Small Extracellular Vesicles Laden Oxygen-Releasing Thermosensitive Hydrogel for Enhanced Antibacterial Therapy against Anaerobe-Induced Periodontitis Alveolar Bone Defect.

Periodontitis is a bacterially induced chronic destructive inflammatory disease that leads to irreversible destruction of the tooth supporting structure, including connective tissue destruction, bone resorption, and even tooth loss. Until now, there has been no effective treatment to repair inflammatory bone loss in periodontitis. Recently, small extracellular vesicles (sEVs) emerged as the essential paracrine factors of mesenchymal stem cells (MSCs) that mediated tissue regeneration. However, limitations of antimicrobial activity associated with the use of sEVs have led to the urgency of new alternative strategies. Currently, we investigated the potential of a biocompatible oxygen-releasing thermosensitive hydrogel laded with sEVs secreted by bone marrow MSCs (BMMSCs) for the alveolar bone defect in periodontitis. The hydrogel composed of different polymers such as chitosan (CS), poloxamer 407 (P407), and cross-linked hyaluronic acid (c-HA) conglomerating is a kind of nanoporous structure material. Then, the gel matrix further encapsulated sEVs and calcium peroxide nanoparticles to realize the control of sEVs and oxygen release. Furthermore, ascorbic acid was added to achieve the REDOX equilibrium and acid-base equilibrium. The experiments in vivo and in vitro proved its good biocompatibility and effectively inhibited the growth of the periodontal main anaerobe, relieved periodontal pocket anaerobic infections, and promoted the periodontal defect regeneration. Therefore, this finding demonstrated that it was a promising approach for combating anaerobic pathogens with enhanced and selective properties in periodontal diseases, even in other bacteria-induced infections, for future clinical application.

Caesarean-section delivery and caries risk of 3-year-old Chinese children: a retrospective cohort study.

BACKGROUND: Caesarean-section (C-section) may influence children's long-term health by affecting bacterial colonization. However, few studies have focused on the association between C-section delivery (CSD) and dental caries, and previous conclusions have been conflicting. This study aimed to explore whether CSD would increase the risk of early childhood caries (ECC) in preschool children in China. METHODS: This study was a retrospective cohort study. Three-year-old children with full primary dentition were included through the medical records system. Children in the nonexposure group were vaginally delivered (VD), while children in the exposure group were delivered through C-section. The outcome was the occurrence of ECC. After agreeing to participate in this study, guardians of included children completed a structured questionnaire on maternal sociodemographic factors, children's oral hygiene and feeding habits. The chi-square test was used to determine differences in the prevalence and severity of ECC between the CSD and VD groups and to analyse the prevalence of ECC according to sample characteristics. Subsequently, potential risk factors for ECC were preliminarily identified through univariate analysis, and the adjusted odds ratios (ORs) were further calculated through multiple logistic regression analysis after controlling for confounding factors. RESULTS: The VD group included 2115 participants while CSD group included 2996 participants. The prevalence of ECC was higher in CSD children than in VD children (27.6% vs. 20.9%, P < 0.05), and the severity of ECC in CSD children was higher (mean number of decayed, missing, and filled teeth, dmft: 2.1 vs. 1.7, P < 0.05). CSD was a risk factor for ECC in 3-year-old children (OR = 1.43, 95% CI = 1.10-2.83). In addition, irregular tooth brushing and always prechewing children's food were risk factors for ECC (P < 0.05). Low maternal educational attainment (high school or below) or socioeconomic status (SES-5) may also increase the prevalence of ECC in preschool children and CSD children (P < 0.05). CONCLUSIONS: CSD would increase the risk of ECC in 3-year-old Chinese children. Paediatric dentists should devote more attention to the development of caries in CSD children. Obstetricians should also prevent excessive and unnecessary CSD.

Laser doppler flowmetry to detect pulp vitality, clinical reference range and coincidence rate for pulpal blood flow in permanent maxillary incisors in Chinese children: a clinical study.

