Biological Function and Potential Applications of Garcinol in Human Dental Pulp Stem Cells.

INTRODUCTION: The regeneration of pulp tissue is crucial for true regenerative endodontic treatment, which requires a reduction in osteogenic differentiation. Garcinol, a histone acetyltransferase inhibitor, is a natural regulator that is known to suppress the osteogenic differentiation of dental pulp stem cells. In this study, the inhibitory effect of garcinol on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) was evaluated using three-dimensional culture under in vitro and in vivo conditions. METHODS: hDPSCs were obtained from caries-free third molars and cultured with 10 µM garcinol for 7 days in an ultra-low attachment plate. The cell stemness and expression of osteogenic differentiation-related genes were analyzed using reverse transcription-polymerase chain reaction and single-cell analysis. A transplantation experiment was performed in mice to investigate whether garcinol-treated hDPSCs showed restrained osteogenic differentiation. RESULTS: hDPSCs cultured in the U-shaped ultra-low attachment plate showed the highest expression of stemness-related genes. Garcinol-treated hDPSCs demonstrated downregulation of osteogenic differentiation, with lower expression of bone sialoprotein, which is related to bone formation, and higher expression of dentin sialophosphoprotein, which is related to dentin formation. However, the garcinol-treated hDPSCs did not show any alterations in their stemness. Consistent results were observed in the transplantation experiment in mice. CONCLUSIONS: Garcinol reduced the osteogenic differentiation of hDPSCs, which can contribute to true regenerative endodontic treatment.

An Endodontic Forecasting Model Based on the Analysis of Preoperative Dental Radiographs: A Pilot Study on an Endodontic Predictive Deep Neural Network.

INTRODUCTION: This study aimed to evaluate the use of deep convolutional neural network (DCNN) algorithms to detect clinical features and predict the three-year outcome of endodontic treatment on preoperative periapical radiographs. METHODS: A database of single-root premolars that received endodontic treatment or retreatment by endodontists with presence of three-year outcome was prepared (n = 598). We constructed a 17-layered DCNN with a self-attention layer (Periapical Radiograph Explanatory System with Self-Attention Network [PRESSAN-17]), and the model was trained, validated, and tested to 1) detect 7 clinical features, that is, full coverage restoration, presence of proximal teeth, coronal defect, root rest, canal visibility, previous root filling, and periapical radiolucency and 2) predict the three-year endodontic prognosis by analyzing preoperative periapical radiographs as an input. During the prognostication test, a conventional DCNN without a self-attention layer (residual neural network [RESNET]-18) was tested for comparison. Accuracy and area under the receiver-operating-characteristic curve were mainly evaluated for performance comparison. Gradient-weighted class activation mapping was used to visualize weighted heatmaps. RESULTS: PRESSAN-17 detected full coverage restoration (area under the receiver-operating-characteristic curve = 0.975), presence of proximal teeth (0.866), coronal defect (0.672), root rest (0.989), previous root filling (0.879), and periapical radiolucency (0.690) significantly, compared to the no-information rate (P < .05). Comparing the mean accuracy of 5-fold validation of 2 models, PRESSAN-17 (67.0%) showed a significant difference to RESNET-18 (63.4%, P < .05). Also, the area under average receiver-operating-characteristic of PRESSAN-17 was 0.638, which was significantly different compared to the no-information rate. Gradient-weighted class activation mapping demonstrated that PRESSAN-17 correctly identified clinical features. CONCLUSIONS: Deep convolutional neural networks can detect several clinical features in periapical radiographs accurately. Based on our findings, well-developed artificial intelligence can support clinical decisions related to endodontic treatments in dentists.

Single-Cell RNA Sequencing Analysis of Human Dental Pulp Stem Cell and Human Periodontal Ligament Stem Cell.

INTRODUCTION: Many studies have investigated the transcriptome profiles of dental stem cells for regenerative medicine. However, such studies use bulk RNA and do not consider cell-level heterogeneity. Here, we investigated the characteristics and heterogeneity of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs) at the single-cell level and examined the differences between them. METHODS: hDPSCs and hPDLSCs were obtained from caries-free premolars (n = 2). Single-cell RNA sequencing of hDPSCs and hPDLSCs was performed using a Single Emulsion Systems (Dolomite Microfluidics, Royston, UK) droplet microfluidic device and the Illumina NextSeq550 system (Illumina, San Diego, CA). Data alignment was performed using STAR v2.4.0, and further analysis was performed using the Seurat, singleR, and clusterProfiler packages in R (R Foundation for Statistical Computing, Vienna, Austria). RESULTS: hDPSCs and hPDLSCs were separated into 3 clusters. hDPSCs mainly exhibited osteogenic and neurogenic cell populations. The main populations of hPDLSCs comprised osteogenic and myofibroblastic populations. hPDLSCs showed high "scores" for osteogenic gene expression, whereas hDPSCs had high neurogenic and endogenic scores. CONCLUSIONS: Single-cell RNA sequencing of hDPSCs and hPDLSCs revealed that the genes were expressed in specific clusters. The results of these analyses can be used as reference databases and valuable resources for further research in dental therapeutics.

