miR-141-3p Targeted SIRT1 to Inhibit Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Purpose: To explore the expression of miR-141-3p during the osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) and its regulatory effect. Methods: Differentiation of BMSCs was induced by dexamethasone. The mRNA expression of miR-141-3p, ALP, RUNX2, and OCN was measured using RT-qPCR. The protein expression was detected via western blot. The target of miR-141-3p was predicted through the TargetScan website and confirmed using luciferase reporter assay. Results: miR-141-3p expression declined during osteogenic differentiation. The relative ALP activities and the mRNA expression of ALP, RUNX2, and OCN were markedly reduced in the miR-141-3p mimic group while increased in the inhibitor group. Cell viability was suppressed in the miR-141-3p mimic group and promoted in the inhibitor group. SIRT1 was predicted to be a downstream gene of miR-141-3p, and this prediction was confirmed via the luciferase reporter assay. The results of the western blot assay demonstrated that SIRT1 expression was decreased in the miR-141-3p mimic group. SIRT1 reversed the inhibitory influence of miR-141-3p on the osteogenic differentiation ability of BMSCs. Conclusion: miR-141-3p targeted SIRT1 to inhibit osteogenic differentiation of BMSCs via the Wnt/ß-catenin signaling pathway.

METTL3 potentiates osteogenic differentiation of bone marrow mesenchymal stem cells via IGF2BP1/m6A/RUNX2.

OBJECTIVE: Maxillofacial bone defect is a critical obstacle for maxillofacial tumors and periodontal diseases. The osteogenic differentiation of bone marrow mesenchymal stem cells BMSCs is critical for maxillofacial osteogenesis and functional reconstruction. Here, our study focused on the functions and mechanism of N6 -methyladenosine during BMSCs osteogenic differentiation BMSCs. SUBJECT AND METHODS: Biofunctions of BMSCs were detected using ALP activity and alizarin red S staining assays. The molecular interaction within RNA/protein was identified by RNA immunoprecipitation and/or methylation immunoprecipitation. RESULTS: Results indicated that m6 A 'writer' METTL3 upregulated during the osteogenic differentiation of BMSCs upon osteogenic induction. Functionally, assays' results revealed that METTL3 overexpression promoted the osteogenic differentiation of BMSC, while METTL3 knockdown repressed the osteogenic differentiation. Mechanistically, results revealed that RUNX2 mRNA was a m6 A-methylated target by METTL3 at its 3'-UTR. Moreover, m6 A reader IGF2BP1 recognized the m6 A site on RUNX2 mRNA to enhance its stability. CONCLUSION: In conclusion, our findings revealed the novel roles of METTL3 in BMSCs osteogenic differentiation via the IGF2BP1/m6 A/RUNX2 signaling axis of m6 A-dependent manner, providing a potential therapeutic target for maxillofacial bone defects treatment.

D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes

Glucose uptake is stimulated by insulin via stimulation of glucose transporter 4 (GLUT4) translocation to the plasma membrane from intracellular compartments in adipose tissue and muscles. Insulin stimulation for prolonged periods depletes GLUT4 protein, particularly in highly insulin-responsive GLUT4 storage vesicles. This depletion mainly occurs via H2O2-mediated retromer inhibition. However, the post-receptor mechanism of insulin activation of oxidative stress remains unknown. Here, we show that phosphatidylcholine-specific phospholipase C (PC-PLC) plays an important role in insulin-mediated downregulation of GLUT4. In the study, 3T3-L1 adipocytes were exposed to a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609), for 30 min prior to the stimulation with 500 nM insulin for 4 h, weakening the depletion of GLUT4. D609 also prevents insulin-driven H2O2 generation in 3T3-L1 adipocytes. Exogenous PC-PLC and its product, phosphocholine (PCho), also caused GLUT4 depletion and promoted H2O2 generation in 3T3-L1 adipocytes. Furthermore, insulin-mediated the increase in the cellular membrane PC-PLC activity was observed in Amplex Red assays. These results suggested that PC-PLC plays an important role in insulin-mediated downregulation of GLUT4 and that PCho may serve as a signaling molecule. (AU)

Preparation of BMP-2/PDA-BCP Bioceramic Scaffold by DLP 3D Printing and its Ability for Inducing Continuous Bone Formation.