BACKGROUND: A laser doppler flowmetry (LDF) test can reflect the pulp vitality caused by the change in pulp blood flow (PBF). This study aimed to investigate the PBF of the permanent maxillary incisors using LDF and to calculate the clinical reference range and coincidence rate for pulp vitality using PBF as an indicator. METHODS: School-age children (7-12 years) were recruited randomly. A total of 455 children (216 female and 239 male) were included in this study. An additional 395 children (7-12 years) who attended the department due to anterior tooth trauma from October 2015 to February 2018 were included to assess the clinical occurrence rate. The PBF was measured using LDF equipment and an LDF probe. RESULTS: The clinical reference range of PBF values for the permanent maxillary incisors (teeth 11, 12, 21, and 22) in children were from 7 to 14 perfusion units (PU), 11 (6.016; 11.900 PU), 12 (6.677; 14.129 PU), 21 (6.043;11.899 PU), and 22 (6.668; 14.174 PU). There was a statistically significant correlation between PBF and children's age (p < 0.000) without any significant gender discrimination (p = 0.395). For all incisors, for any age group, the PBF detection value of the lateral incisors was significantly higher than that of the central incisors (p < 0.05). The clinical coincidence rate of detecting PBF in the traumatic teeth was 90.42% and the sensitivity and specificity were 36.99% and 99.88%, respectively. CONCLUSIONS: The determination of the PBF clinical reference range and clinical coincidence rate for the permanent maxillary incisors in children using LDF provided a promising theoretical basis for clinical applications.

Inhibitory effects of Stevioside on Streptococcus mutans and Candida albicans dual-species biofilm.

Introduction: Streptococcus mutans is the most prevalent biofilm-forming pathogen in dental caries, while Candida albicans is often detected in the presence of S. mutans. Methods: We aimed to evaluate the anti-caries effect of stevioside in medium trypticase soy broth (TSB) with or without sucrose supplementation compared with the same sweetness sucrose and xylitol in a dual-species model of S. mutans and C. albicans, based on planktonic growth, crystal violet assay, acid production, biofilm structural imaging, confocal laser scanning microscopy, and RNA sequencing. Results: Our results showed that compared with sucrose, stevioside significantly inhibited planktonic growth and acid production, changed the structure of the mixed biofilm, and reduced the viability of biofilm and the production of extracellular polysaccharides in dual-species biofilm. Through RNA-seq, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway impact analysis showed that stevioside decreased sucrose metabolism and increased galactose and intracellular polysaccharide metabolism in S. mutans, and decreased genes related to GPI-modified proteins and secreted aspartyl proteinase (SAP) family in C. albicans. In contrast to xylitol, stevioside also inhibited the transformation of fungal morphology of C. albicans, which did not form mycelia and thus had reduced pathogenicity. Stevioside revealed a superior suppression of dual-species biofilm formation compared to sucrose and a similar anti-caries effect with xylitol. However, sucrose supplementation diminished the suppression of stevioside on S. mutans and C. albicans. Conclusions: Our study is the first to confirm that stevioside has anticariogenic effects on S. mutans and C. albicans in a dual-species biofilm. As a substitute for sucrose, it may help reduce the risk of developing dental caries.

The potential therapeutic role of extracellular vesicles in critical-size bone defects: Spring of cell-free regenerative medicine is coming.

In recent years, the incidence of critical-size bone defects has significantly increased. Critical-size bone defects seriously affect patients' motor functions and quality of life and increase the need for additional clinical treatments. Bone tissue engineering (BTE) has made great progress in repairing critical-size bone defects. As one of the main components of bone tissue engineering, stem cell-based therapy is considered a potential effective strategy to regenerate bone tissues. However, there are some disadvantages including phenotypic changes, immune rejection, potential tumorigenicity, low homing efficiency and cell survival rate that restrict its wider clinical applications. Evidence has shown that the positive biological effects of stem cells on tissue repair are largely mediated through paracrine action by nanostructured extracellular vesicles (EVs), which may overcome the limitations of traditional stem cell-based treatments. In addition to stem cell-derived extracellular vesicles, the potential therapeutic roles of nonstem cell-derived extracellular vesicles in critical-size bone defect repair have also attracted attention from scholars in recent years. Currently, the development of extracellular vesicles-mediated cell-free regenerative medicine is still in the preliminary stage, and the specific mechanisms remain elusive. Herein, the authors first review the research progress and possible mechanisms of extracellular vesicles combined with bone tissue engineering scaffolds to promote bone regeneration via bioactive molecules. Engineering modified extracellular vesicles is an emerging component of bone tissue engineering and its main progression and clinical applications will be discussed. Finally, future perspectives and challenges of developing extracellular vesicle-based regenerative medicine will be given. This review may provide a theoretical basis for the future development of extracellular vesicle-based biomedicine and provide clinical references for promoting the repair of critical-size bone defects.