The Effect of Mechanical Vibration on Osteogenesis of Periodontal Ligament Stem Cells.

INTRODUCTION: Appropriate occlusal forces can prevent ankylosis after tooth replantation or transplantation. However, the "proper occlusal forces" on periodontal ligament (PDL) healing have not yet been defined due to insufficient in vitro studies and uncertain in vitro models. Herein, we presented a mechanical vibration device as an in vitro model to determine such favorable occlusal forces. METHODS: Human periodontal ligament stem cells (hPDLSCs) were exposed to mechanical vibration force with 4 frequencies (30, 90, 150, and 210 rpm). Cell viability and the expression of osteogenic differentiation-related genes and proteins were tested in vitro. The calvarial transplantation experiment was performed to assess the bone formation ability of 150 rpm mechanical vibration stimulation (MVS). RESULTS: MVS at 150 and 210 rpm significantly reduced cell viability in the early stages. The 150-rpm MVS decreased osteogenic marker expression at the early time point (3 days) but had no harmful effects at the late time point (14 days). Furthermore, hPDLSC cell sheets treated with 150-rpm MVS had potential to decrease bone formation in rat calvarial defects serendipitously and facilitated functional PDL-like tissue formation. CONCLUSIONS: We found that MVS at a frequency of 150 rpm could provide a strategy for a transient reduction in the osteogenic potential of hPDLSCs and promote PDL-like tissue formation. Thus, 150-rpm MVS could be used as a controllable proper occlusal force to prevent ankylosis and promote PDL healing after tooth replantation or transplantation.

Comparison of Biocompatibility of Calcium Silicate-Based Sealers and Epoxy Resin-Based Sealer on Human Periodontal Ligament Stem Cells.

The aim of this study was to evaluate the biocompatibility of calcium silicate-based sealers (CeraSeal and EndoSeal TCS) and epoxy resin-based sealer (AH-Plus) in terms of cell viability, inflammatory response, expression of mesenchymal phenotype, osteogenic potential, cell attachment, and morphology, of human periodontal ligament stem cells (hPDLSCs). hPDLSCs were acquired from the premolars (n = 4) of four subjects, whose ages extended from 16 to 24 years of age. Flow cytometry analysis showed stemness of hPDLSCs was maintained in all materials. In cell viability test, AH-Plus showed the lowest cell viability, and CeraSeal showed significantly higher cell viability than others. In ELISA test, AH-Plus showed higher expression of IL-6 and IL-8 than calcium silicate-based sealers. In an osteogenic potential test, AH-Plus showed a lower expression level than other material; however, EndoSeal TCS showed a better expression level than others. All experiments were repeated at least three times per cell line. Scanning electronic microscopy studies showed low degree of cell proliferation on AH-Plus, and high degree of cell proliferation on calcium silicate-based sealers. In this study, calcium silicate-based sealers appear to be more biocompatible and less cytotoxic than epoxy-resin based sealers.

Differential Protein Expression in Human Dental Pulp: Comparison of Healthy, Inflamed, and Traumatic Pulp.

Trauma or injury to the dental pulp causes inflammation. This study compared the proteome of healthy pulp with inflamed pulp and traumatic pulp to identify the differentially expressed proteins in the diseased state. Five participants were grouped based on the pulpal status of the teeth: healthy, inflamed, or traumatic pulp. Pulp was extirpated and stored immediately in liquid nitrogen. Pulp tissues were subjected to 2-dimensional gel electrophoresis, and spot selection was performed. The selected spots were analyzed using liquid chromatography-tandem mass spectrometry and identified by correlating mass spectra to the proteomic databases. Fifteen spots showed increased expression in the inflamed and traumatic pulp. Annexin V, type II keratin, and hemoglobin levels were increased two-fold in the inflamed and traumatic pulp group and annexin V, mutant beta-actin, and hemoglobin were increased by ten-fold in the inflamed or traumatic pulp group, compared to levels in the healthy pulp group. Annexin V constituted two out of fifteen protein spots, and seemed to play a critical role in inhibiting inflammation and promoting the immune reaction. Further studies on this protein concerning its role in pulp repair are necessary to elucidate the underlying mechanisms.

In Vitro Comparison of Biocompatibility of Calcium Silicate-Based Root Canal Sealers.