Digital light processing (DLP)-based 3D printing is suitable to fabricate bone scaffolds with small size and high precision. However, the published literature mainly deals with the fabrication procedure and parameters of DLP printed bioceramic scaffold, but lacks the subsequent systematic biological evaluations for bone regeneration application. In this work, a biphasic calcium phosphate (BCP) macroporous scaffold was constructed by DLP-based 3D printing technique. Furthermore, bone morphogenetic protein-2 (BMP-2) was facilely incorporated into this scaffold through a facile polydopamine (PDA) modification process. The resultant scaffold presents an interconnected porous structure with pore size of ∼570 µm, compressive strength (∼3.6 MPa), and the self-assembly Ca-P/PDA nanocoating exhibited excellent sustained-release property for BMP-2. Notably, this BMP-2/PDA-BCP scaffold presents favorable effects on the adhesion, proliferation, osteogenic differentiation, and mineralization of bone marrow stromal cells (BMSCs). Furthermore, in vivo experiments conducted on rats demonstrated that the scaffolds could induce cell layer aggregation adjacent to the scaffolds and continuous new bone generation within the scaffold. Collectively, this work demonstrated that the BMP-2/PDA-BCP scaffold is of immense potential to treat small craniofacial bone defects in demand of high accuracy.

D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes.

Glucose uptake is stimulated by insulin via stimulation of glucose transporter 4 (GLUT4) translocation to the plasma membrane from intracellular compartments in adipose tissue and muscles. Insulin stimulation for prolonged periods depletes GLUT4 protein, particularly in highly insulin-responsive GLUT4 storage vesicles. This depletion mainly occurs via H2O2-mediated retromer inhibition. However, the post-receptor mechanism of insulin activation of oxidative stress remains unknown. Here, we show that phosphatidylcholine-specific phospholipase C (PC-PLC) plays an important role in insulin-mediated downregulation of GLUT4. In the study, 3T3-L1 adipocytes were exposed to a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609), for 30 min prior to the stimulation with 500 nM insulin for 4 h, weakening the depletion of GLUT4. D609 also prevents insulin-driven H2O2 generation in 3T3-L1 adipocytes. Exogenous PC-PLC and its product, phosphocholine (PCho), also caused GLUT4 depletion and promoted H2O2 generation in 3T3-L1 adipocytes. Furthermore, insulin-mediated the increase in the cellular membrane PC-PLC activity was observed in Amplex Red assays. These results suggested that PC-PLC plays an important role in insulin-mediated downregulation of GLUT4 and that PCho may serve as a signaling molecule.

TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis.

TRK-fused gene (TFG) is known to be involved in protein secretion and plays essential roles in an antiviral innate immune response. However, its function in LPS-induced inflammation and pyroptotic cell death is still unknown. Here, we reported that TFG promotes the stabilization of Unc-51 like autophagy activating kinase (ULK1) and participates in LPS plus nigericin (Ng) induced pyroptotic cell death. Our results showed that TFG-deficient THP-1 macrophages exhibit higher mitochondrial ROS production. LPS/Ng stimulation triggers a much higher level of ROS and induces pyroptotic cell death. ULK1 undergoes a rapid turnover in TFG-deficient THP-1 cells. TFG forms complex with an E3 ligase, tumor necrosis factor receptor-associated factor 3 (TRAF3), and stabilizes ULK1 via disturbing ULK1-TRAF3 interaction. Knockdown of TFG facilitates the interaction of ULK1 with TRAF3 and subsequent K48-linked ULK1 ubiquitination and proteasome degradation. Rescue of ULK1 expression blocks LPS/Ng-induced cell death in TFG-deficient THP-1 macrophages. Taken together, TFG plays an essential role in LPS/Ng-induced pyroptotic cell death via regulating K48-linked ULK1 ubiquitination in macrophages.

