E-Cadherin and enhanced expressions of N-Cadherin, Snail and Slug were observed in SAS (poorly differentiated OSCC), whereas E-Cadherin expression was enhanced in Ca9-22 (well differentiated OSCC) and variable depending on TGF-β1 concentration in HSC-3 (lymph node metastatic OSCC). Expressions of MMPs were enhanced in all three OSCC cell lines. TGF-β1 increases proliferation and invasion capacity of OSCC cell lines, while TGF-β signaling inhibition suppresses proliferation. TGF-β1 treatment may induce EMT by enhancing transcription activity, suppressing epithelial markers and augmenting mesenchymal markers in poorly differentiated OSCC, but induce both mesenchymal-epithelial transition (MET) and EMT depending on TGF-β1 concentration in lymph node metastatic OSCC. TGF-β1-induced enhancement of MMP expression may be associated with increased invasion capacity.

Our aim was to update a standard technique for embedding bone samples, with or without implants, using methyl methacrylate (MMA) to obtain reliable optical microscopy images from mineralized bone tissues and bone-implant interfaces. In addition, comparative studies were carried out using different temperatures throughout the polymerization process. Twenty-two New Zealand rabbit calvaria and femur bone samples with or without implants were used. The samples were fixed in 10% buffered formalin, dehydrated in an ascending alcohol series, and infiltrated in methyl methacrylate solutions (I, II and III). The specimens were divided into three groups. Groups 1 and 2 were polymerized at 24°C ± 5°C, while Group 3 was polymerized at 60°C. Group 1 and 2 obtained homogeneous, transparent, crystalline polymerization. Group 3 presented multiple bubbles (ø 2 – 3 mm), depressions, folds and even rupture of the vial container. The MMA bone samples polymerized at room temperature, showed good embedded, low hardness index, thin cuts, and effective staining.

Increasing evidence supports the important role of necroptosis in podocyte injury during diabetic nephropathy. Ubiquitin-specific peptidase 22 (USP22) is known to be involved in the regulation of necroptosis and high glucose (HG)-induced podocyte injury. This study aimed to indicate whether USP22 contributes to HG-induced podocyte injury via necroptosis. After exposing podocytes to HG for 24, 48, and 72 h, the morphology and viability of podocytes were respectively observed by a microscope and the cell counting kit-8 (CCK-8) assay. Reverse transcription polymerase chain reaction (RT-PCR) was exploited to detect the expression of USP22 in HG-treated podocytes with diverse transfections. Meanwhile, Western blotting was employed to determine the levels of USP22, key molecules of necroptosis (RIPK3 and MLKL), podocyte cell markers (Nephrin and Synaptopodin), and epithelial–mesenchymal transition (EMT)-related proteins (P-cadherin and N-cadherin). The expression of Nephrin was also detected using immunofluorescence. Co-immunoprecipitation (Co-IP) and ubiquitination assays were carried out to explore the mechanism underlying the role of USP22 in necroptosis. The impaired cell viability and the upregulation of USP22, RIPK3, and MLKL were observed in podocytes after HG stimulation. USP22 depletion could restrain HG-induced injury, necroptosis, and EMT in podocytes. Necroptosis inhibitor (necrostatin-1) reversed the decreased cell viability, injury, and EMT induced by USP22 overexpression in HG-treated podocytes. Co-IP assay demonstrated an interaction between USP22 and RIPK3. Furthermore, silencing of USP22 increased the ubiquitination level of RIPK3 in podocytes. Our findings revealed that USP22 inhibition attenuated HG-induced podocyte injury, necroptosis, and EMT by increasing the ubiquitination level of RIPK3.

Owing to the high incidence of oral squamous cell carcinoma (OSCC), elucidation of its diagnostic and prognostic factors is of great importance. Spindle pole body component 25 (SPC25) is known to play vital roles in different types of cancer. Nevertheless, the role of SPC25 in OSCC has not been elucidated yet. This study aimed to investigate the potential role of SPC25 in OSCC progression via modulating the transforming growth factor beta-1 (TGF-β1) signaling pathway. Data showed that SPC25 was highly expressed in oral cancer tumors and cells, which was associated with staging and poor survival of OSCC patients. Silencing of SPC25 was found to inhibit proliferation, colony formation, stemness, and epithelial-mesenchymal transition (EMT) of HSC-3 cells. Further analysis revealed that SPC25 overexpression promoted proliferation, colony formation, stemness, and EMT of OCEM-1 cells. In vivo analysis showed that silencing of SPC25 reduced tumor size, volume, and weight; it also suppressed the expression levels of Ki27, CDK2, vimentin, and TGF-β1. Our findings indicated that SPC25 is crucial for the progression of OSCC, and it modulates proliferation, stemness, and EMT of OSCC cells via regulating the TGF-β1 signaling pathway and its associated proteins. This study provided novel insights into the role of SPC25 in the pathogenesis of OSCC, and SPC25 could be a potential therapeutic target for the treatment of OSCC.