Impaired autophagy flux by lncRNA NEAT1 is critical for inflammation factors production in human periodontal ligament stem cells with nicotine treatment.

BACKGROUND AND OBJECTIVES: Periodontitis is the top reason for tooth loss, and smoking significantly increases severe periodontitis risk. Defective autophagy has been reported to play a vital role in periodontitis. This study aimed to elucidate the relationship between autophagy and inflammation factors production in nicotine-treated periodontal ligament stem cells (PDLSCs) and the underlying mechanism. METHODS: In this study, transmission electron microscopy, immunofluorescence, and the mCherry-GFP-LC3 plasmid were used to study autophagy flux. The gene levels of inflammation factors and long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) were detected by quantitative real-time PCR (qRT-PCR). Western blot was performed to assess the protein levels of autophagic markers and α7 nicotinic acetylcholine receptor (α7nAChR). RESULTS: We found that nicotine impaired autophagosome-lysosome fusion and lysosome functions to block autophagy flux, contributing to inflammatory factors production in nicotine-treated PDLSCs. Moreover, nicotine upregulated NEAT1 by activating α7nAChR. NEAT1 decreased autophagy flux by downregulating syntaxin 17 (STX17). CONCLUSION: Our data indicate that NEAT1-decreased autophagy flux is pivotal for inflammation factors production in nicotine-treated PDLSCs.

Enhanced conservation of vital pulp and apical tissues by the application of crown rotation surgery for inversely impacted central incisors: A follow-up analysis of two patients over 4 years.

AIM: To describe a novel surgical method (crown rotation surgery) to manage inversely impacted central incisors with immature roots. METHODOLOGY: Two young patients each presented with an inversely impacted maxillary central incisor. To protect the apical tissues, the two impacted incisors were rotated downwards to a relatively normal position without extraction from their bony sockets. RESULTS: After crown rotation surgery, spontaneous eruption, continuous root development, and periodontal healing of the rotated incisors were observed. The pulp retained vitality and blood flow was normal. Moreover, there were no obvious signs of pulp canal obliteration (PCO), as indicated by Cone Beam Computed Tomography (CBCT) imaging. CONCLUSIONS: By optimising protection of the vital pulp and apical tissues, crown rotation surgery represents a minimally invasive, conservative, and practical surgical technique for treating inversely impacted incisors with developing roots. In contrast to existing surgical methods, crown rotation surgery may avoid certain complications, including PCO and abnormal or arrested root development. KEY LEARNING POINTS: By optimizing protection of the vital pulp and apical tissues, crown rotation surgery represents a minimally invasive, conservative and practical surgical technique for treating inversely impacted incisors with developing roots. In contrast to existing surgical methods, crown rotation surgery may avoid certain complications, including PCO and abnormal or arrested root development.

Development of gestational diabetes mellitus in women with periodontitis in early pregnancy: A population-based clinical study.