Numerical simulation and analysis of midfacial impacts and traumatic brain injuries.

BACKGROUNDS: The maxillofacial region is the exposed part of the human body and is susceptible to injury due to the limited protective equipment. Due to anatomic proximity of the maxillofacial skeleton and cranium, the force can be transmitted directly to the brain in case of maxillofacial impact, maxillofacial injuries are often accompanied with craniocerebral trauma. Therefore, it is necessary to study the biomechanical response mechanism of trauma to improve prevention of traumatic brain injury (TBI). METHODS: To investigate the biomechanical mechanism between the two injuries, a finite element (FE) head model including skull, midfacial bones and detailed anatomical intracranial features was successfully developed based on CT/MRI data. The model was validated by comparing it with one classical cadaver experiment. During the simulations, three different load forces were used to simulate common causes of injury seen in the clinic including boxing-type impact injury and car accident-type impact injury, and four locations on the model were considered as common injury sites in the midface. RESULTS: Twelve common impact scenarios were reproduced by FE simulation successfully. Simulations showed that there was a linear relationship between the severity of TBI and the collision energy. The location of TBI was directly related to the location of the impact site, and a lateral impact was more injurious to the brain than an anterior-posterior impact. The relative movement between the skull and brain could cause physical damage to the brain. The study indicated that the midfacial bones acted as a structure capable of absorbing energy and protecting the brain from impact. CONCLUSIONS: This biomechanical information may assist surgeons better understand and diagnose brain injuries accompanied by midfacial fractures.

Helmet chinstrap protective role in maxillofacial blast injury.

BACKGROUND: The protective role of helmet accessories in moderating stress load generated by explosion shock waves of explosive devices is usually neglected. OBJECTIVE: In the presented study, the protective role of the helmet chinstrap against the impulse and overpressure experienced by the maxillofacial region were examined. METHODS: The explosion shock wave and skull interaction were investigated under three different configurations: (1) unprotected skull, (2) skull with helmet (3) skull with helmet and chinstrap. For this purpose, a 3D finite element model (FEM) was constructed to mimic the investigated biomechanics module. Three working conditions were set according to different explosive charges and distances to represent different load conditions. Case 1: 500 mg explosive trinitrotoluene (TNT), 3 cm, case 2: 1000 mg TNT, 3 cm, and case 3: 1000 mg TNT and 6 cm distance to the studied object. The explosion effect was discussed by examining the shock wave stress flow pattern. Three points were selected on the skull and the stress curve of each point position were illustrated for each case study. RESULTS: The results showed that the helmet chinstrap can reduce the explosive injuries and plays a protective role in the maxillofacial region, especially for the mandible.

Associations between osteoporosis and risk of periodontitis: A pooled analysis of observational studies.

BACKGROUND: Periodontitis and osteoporosis are most popular among aging population and both conditions might be linked, even though, this suggestion still until now debated. OBJECTIVES: A meta-analysis on previous investigations has been used to evaluate the correlation between periodontitis and osteoporosis to determine whether osteoporosis is a local indicator of bone loss, or whether it is depending on or related to periodontitis causes. METHODS: The literature database, including but not excluding, The Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, CINAHL, and Science Citation Index Expanded, was searched in this work during Feb, 2020. We conducted the investigations contain cohort studies, cross-sectional studies, as well as case-control studies with relative risk (RR) or odds ratio (OR) and 95% confidence intervals (CIs). Subgroup and Sensitivity analysis were also applied to identify heterogeneity sources. RESULTS: 23 observational studies with 12 cohorts, 7 cross-sectional and 4 case-control studies, were included, together with 2,157,037 participants. Osteoporosis patients were more exposed to periodontitis (OR, 1.96; 95% CI, 1.50-2.54). Subgroup analyses showed that the higher risk of osteoporosis in periodontitis patients exists in both cross-sectional studies (OR, 2.17; 95% CI, 1.80-2.61) and case-control studies (OR 2.63; 95% CI, 1.69-4.09), and marginally in cohort studies (OR, 1.70; 95% CI, 1.16-2.49). CONCLUSION: Review analyses have shown that osteoporosis is closely related to the increased risk of periodontitis in the future. Dental specialists better to understand the potential association between periodontitis and osteoporosis.

