The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly ...understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.
The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation ...of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca2+ channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K–Akt signaling. Panx3 also formed gap junctions and propagated Ca2+ waves between cells. Blocking the Panx3 Ca2+ channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca2+ channel and a hemichannel, and by forming gap junctions.
Recently, it was suggested that the nitrite (NO
) produced from NO
by oral bacteria might contribute to oral and general health. Therefore, we aimed to clarify the detailed information about the ...bacterial NO
-production in the oral biofilm. Dental plaque and tongue-coating samples were collected, then the NO
-producing activity was measured. Furthermore, the composition of the NO
-producing bacterial population were identified using the Griess reagent-containing agar overlay method and molecular biological method. NO
-producing activity per mg wet weight varied among individuals but was higher in dental plaque. Additionally, anaerobic bacteria exhibited higher numbers of NO
-producing bacteria, except in the adults' dental plaque. The proportion of NO
-producing bacteria also varied among individuals, but a positive correlation was found between NO
-producing activity and the number of NO
-producing bacteria, especially in dental plaque. Overall, the major NO
-producing bacteria were identified as Actinomyces, Schaalia, Veillonella and Neisseria. Furthermore, Rothia was specifically detected in the tongue coatings of children. These results suggest that dental plaque has higher NO
-producing activity and that this activity depends not on the presence of specific bacteria or the bacterial compositions, but on the number of NO
-producing bacteria, although interindividual differences were detected.
Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to ...differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting β-catenin degradation through GSK3β activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca2+ channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways.
Background: The mechanism of the transition from osteoprogenitor cell proliferation to differentiation is unclear.
Results: Panx3 inhibits osteoprogenitor proliferation by blocking canonical Wnt signaling and promoting p21 activation.
Conclusion: A Panx3 hemichannel induces multiple Panx3 signaling pathways critical for the cell cycle exit.
Significance: Our findings reveal that Panx3 is a new regulator to switch the stage from proliferation to differentiation in osteoprogenitor cells.
Objectives
To investigate the detailed ultrastructural patterns of dental abnormalities affected by Axenfeld‐Rieger syndrome (ARS) with a heterozygous microdeletion involving paired‐like homeodomain ...2 (PITX2) and explored the underlying molecular mechanisms driving enamel defects.
Subjects and methods
Sanger sequencing, genomic quantitative PCR analysis, and chromosomal microarray analysis (CMA) were used to screen the disease‐causing mutation in one ARS proband. An exfoliated tooth from an ARS patient was analyzed with scanning electron microscopy and micro–computerized tomography. A stable Pitx2 knockdown cell line was generated to simulate PITX2 haploinsufficiency. Cell proliferation and ameloblast differentiation were analyzed, and the role of the Wnt/β‐catenin pathway in proliferation of ameloblast precursor cells was investigated.
Results
An approximately 0.216 Mb novel deletion encompassing PITX2 was identified. The affected tooth displayed a thinner and broken layer of enamel and abnormal enamel biomineralization. PITX2 downregulation inhibited the proliferation and differentiation of inner enamel epithelial cells, and LiCl stifmulation partially reversed the proliferation ability after Pitx2 knockdown.
Conclusions
Enamel formation is disturbed in some patients with ARS. Pitx2 knockdown can influence the proliferation and ameloblast differentiation of inner enamel epithelial cells, and PITX2 may regulate cell proliferation via Wnt/β‐catenin signaling pathway.
A subpopulation of mesenchymal stem cells, developmentally derived from multipotent neural crest cells that form multiple facial tissues, resides within the dental pulp of human teeth. These stem ...cells show high proliferative capacity in vitro and are multipotent, including adipogenic, myogenic, osteogenic, chondrogenic, and neurogenic potential. Teeth containing viable cells are harvested via minimally invasive procedures, based on various clinical diagnoses, but then usually discarded as medical waste, indicating the relatively low ethical considerations to reuse these cells for medical applications. Previous studies have demonstrated that stem cells derived from healthy subjects are an excellent source for cell-based medicine, tissue regeneration, and bioengineering. Furthermore, stem cells donated by patients affected by genetic disorders can serve as in vitro models of disease-specific genetic variants, indicating additional applications of these stem cells with high plasticity. This review discusses the benefits, limitations, and perspectives of patient-derived dental pulp stem cells as alternatives that may complement other excellent, yet incomplete stem cell models, such as induced pluripotent stem cells, together with our recent data.