AIM: This study aimed to determine whether periodontitis in early pregnancy and periodontal therapy during gestation affect the incidence of gestational diabetes mellitus (GDM) through a population-based clinical study. MATERIALS AND METHODS: Subjects without periodontitis at 1-4 weeks of gestation who met our inclusion criteria were enrolled in the non-periodontitis group. Periodontitis patients who agreed or refused to receive periodontal therapy during pregnancy were separately enrolled in the periodontitis treated or untreated group. At 12-16 weeks of gestation, gingival crevicular fluid (GCF) and venous blood were collected for analyses of bacterial species and serum inflammatory mediators, respectively. At 24-28 weeks of gestation, GDM patients were identified by oral glucose tolerance tests. The association tests were performed using Chi-squared statistics and regression analyses. RESULTS: The complete data of 3523 pregnant women were recorded during the study period. GDM incidence among the untreated periodontitis participants (84/749, 11.21%) was significantly higher than that among the non-periodontitis participants (108/2255, 4.79%) (p < .05), and periodontal treatment during gestation reduced the incidence from 11.21% (untreated group) to 7.32% (38/519, treated group) (p < .05). Based on multiple logistic regression analyses, it was found that periodontitis in early pregnancy was associated with GDM, and three-step regression analyses showed that Porphyromonas gingivalis (P. gingivalis) and the serum TNF-α and IL-8 levels played a role in the association between untreated periodontitis and GDM. Furthermore, Pearson's correlation test indicated that the existence of P. gingivalis in GCF was positively correlated with high serum levels of these two inflammatory mediators. CONCLUSIONS: This study establishes a connection between periodontitis in early pregnancy and GDM and demonstrates that the presence of P. gingivalis is associated with high levels of inflammatory mediators in serum, and thereby may contribute to the development of GDM. In-depth mechanistic studies are needed to further support these findings.

Nicotine regulates autophagy of human periodontal ligament cells through α7 nAchR that promotes secretion of inflammatory factors IL-1ß and IL-8.

BACKGROUND: Nicotine is an important risk factor and the main toxic component associated with periodontitis. However, the mechanism of nicotine induced periodontitis is not clear. To investigated the mechanism through which nicotine regulates autophagy of human periodontal ligament cells (hPDLCs) through the alpha7 nicotinic acetylcholine receptor (α7 nAChR) and how autophagy further regulates the release of IL-1ß and IL-8 secretion in hPDLCs. METHODS: HPDLCs were obtained from root of extracted teeth and pre-incubated in alpha-bungarotoxin (α-BTX) or 3-Methyladenine (3-MA), followed by culturing in nicotine. We used a variety of experimental detection techniques including western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM) and RT-qPCR to assess the expression of the LC3 protein, autolysosome, and release of IL-1ß and IL-8 from hPDLCs. RESULTS: Western blots, immunofluorescence and TEM results found that the nicotine significantly increased the autophagy expression in hPDLCs that was time and concentration dependent and reversed by α-BTX treatment (p < 0.05). RT-qPCR and ELISA results revealed a noticeable rise in the release of inflammatory factors IL-1ß and IL-8 from hPDLCs in response to nicotine. RT-qPCR and ELISA results showed that nicotine can significantly up-regulate the release of inflammatory factors IL-1ß and IL-8 in hPDLCs, and this effect can be inhibited by 3-MA (p < 0.05). CONCLUSIONS: Nicotine regulated autophagy of hPDLCs through α7 nAChR and in turn the regulation of the release of inflammatory factors 1L-1ß and 1L-8 by hPDLCs.

Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence.

Design of endogenous stimuli-responsive amino acids allows for precisely modulating proteins or peptides under a biological microenvironment and thereby regulating their performance. Herein we report a noncanonical amino acid 2-nitroimidazol-1-yl alanine and explore its functions in creation of the nitroreductase (NTR)-responsive peptide-based supramolecular probes for efficient hypoxia imaging. On the basis of the reduction potential of the nitroimidazole unit, the amino acid was synthesized via the Mitsunobu reaction between 2-nitroimidazole and a serine derivate. We elucidated the relationship between the NTR-responsiveness of the amino acid and the structural feature of peptides involving a series of peptides. This eventually facilitates development of aromatic peptides undergoing NTR-responsive self-assembly by rationally optimizing the sequences. Due to the intrinsic role of 2-nitroimidazole in the fluorescence quench, we created a morphology-transformable supramolecular probe for imaging hypoxic tumor cells based on NTR reduction. We found that the resulting supramolecular probes penetrated into solid tumors, thus allowing for efficient fluorescence imaging of tumor cells in hypoxic regions. Our findings demonstrate development of a readily synthesized and versatile amino acid with exemplified properties in creating fluorescent peptide nanostructures responsive to a biological microenvironment, thus providing a powerful toolkit for synthetic biology and development of novel biomaterials.

In situ rotation surgery for correction of growing, inversely impacted maxillary central incisors.