Spherical-Patches Extraction for Deep-Learning-Based Critical Points Detection in 3D Neuron Microscopy Images.

Digital reconstruction of neuronal structures is very important to neuroscience research. Many existing reconstruction algorithms require a set of good seed points. 3D neuron critical points, including terminations, branch points and cross-over points, are good candidates for such seed points. However, a method that can simultaneously detect all types of critical points has barely been explored. In this work, we present a method to simultaneously detect all 3 types of 3D critical points in neuron microscopy images, based on a spherical-patches extraction (SPE) method and a 2D multi-stream convolutional neural network (CNN). SPE uses a set of concentric spherical surfaces centered at a given critical point candidate to extract intensity distribution features around the point. Then, a group of 2D spherical patches is generated by projecting the surfaces into 2D rectangular image patches according to the orders of the azimuth and the polar angles. Finally, a 2D multi-stream CNN, in which each stream receives one spherical patch as input, is designed to learn the intensity distribution features from those spherical patches and classify the given critical point candidate into one of four classes: termination, branch point, cross-over point or non-critical point. Experimental results confirm that the proposed method outperforms other state-of-the-art critical points detection methods. The critical points based neuron reconstruction results demonstrate the potential of the detected neuron critical points to be good seed points for neuron reconstruction. Additionally, we have established a public dataset dedicated for neuron critical points detection, which has been released along with this article.

Neuron Image Segmentation via Learning Deep Features and Enhancing Weak Neuronal Structures.

Neuron morphology reconstruction (tracing) in 3D volumetric images is critical for neuronal research. However, most existing neuron tracing methods are not applicable in challenging datasets where the neuron images are contaminated by noises or containing weak filament signals. In this paper, we present a two-stage 3D neuron segmentation approach via learning deep features and enhancing weak neuronal structures, to reduce the impact of image noise in the data and enhance the weak-signal neuronal structures. In the first stage, we train a voxel-wise multi-level fully convolutional network (FCN), which specializes in learning deep features, to obtain the 3D neuron image segmentation maps in an end-to-end manner. In the second stage, a ray-shooting model is employed to detect the discontinued segments in segmentation results of the first-stage, and the local neuron diameter of the broken point is estimated and direction of the filamentary fragment is detected by rayburst sampling algorithm. Then, a Hessian-repair model is built to repair the broken structures, by enhancing weak neuronal structures in a fibrous structure determined by the estimated local neuron diameter and the filamentary fragment direction. Experimental results demonstrate that our proposed segmentation approach achieves better segmentation performance than other state-of-the-art methods for 3D neuron segmentation. Compared with the neuron reconstruction results on the segmented images produced by other segmentation methods, the proposed approach gains 47.83% and 34.83% improvement in the average distance scores. The average Precision and Recall rates of the branch point detection with our proposed method are 38.74% and 22.53% higher than the detection results without segmentation.

NNK-mediated upregulation of DEPDC1 stimulates the progression of oral squamous cell carcinoma by inhibiting CYP27B1 expression.