Physical failure analysis (PFA) specifies layout designs that affect large-scale integration (LSI) failure. Because of their capability and cost-effectiveness, convolutional neural networks (CNNs) ...have been applied to LSI layout analysis. However, the information on failure for root cause analyses is generally limited. Moreover, information over a large area, which includes many geometries, is required to understand the effects of a layout on failure. These problems pose challenges in training the CNN models to achieve high accuracy for layout segment classification. In this study, we propose a scheme for layout segment classification that uses CNN to analyze the effects of layout on failure. Thus, multiple segment images of LSI layouts are used as inputs, and the outputs of CNN models are used to classify the input images as either risk or non-risk segments. First, we evaluated a simple 3-layer model, and then a 15-layer model fine-tuned using the transfer learning of the VGG16 model. The 15-layer model outperformed the 3-layer model for classifying the LSI layout segments. From a cross-validation of the 15-layer model, a true positive rate of >80% and a false positive rate of <10% are obtained for extracting layout regions related to actual defects. The outputs of the CNN models from the input layout segment demonstrate similarity with the defective layouts. In the regional layouts across the LSI chips, the contour plot of model outputs is visualized as a hazard map of failures. This information is necessary for additional failure cause analyses.
Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical ...forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation.
Root cause analysis (RCA) of failures is mandatory to obtain the reliability and productivity of LSIs. Although analyzing layout-induced defects is crucial to optimize design rules and to predict ...unknown defects, it is a challenging task due to the difficulty in explaining the relationship between defects and circuit layouts. We applied convolutional neural networks (CNNs) to classify LSI layout images to perform the RCA of layout-induced defects in a previous study. However, due to the low resolution of images, actual defect positions were not clearly distinguished. In the present study, we use image clips of different sizes and resolutions for the CNN classification. Experimental results indicate that the validity of the extracted layout features depends on the resolution of image clips. Using the visual explanation technique GradCAM++, the features of defective layouts can be accurately captured in local areas when CNN models are trained on smaller image clips with higher resolution. These layout features included a group of patterns with their surroundings. Conversely, utilizing smaller-size clips deteriorates the classification accuracy due to the incorporation of less information from the images. In the conducted experiments, even in the case of using smaller clips, acceptable performance (the detection rate of defect positions DTR <inline-formula> <tex-math notation="LaTeX">\cong ~90 </tex-math></inline-formula>%, and the risk-image classification rate RCR <inline-formula> <tex-math notation="LaTeX">\cong ~10 </tex-math></inline-formula>%) can be obtained by increasing the size of training datasets. Partial layouts extracted as features of defective layouts can then be used in RCA and in designing future products.
Dental enamel is hardest tissue in the body and is produced by dental epithelial cells residing in the tooth. Their cell fates are tightly controlled by transcriptional programs that are facilitated ...by fate determining transcription factors and chromatin regulators. Understanding the transcriptional program controlling dental cell fate is critical for our efforts to build and repair teeth. In this review, we describe the current understanding of these regulators essential for regeneration of dental epithelial stem cells and progeny, which are identified through transgenic mouse models. We first describe the development and morphogenesis of mouse dental epithelium in which different subpopulations of epithelia such as ameloblasts contribute to enamel formation. Then, we describe the function of critical factors in stem cells or progeny to drive enamel lineages. We also show that gene mutations of these factors are associated with dental anomalies in craniofacial diseases in humans. We also describe the function of the master regulators to govern dental lineages, in which the genetic removal of each factor switches dental cell fate to that generating hair. The distinct and related mechanisms responsible for the lineage plasticity are discussed. This knowledge will lead us to develop a potential tool for bioengineering new teeth.
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