Treatment of an impacted incisor with a dilacerated root is challenging for clinicians because of the position of the impacted incisor, the abnormality of the root, unfavorable prognosis, and, especially, the long treatment duration. We report on 2 young patients who had inversely impacted maxillary central incisors with developing labially dilacerated roots. Both patients were treated by a novel surgical approach, in situ rotation, by which the crowns of the inversely impacted incisors were carefully rotated to a relatively normal position, whereas the apical location remained relatively unchanged. About 2 weeks after surgery, spontaneous eruption of the treated incisors was observed. Three months later, the postoperative central incisors were further aligned into the maxillary arch with a fixed orthodontic appliance. Follow-up visits 2 or 3 years after surgery indicated that the positions of the dilacerated incisors maintained stability with good gingival esthetics, and the pulpal vitality was favorable. The roots grew further in a relatively normal direction of the incisor's longitudinal axis, which was different from the initial curvature angle. Moreover, with the in situ rotation surgery, treatment time was greatly reduced and resulted in a favorable prognosis compared with conventional treatment.

Treatment of a Young Maxillary Central Incisor with Two Root Canals: A Case Report.

Currently, the common treatment for pulpitis and periapical diseases is root canal treatment. However, the complex variations of root canal system often affect the effect of root canal treatment and even lead to treatment failure. Therefore, it is of great significance for dentists to emphasize the diversity of root canal morphology and to be familiar with the anatomical morphology of root canal to improve the success rate of clinical diagnosis and treatment. We reported a case of an eight-year-old female patient of type V root canal in a young maxillary middle incisor. The young maxillary central incisor was diagnosed with irreversible pulpitis by radiography and CBCT examination. The present case report described the entire treatment procedure of a young maxillary central incisor after trauma, including successive apical examination, CBCT examination and multiple root canal filling, and finally the treatment was completed with the aid of microscope. The root canals were filled with the vertical compression technique. Z350 composite resin was used to repair the tooth. The patient was followed-up at three, six, and 12 months after endodontic treatment. All of the examinations were normal. Finding the anatomical configuration of root canal by various ways will help to improve the success rate of root canal therapy.

Simultaneous incorporation of PTH(1-34) and nano-hydroxyapatite into Chitosan/Alginate Hydrogels for efficient bone regeneration.

Tissue regeneration based on the utilization of artificial soft materials is considered a promising treatment for bone-related diseases. Here, we report cranial bone regeneration promoted by hydrogels that contain parathyroid hormone (PTH) peptide PTH(1-34) and nano-hydroxyapatite (nHAP). A combination of the positively charged natural polymer chitosan (CS) and negatively charged sodium alginate led to the formation of hydrogels with porous structures, as shown by scanning electron microscopy. Rheological characterizations revealed that the mechanical properties of the hydrogels were almost maintained upon the addition of nHAP and PTH(1-34). In vitro experiments showed that the hydrogel containing nHAP and PTH(1-34) exhibited strong biocompatibility and facilitated osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) via the Notch signaling pathway, as shown by the upregulated expression of osteogenic-related proteins. We found that increasing the content of PTH(1-34) in the hydrogels resulted in enhanced osteogenic differentiation of BMSCs. Implantation of the complex hydrogel into a rat cranial defect model led to efficient bone regeneration compared to the rats treated with the hydrogel alone or with nHAP, indicating the simultaneous therapeutic effect of nHAP and PTH during the treatment process. Both the in vitro and in vivo results demonstrated that simultaneously incorporating nHAP and PTH into hydrogels shows promise for bone regeneration, suggesting a new strategy for tissue engineering and regeneration in the future.

Advancing application of mesenchymal stem cell-based bone tissue regeneration.