Oral squamous cell carcinoma (OSCC) is a prevalent and malignant cancer. However, the molecular mechanism of OSCC progression is not fully understood. In this study, we observed that the DEP domain containing 1 (DEPDC1) protein was overexpressed in OSCC tissues and that the increased expression of DEPDC1 was closely associated with tumor size and poor clinical outcomes in OSCC patients. The results of functional investigations demonstrated that DEPDC1 stimulates OSCC cell proliferation by inhibiting cytochrome P450 family 27 subfamily B member (CYP27B1) expression. Furthermore, we observed that upregulated DEPDC1 expression was closely associated with smoking status in OSCC patients. The results of in vitro experiments showed that the tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) stimulates DEPDC1 expression by promoting the methylation of its gene body by increasing DNMT1 expression in OSCC cells. Notably, the silencing of DEPDC1 dramatically inhibited OSCC growth by inhibiting cell proliferation and inducing apoptosis in vivo. These findings suggest that smoking causes DEPDC1 overexpression in OSCC through DNMT1-regulated DNA methylation and that upregulated DEPDC1 stimulates OSCC cell proliferation by inhibiting CYP27B1 expression. Our results establish a new mechanism of OSCC progression and highlight DEPDC1 as a candidate prognostic biomarker and therapeutic target in OSCC.

RCOR1 directly binds to MED28 and weakens its inducing effect on cancer stem cell-like activity of oral cavity squamous cell carcinoma cells.

BACKGROUND: Mediator is a multiprotein complex that acts as an essential transcriptional coactivator in eukaryotic cells for successful transcription. In this study, we aimed to explore the expression profile of 33 mediator subunit genes in oral cavity squamous cell carcinoma (OCSCC) and the functional role of MED28 in cellular behaviors of OCSCC cells. METHODS: Single-cell (sc)RNA-seq data from OCSCC cells (Puram 2017's dataset) and bulk-seq data of the OCSCC subgroup of TCGA-head and neck squamous cell carcinoma (HNSC) were used for bioinformatic analysis. SCC9 cells were used in in-vitro and in-vivo analysis. RESULTS: Among the 33 genes subjected to screening, MED28 showed the best prognostic value and its upregulation might independently predict shorter OS (HR: 3.699, 95% CI: 1.383-9.892, P = .009) and PFS (HR: 2.769, 95% CI: 1.462-5.244, P = .002). MED28 expression was positively correlated with cancer stem cell (CSC)-like properties of SCC9 cells, including colony/sphere formation, and the expression of CSC markers (CD44, KLF4, NANOG, and OCT4). RCOR1 could suppress the CSC-like properties of SCC9 cells and had direct interaction with MED28. Its overexpression partly abrogated MED28-induced expression of CSC markers. RCOR1 expression was associated with promoter hypermethylation, while MED28 expression was positively correlated with its MED28 copy number (Pearson's r = .44) in OCSCC tissues. CONCLUSION: Among the mediator complex subunits, MED28 might serve as a potential biomarker of unfavorable survival. Its overexpression increased CSC-like activity of OCSCC cells, the effect of which could be abrogated by RCOR1 via direct interaction.

Effect of Two Dental Restorative Materials on Adhesion and Molecular Structure of Oral Bacteria.

Dental restorative materials are widely used to repair teeth and dentition defects. However, the dental restorative materials tend to react with oral bacteria when they are exposed to oral conditions, which leads to a change in the oral microecology. Herein, we have employed molecular dynamics simulations to investigate the interaction between different dental restorative materials and oral bacteria. It was found that the staphylococcal protein A (SPA) is more likely to attach on the surface of silicon carbide (SiC) substrate than hematite (Fe2O3) substrate surface. Furthermore, the tightly adhesion and accumulation of SPA on SiC surface changes the molecular structure of SPA, which will induce a change in the oral microecology. This study has demonstrated that the adhesion and molecular structure of oral bacteria is strongly dependent on dental restorative materials by molecular dynamics simulations, and Fe2O3 is more suitable to be a dental restorative material. It is therefore believed that molecular dynamics simulations can be used to further screen suitable materials for oral rehabilitation.

Transcriptome analysis of human periodontal ligament fibroblasts exposed to Porphyromonas gingivalis LPS.