The aim of this study was to assess the effect of three calcium silicate-based sealers (EndoSeal MTA, Nano-ceramic Sealer, and Wellroot ST) and two epoxy resin-based sealers (AH-Plus, AD Seal) on various aspects, such as cell viability, inflammatory response, and osteogenic potential, of human periodontal ligament stem cells (hPDLSCs). AH-Plus showed the lowest cell viability on hPDLSCs in all time periods in fresh media. In set media, hPDLSCs showed no significant differences in cell viability among all the tested materials. Wellroot ST showed the highest level of cell adhesion and the morphology of attached cells. AH-plus presented a significantly higher expression of IL-6 and IL-8 than the other sealers. AD Seal and three calcium silicate sealers showed high expression of the mesenchymal stem cell markers. ALP mRNA expression showed a significant increase in time-dependent manner on all of three calcium silicate-based sealers, which do not seem to interfere with the differentiation of hPDLSCs into osteoblasts. Based on the results from this study, calcium silicate-based sealers appear to be more biocompatible and less cytotoxic than epoxy resin-based sealers. Meanwhile, further and long-term clinical follow-up studies are required.

Development of a mouse model for pulp-dentin complex regeneration research: a preliminary study.

OBJECTIVES: To achieve pulp-dentin complex regeneration with tissue engineering, treatment efficacies and safeties should be evaluated using in vivo orthotopic transplantation in a sufficient number of animals. Mice have been a species of choice in which to study stem cell biology in mammals. However, most pulp-dentin complex regeneration studies have used large animals because the mouse tooth is too small. The purpose of this study was to demonstrate the utility of the mouse tooth as a transplantation model for pulp-dentin complex regeneration research. MATERIALS AND METHODS: Experiments were performed using 7-week-old male Institute of Cancer Research (ICR) mice; a total of 35 mice had their pulp exposed, and 5 mice each were sacrificed at 1, 2, 4, 7, 9, 12 and 14 days after pulp exposure. After decalcification in 5% ethylenediaminetetraacetic acid, the samples were embedded and cut with a microtome and then stained with hematoxylin and eosin. Slides were observed under a high-magnification light microscope. RESULTS: Until 1 week postoperatively, the tissue below the pulp chamber orifice appeared normal. The remaining coronal portion of the pulp tissue was inflammatory and necrotic. After 1 week postoperatively, inflammation and necrosis were apparent in the root canals inferior to the orifices. The specimens obtained after experimental day 14 showed necrosis of all tissue in the root canals. CONCLUSIONS: This study could provide opportunities for researchers performing in vivo orthotopic transplantation experiments with mice.

Effects of Different Calcium Silicate Cements on the Inflammatory Response and Odontogenic Differentiation of Lipopolysaccharide-Stimulated Human Dental Pulp Stem Cells.

This study aimed to analyze the effects of different calcium silicate cements (CSCs) on the inflammatory response and odontogenic differentiation of lipopolysaccharide-stimulated human dental pulp stem cells. Human dental pulp stem cells (hDPSCs) were stimulated with lipopolysaccharide (LPS) to induce inflammation. These LPS-induced dental pulp stem cells (LDPSCs) were cultured with ProRoot MTA, Biodentine, Retro MTA, and Dycal. Cell viability was evaluated using the Cell Counting Kit-8 assay. Interleukin (IL)-6, IL-8, and transforming growth factor (TGF)-ß1 cytokine levels were assessed using the enzyme-linked immunosorbent assay. The expressions of alkaline phosphatase (ALP), osteocalcin, and runt-related transcription factor 2 (RUNX2) were analyzed through real-time polymerase chain reaction. ProRoot MTA, Biodentine, and Retro MTA did not significantly decrease the cell viability of LDPSCs for up to 48 h (p < 0.05). Retro MTA significantly decreased the expression of IL-6 and IL-8 by LDPSCs. ProRoot MTA and Biodentine significantly reduced TGF-ß expression by LDPSCs (p < 0.05). Regarding odontogenic differentiation, all CSCs had no effect on ALP expression but increased the production of RUNX2 at 12 h.

Evaluation of the Biodistribution of Human Dental Pulp Stem Cells Transplanted into Mice.

INTRODUCTION: Several studies have attempted to use human dental pulp stem cells (hDPSCs) for pulp-dentin complex regeneration in vitro. However, the safety of such applications should be first evaluated in vivo before their use in clinical trials. The purpose of this study was to investigate the in vivo fate of intrapulpally transplanted hDPSCs. METHODS: hDPSCs were isolated and cultured from impacted third molars. In vivo experiments were performed using 7-week-old male BALB/c nude mice. Under deep anesthesia, 1 × 105 hDPSCs were transplanted in mice via the tail vein for intravenous injection or into the pulp chamber for intrapulpal transplantation. A total of 56 mice, 28 per group, were used. Mice were sacrificed at different time points, and the numbers of hDPSCs in the organs were analyzed quantitatively. In addition, qualitative analysis was performed to detect intrapulpally transplanted hDPSCs. RESULTS: Intravenously injected hDPSCs were mostly distributed to the lungs and rarely detected in other organs at all observed time points. The hDPSCs transplanted into the pulp chamber rarely migrated to other organs over time. CONCLUSIONS: These data indicate a differential distribution of transplanted hDPSCs between the intravenous and intrapulpal route and show the safety of pulpal transplantation of hDPSCs.