Reconstruction of bone defects, especially the critical-sized defects, with mechanical integrity to the skeleton is important for a patient's rehabilitation, however, it still remains challenge. Utilizing biomaterials of human origin bone tissue for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural bone tissue with regard to its properties. However, not only efficacious and safe but also cost-effective and convenient are important for regenerative biomaterials to achieve clinical translation and commercial success. Advances in our understanding of regenerative biomaterials and their roles in new bone formation potentially opened a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multicomponent construction of native extracellular matrix (ECM) for cell accommodation, the ECM-mimicking biomaterials and the naturally decellularized ECM scaffolds were used to create new tissues for bone restoration. On the other hand, with the going deep in understanding of mesenchymal stem cells (MSCs), they have shown great promise to jumpstart and facilitate bone healing even in diseased microenvironments with pharmacology-based endogenous MSCs rescue/mobilization, systemic/local infusion of MSCs for cytotherapy, biomaterials-based approaches, cell-sheets/-aggregates technology and usage of subcellular vesicles of MSCs to achieve scaffolds-free or cell-free delivery system, all of them have been shown can improve MSCs-mediated regeneration in preclinical studies and several clinical trials. Here, following an overview discussed autogenous/allogenic and ECM-based bone biomaterials for reconstructive surgery and applications of MSCs-mediated bone healing and tissue engineering to further offer principles and effective strategies to optimize MSCs-based bone regeneration.

Inflammation has synergistic effect with nicotine in periodontitis by up-regulating the expression of α7 nAChR via phosphorylated GSK-3ß.

Periodontitis is the leading cause of adult tooth loss, and those who smoke are at an increased risk of developing periodontitis. α7 nicotinic acetylcholine receptor (α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking-related periodontitis. However, this has not been experimentally demonstrated. We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT-qPCR and Western blot analyses. Besides, immunohistochemical staining, micro-CT analysis and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Inflammation up-regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up-regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation-induction of PDLSCs. Moreover, the inflammation-induced up-regulation of α7 nAChR was partially dependent on the level of phosphorylated GSK-3ß. This study provides experimental evidence for the pathological development of smoking-related periodontitis and sheds new light on developing inflammation and α7 nAChR-targeted therapeutics to treat and prevent the disease.

Reactive Oxygen Species-Responsive Adaptable Self-Assembly of Peptides toward Advanced Biomaterials.

Precise control over self-assembly of peptides into adaptable nanostructures allows for emulating the dynamic organization of natural proteins and their sophisticated biological functions. Utilization of disease biomarkers as internal stimuli for manipulating the self-assembly of peptides bestows their adaptable features with a spatiotemporal resolution to satisfy the requirement of the performance of biomaterials. Reactive oxygen species (ROS) consisting of reactive ions or free radicals are overexpressed by pathological lesions and have been recognized as one of conventional biomarkers for disease progression. Despite the progress made over the past decade in stimulus-responsive self-assembly of peptides as well as the summarization of this progress, the specific reviews focusing on ROS-responsive self-assembly of peptides remain scarce. This review summarizes the progress achieved over the past decade of the ROS-responsive self-assembly of peptides into adaptable nanostructures and their applications in biomaterials. We focus on the chemical sources responsible for the ROS-sensitive behavior of peptides, in which the chemical moieties are incorporated into peptides as side chains, terminal groups, or backbone linkages. The ROS-responsive self-assembly of peptides into nanostructures with morphologies adaptable to ROS-oxidation and their applications in enzymatic catalysis, chemosensing, drug delivery, and tissue engineering will be highlighted. Understanding the established ROS-responsive peptide self-assembling systems allows us to provide perspectives for their further development and thereby elucidate their great potential in development of advanced biomaterials.

Mechanical Stress Modulates the RANKL/OPG System of Periodontal Ligament Stem Cells via α7 nAChR in Human Deciduous Teeth: An In Vitro Study.