OBJECTIVE: Porphyromonas gingivalis (P. gingivalis)-derived LPS is a major mediator of inflammation and can promote the resorption of alveolar bone in chronic periodontitis. Although the effect of P. gingivalis LPS on human periodontal ligament fibroblasts (hPDLFs) was already investigated by numerous studies, the change of whole transcriptional profile remains undefined. The aim of this study was to investigate P. gingivalis LPS induced whole transcriptional profile in hPDLFs and the expression of the genes associated with the periodontitis. MATERIALS AND METHODS: RNA-seq was performed on hPDLFs treated with or without P. gingivalis LPS. Moreover, the expression of selected inflammatory cytokines, chemokines and matrix metalloproteinases (MMPs), which contribute to periodontitis, were evaluated by quantitative RT-PCR and further measured by ELISA. RESULTS: We found that an average of 12,752 genes were detected among the different groups, and 1374 differentially expressed genes (DEGs) were identified between groups with or without P. gingivalis LPS stimulation for 24 h. However, only 36 DEGs were examined in hPDLFs exposed to P. gingivalis LPS for 24 h or 72 h. Furthermore, the mRNA levels and concentrations of interleukin 8 (IL-8), IL-6, monocyte chemotactic protein 1 (MCP-1), chemokine (CXC motif) ligand 5 (CXCL5), MMP1 and MMP3 were significantly higher in hPDLFs exposed to P. gingivalis LPS for 24 h compared to the untreated hPDLFs. CONCLUSIONS: The entire transcriptional profile of P. gingivalis LPS stimulation of hPDLFs was presented for the first time, which could provide an important basis and experimental direction for further research into the mechanisms of periodontitis.

DeepBranch: Deep Neural Networks for Branch Point Detection in Biomedical Images.

Morphology reconstruction of tree-like structures in volumetric images, such as neurons, retinal blood vessels, and bronchi, is of fundamental interest for biomedical research. 3D branch points play an important role in many reconstruction applications, especially for graph-based or seed-based reconstruction methods and can help to visualize the morphology structures. There are a few hand-crafted models proposed to detect the branch points. However, they are highly dependent on the empirical setting of the parameters for different images. In this paper, we propose a DeepBranch model for branch point detection with two-level designed convolutional networks, a candidate region segmenter and a false positive reducer. On the first level, an improved 3D U-Net model with anisotropic convolution kernels is employed to detect initial candidates. Compared with the traditional sliding window strategy, the improved 3D U-Net can avoid massive redundant computations and dramatically speed up the detection process by employing dense-inference with fully convolutional neural networks (FCN). On the second level, a method based on multi-scale multi-view convolutional neural networks (MSMV-Net) is proposed for false positive reduction by feeding multi-scale views of 3D volumes into multiple streams of 2D convolution neural networks (CNNs), which can take full advantage of spatial contextual information as well as fit different sizes. Experiments on multiple 3D biomedical images of neurons, retinal blood vessels and bronchi confirm that the proposed 3D branch point detection method outperforms other state-of-the-art detection methods, and is helpful for graph-based or seed-based reconstruction methods.

Associations Between Poor Oral Health and Risk of Squamous Cell Carcinoma of the Head and Neck: A Meta-Analysis of Observational Studies.

