Bisphenol A exposure exacerbates tracheal inflammatory injury in selenium-deficient chickens by regulating the miR-155/TRAF3/ROS pathway.

Bisphenol A (BPA) is an endocrine disruptor. Excessive BPA intake can damage the structure and function of the respiratory tract. Dietary selenium (Se) deficiency may also cause immune tissue damage. To investigate the potential mechanism of BPA on tracheal damage in selenium-deficient chickens and the role of microRNAs (miRNAs) in this process, we established in vitro and in vivo Se deficiency and BPA exposure models and screened out miR-155 for follow-up experiments. We further predicted and confirmed the targeting relationship between miR-155 and TRAF3 using TargetScan and dual luciferase assays and found that miR-155 was highly expressed and caused inflammatory damage. Further studies showed that BPA exposure increased airway oxidative stress, activated the NF-κB pathway, and caused inflammation and immune damage in selenium-deficient chickens, but down-regulating miR-155 and NAC treatment could reverse this phenomenon. This suggested that these pathways are regulated by the miR-155/TRAF3/ROS axis. In conclusion, BPA exposure aggravates airway inflammation in selenium-deficient chickens by regulating miR-155/TRAF3/ROS. This study revealed the mechanism of BPA exposure combined with Se deficiency in tracheal inflammatory injury in chickens and enriched the theoretical basis of BPA injury in poultry.

SMAD2/3 Phosphorylation is correlated with matrix metalloproteinase 9 expression in human periapical lesions.

OBJECTIVE: The aim of this study was to investigate the presence of phosphorylated SMAD2/3 (P-SMAD2/3) in periapical lesions in humans and its possible correlation with matrix metalloproteinase 9 (MMP9) during the development of apical periodontitis. DESIGN: In this study, a total of 38 samples were collected, consisting of 16 healthy controls and 22 periapical lesions. These samples underwent fixation, dehydration, and embedding for further histologic and immunochemical analysis. The expression of phosphorylated SMAD2/3 and MMP9 was quantified using the average integrated optical density. Additionally, immunofluorescence analysis was conducted to investigate the colocalization of phosphorylated SMAD2/3 and MMP9. RESULTS: The study found that periapical lesions exhibited a stronger expression of MMP9 compared to healthy controls. Additionally, the expression of phosphorylated SMAD2/3 was observed to increase in the periapical granulomas and radicular cysts group, as compared to the normal group (P < 0.01). The results of the immunofluorescence test showed that phosphorylated SMAD2/3 was colocalized with MMP9. CONCLUSIONS: The study found that SMAD2/3 phosphorylation is correlated with matrix metalloproteinase 9 expression in human periapical lesions, suggesting its potential involvement in tissue destruction and immune cell infiltration in periapical lesions.

Bisphenol A aggravate selenium deficiency-induced apoptosis via miR-215-3p/Dio1 to activate ROS/PI3K/AKT pathway in chicken arterial.

Both bisphenol A (BPA) and selenium (Se) deficiency can affect the expression of microRNAs (miRNAs), which can specifically regulate its target mRNA and induce apoptosis, and play a significant role in cardiovascular injury diseases. To explore the mechanism of apoptosis induced by BPA and Se deficiency in chicken arterial endothelial tissue and the role of miRNAs in this process, the model of BPA exposure/Se deficiency in chicken and PAEC cells have been employed. The targeting relationship between miR-215-3p and iodothyronine deiodinase 1 (Dio1) in PAEC was verified by double luciferase gene report. The level of miR-215-3p was detected by qRT-PCR. The oxidative stress level of arterial endothelial cells was detected by oxidative stress kit and DCFH-DA probe method. The PI3K/AKT pathway, mitochondrial dynamics, and apoptosis-related genes were detected by qRT-PCR and western blot. The mitochondrial ATP level and nitric oxide synthases (NOSs) level were detected with the kit. TUNEL, acridine orange/ethidium bromide, and flow cytometry were used to detect the level of apoptosis. The results showed that BPA exposure and Se deficiency led to overexpression of miR-215-3p, aggravated oxidative stress, inhibited activation of PI3K/AKT pathway, promoted mitochondrial division, increased expression of apoptosis related genes, and finally led to apoptosis of chicken arterial endothelial cells. We also established knockdown/overexpression models of miR-215-3p and Dio1 in vitro, and found that overexpression of miR-215-3p and knockout of Dio1 can induce apoptosis. Interestingly, miR-215-3p-Inhibitor and N-acetyl- l-cysteine (NAC) partially prevented apoptosis caused by BPA exposure and Se deficiency, and LY294002 aggravated apoptosis. These results suggest that BPA exposure aggravates the apoptosis of Se deficient arterial endothelial cells in chickens by regulating the ROS/PI3K/AKT pathway activated by miR-215-3p/Dio1. The miR-215-3p/Dio1 axis provides a new way to understand the toxic mechanism of BPA exposure and Se deficiency, and reveals a new regulatory model of apoptosis damage in vascular diseases.

Understanding the Farmland Rights Confirmation Policy from the Perspective of Farmers: Evidence from Jiangxi, China.