The aim of this study was to investigate the mechanism by which periodontal ligament stem cells (PDLSCs) modulate root resorption of human deciduous teeth under mechanical stress. In this investigation, the PDLSCs were derived from deciduous and permanent teeth at different stages of root resorption. A cyclic hydraulic pressure was applied on the PDLSCs to mimic chewing forces in the oral environment. The cultured cells were characterized using osteogenic and adipogenic differentiation assays, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting analysis. The PDLSCs exhibited the ability to induce osteoclast differentiation under certain mechanical stresses. As the expressions of RUNX2, alkaline phosphatase (ALP), and osteoprotegerin (OPG) were significantly reduced, the receptor activator of the nuclear factor kappa-B ligand (RANKL) was upregulated increasing the RANKL/OPG ratio. Under hydrodynamic pressure at 0-135 kPa, the expressions of alpha 7 nicotinic acetylcholine receptors (α7 nAChR), p-GSK-3ß, and active-ß-catenin were markedly upregulated in PDLSCs from unresorbed deciduous teeth. Treatment with the α7 nAChR inhibitor alpha-bungarotoxin (α-BTX) and the Wnt pathway inhibitor DKK1 may reverse the mechanical stress inducing upregulation of RANKL and reduction of RUNX2, ALP, and OPG. Alizarin red staining confirmed these results. The mechanical stress applied on the deciduous tooth PDLSCs can induce osteoclastic effects through upregulation of α7 nAChR and activation of the canonical Wnt pathway. It can be suggested that chewing forces may play a major role at the beginning of the physiological root resorption of deciduous teeth.

[Mechano-growth factor regulated cyclic stretch-induced osteogenic differentiation and MMP-1, MMP-2 expression in human periodontal ligament cells by activating the MEK/ERK1/2 pathway].

PURPOSE: To explore the role and mechanism of mechano-growth factor (MGF) in cyclic stretch (CS)-induced osteogenic differentiation and MMP-1, MMP-2 expression in human periodontal ligament cells (hPDLCs). METHODS: HPDLCs were isolated and transfected with si-MGF, or stimulated with MGF or MEK/ERK pathway inhibitor U0126. Cells were cultured in Flexercell system with 10% elongation at 0.1 Hz. An alkaline phosphatase (ALP) kit was used to detect ALP activity. QRT-PCR assay was performed to determine the transcript levels of MGF and osteogenic genes, including ALP, runt-related transcription factor 2 (Runx2) and osteopontin (OPN). Western bot was used to evaluate the effect on MEK/EKR1/2 signaling. Statistical analysis was performed using SPSS 19.0 software package. RESULTS: CS induced the expression of MGF in hPDLCs in a time-dependent manner (P＜0.05). In contrast to the control group, transfection with si-MGF inhibited the expression of MGF in hPDLCs (P＜0.05). Moreover, cessation of MGF dramatically suppressed ALP activity (P＜0.05) and the expression of osteogenic gene ALP, Runx2 and OPN (P＜0.05) in hPDLCs. Furthermore, down-regulation of MGF restrained the expression of MMP-1 and MMP-2, in contrast to CS group (P＜0.05). Conversely, stimulation with MGF further enhanced the effects of CS on osteogenic differentiation of hPDLCs and MMP-1, MMP-2 expression (P＜0.05). Additionally, MGC silencing abrogated CS-induced expression of p-ERK (P＜0.05), which was further enhanced following MGF treatment (P＜0.05). Simultaneously, precondition with U0126 antagonized MGF-enhanced effects on CS-triggered osteogenic differentiation and MMP-1, MMP-2 expression (P＜0.05). CONCLUSIONS: Mechano-growth factor regulates cyclic stretch-induced osteogenic differentiation and MMP-1, MMP-2 expression in human periodontal ligament cells by activating MEK/ERK1/2 signaling pathway.

Facile large-area autofocusing Raman mapping system for 2D material characterization.

Two-dimensional (2D) materials have attracted tremendous research interests due to their intriguing properties and promising applications. As one of the most typical 2D material characterization methods, however, the conventional Raman mapping only works within few-hundreds micrometers range at a time due to the focus depth constraint and the non-ideal level of the substrate. To implement wafer-scale Raman scanning, large-area autofocusing Raman mapping (LARM) is highly desirable. Here, we present a modified centroid method to build a facile LARM system in which the Raman excitation laser is employed as the focus laser, reducing the system cost and complexity. Based on identifying the shape of the semicircle laser reflection image, a self-written autofocusing algorithm allows a real-time adjusting the focus position during the large-scale scanning. As a state-of-the-art demonstration, the thickness distribution of both few layer WS2 triangle domains sparsely located in sub-millimeter range and polycrystalline continuous MoS2 film up to 2-inch scale can be well-revealed. Our results may shed light on wafer-scale nondestructive optical characterization of 2D materials.