PURPOSE: Many epidemiologic studies have reported an association of poor oral health, especially periodontal disease (PD) and tooth loss, with the risk of squamous cell carcinoma of the head and neck (SCCHN). However, these studies have yielded inconsistent results. Therefore, the present study investigated whether poor oral health is an independent predictor of SCCHN through a meta-analysis of observational studies. MATERIALS AND METHODS: The PubMed, EMBASE, and Cochrane Library databases were systematically searched for relevant observational studies of the association between oral health and risk of SCCHN conducted up to October 2017. The meta-analysis was conducted using STATA 12.0 (StataCorp, College Station, TX). A fixed- or random-effects model was applied to evaluate pooled risk estimates, and sensitivity and subgroup analyses were performed to identify sources of heterogeneity and pooled estimation. Publication bias was assessed using the Begg test, the Egger test, and funnel plots. RESULTS: Twenty-seven relevant observational studies were identified, consisting of 24 case-and-control studies, 2 prospective studies, and 1 cross-sectional study, with 26,750 participants. Notably, oral health correlated meaningfully with SCCHN (odds ratio [OR] = 2.24; 95% confidence interval [CI], 1.77-2.82). In subgroup analyses, participants with PD (OR = 2.52; 95% CI, 1.43-4.44) had a higher risk of developing SCCHN than those with tooth loss (OR = 2.13; 95% CI, 1.63-2.78). The risk estimates exhibited substantial heterogeneity. Evidence of publication bias was limited. CONCLUSIONS: The results of this meta-analysis suggest that patients with tooth loss or PD might face a substantial and independent risk of SCCHN, even after adjusting for smoking and alcohol consumption. However, the pooled estimates from observational studies could not establish a causative relation among PD, tooth loss, and SCCHN. Additional investigations of this correlation are warranted.

[Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells].

OBJECTIVE: This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells. METHODS: iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferation was detected by CCK8, and the expression levels of ameloblastin (AMBN), cytokeratin (CK) 14, dentin sialophospho-protein (DSPP), bone sialoprotein (BSP), and Runx2 mRNA were detected by quantitative polymerase chain reaction. Alkaline phosphatase (ALP) activity was used to detect the degree of cell differentiation. RESULTS: Compared with blank and empty-vector groups, proliferation activity and ALP activity of BMP4-overexpressing group obvious increased (P<0.05), BMP4, AMBN, CK14, DSPP, BSP, Runx2 mRNA expression also increased (P<0.05). CONCLUSIONS: BMP4 can significantly promote the odontogenic differentiation of iPS.

Calcitonin gene­related peptide reduces Porphyromonas gingivalis LPS­induced TNF­α release and apoptosis in osteoblasts.

Periodontal diseases comprise mixed bacterial infections mainly caused by Gram­negative anaerobic bacteria. Lipopolysaccharides (LPS) are important virulence factors and periodontal pathogens, which change local cytokine levels and promote osteoblast apoptosis, thereby leading to an imbalance in bone remodeling mechanisms and accelerating bone loss. Calcitonin gene­related peptide (CGRP) is a vasoactive neuropeptide that is released from sensory nerves and has a positive effect on osteoblast proliferation and differentiation. In addition, this small molecule peptide is an important immune regulator in the inflammatory response. The aim of the present study was to assess the in vitro effects of CGRP on Porphyromonas gingivalis (Pg)LPS­induced osteoblast apoptosis. Osteoblast cultures were stimulated either with various concentrations of PgLPS (0, 25, 50, 100, 500 and 1,000 ng/ml) for 48 h or with 500 ng/ml PgLPS for various lengths of time (0, 6, 12, 24, 48 and 72 h). The PgLPS­stimulated cells were pretreated with different concentrations of CGRP (0, 1, 10, 100 and 1,000 nM) and cell viability and apoptotic rates were measured by Cell Counting kit­8 assays and flow cytometry, respectively. CGRP, cleaved (c)­Caspase­8 and c­Caspase­3 protein expression levels were analyzed by western blotting. Changes in cytokine expression levels, which included tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, IL­6, monocyte chemotactic protein (MCP)­1 and MCP­2, were measured by ELISA. PgLPS was demonstrated to inhibit osteoblast viability and promote apoptosis in a time­ and concentration­dependent manner. CGRP expression was revealed to reduce PgLPS­induced cytostatic activity and apoptosis in osteoblasts. CGRP also suppressed the PgLPS­induced release of TNF­α and inhibited the activation of c­Caspase­3 and c­Caspase­8, thus preventing apoptosis in osteoblasts. CGRP may be an important neuropeptide in bone remodeling and may reduce osteoblast apoptosis in inflammatory conditions. These results may provide a solid foundation for CGRP to serve as a new target for the treatment of periodontitis.