Since the 1970s, although the Chinese central government has constructed farmland rights confirmation policy to stimulate the vitality of rural land elements, it is rare to discuss the effectiveness of the policy from the perspective of farmers. This paper applies the deep interview and questionnaire to present an analysis framework consisting of "policy implementation-input behavior in agricultural production-farmland transfer" and testifies the framework with the mediation model. The findings show that, in general, the farmland rights confirmation policy (FRCP) has a significant negative impact on leasing out farmland and a significant positive impact on leasing in farmland. In particular, for farmland leasing out, the mediating effects of agricultural capital input and agricultural time input account for 15.504% and 14.536% of the total effect, respectively. In addition, for leasing farmland in, these two mediating effects accordingly account for 13.798% and 12.155% of the total effect. It is worth noting that in the future, we should understand FRCP based on the consideration of farmers' behavior in a given local context, while also focusing on policy implementation as well as policy design.

Expression of Matrix Metalloproteinases and Tissue Inhibitor of Matrix Metalloproteinases during Apical Periodontitis Development.

INTRODUCTION: Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are considered important mediators of the periapical immune response to infection. This study aimed to clarify the putative relationship between MMPs and TIMPs by elucidating the activity of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 in the temporal development of apical periodontitis (AP) in mice. METHODS: AP was induced in the lower first molars of 30 male Kunming mice. The animals were randomly killed at 0, 7, 14, 28, 60, and 90 days after pulp exposure. The jaws were removed and subjected to quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. RESULTS: The MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 messenger RNA and protein expression levels increased with periapical inflammation progression (P < .05). The MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 messenger RNA and protein expression levels increased during the acute and chronic stages of periapical lesions, with less MMP-2 and MMP-9 expression levels at the chronic stage (P < .05). The MMP-8 expression increased at the chronic stage of inflammation (P < .05) but not at the acute stage. Immunostained MMP-2 and TIMP-1 were observed in all experimental periods. CONCLUSIONS: MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were expressed in all periapical samples with varying levels between them. MMP expression could be related to TIMP expression in the temporal development of AP.

Anti-diabetic effects of astaxanthin on an STZ-induced diabetic model in rats.

Type 2 diabetes mellitus (T2DM), which is characterized by insulin resistance and relative insulin insufficiency, has become the most common chronic metabolic disease threatening global health. The preferred therapies for T2DM include lifestyle interventions and the use of anti-diabetic drugs. However, considering their adverse reactions, it is important to find a low-toxicity and effective functional food or drug for diabetes prevention and treatment. Astaxanthin is a potent antioxidant carotenoid found in marine organisms has been reported to prevent diet-induced insulin resistance and hepatic steatosis. To investigate the anti-diabetic effects of astaxanthin, male Wistar rats were fed a high-energy diet for 4 weeks, followed by a low dose streptozotocin (STZ) injection to induce the diabetes model, and the rats were then fed an astaxanthin-containing diet for another 3 weeks. Astaxanthin significantly decreased blood glucose and total cholesterol (TC) levels, and increased blood levels of high density lipoprotein cholesterol (HDL-C) in STZ-induced diabetic rats in a dose dependent manner. These results were associated with increased expression of insulin sensitivity related genes (adiponectin, adipoR1, and adipoR2) in vivo, thereby attenuating STZ-induced diabetes. In addition, we also compared the anti-diabetic effects of astaxanthin and monacolin K, which has been reported to downregulate hyperlipidemia and hyperglycemia. The results revealed that astaxanthin and monacolin K showed similar anti-diabetic effects in STZ-induced diabetic rats. Therefore, astaxanthin may be developed as an anti-diabetic agent in the future.

Comparison of the Therapeutic Effects of Tension Band with Cannulated Screw and Tension Band with Kirschner Wire on Patella Fracture.

BACKGROUND: Patella fracture accounts for 1% of bone injury, of which anatomical reduction is of great significance to the recovery. Tension band with cannulated screw and Kirschner wire is commonly used methods for the treatment of displaced patella fracture. However, there is still some controversy on the clinical efficacy of the two treatment methods. OBJECTIVE: This study aimed at comparing the therapeutic effects of the cannulated screw and Kirschner wire tension bands on patella fracture and at providing more data basis for clinical selection of treatment methods for patella fracture. METHODS: Altogether, 146 patients with displaced patella fracture admitted to our hospital from March 2016 to February 2018 were selected and divided into two groups according to the different treatment methods. Among them, 71 patients received tension band with a cannulated screw (TBWCS group) and 75 patients received tension band with Kirschner wire (TBWKW group). Two groups of patients were compared in terms of surgical treatment effect after one year of treatment, complications within six months after the operation and operation-related indexes. The pain visual analogue scale (VAS) score, knee flexion degree, Lysholm score, and Bostman score were recorded at 1, 3, 6, and 12 months after operation, and the activity of daily living scale (ADL) score was evaluated at the last follow-up. RESULTS: During the operation of patella fracture patients, the intraoperative blood loss, hospitalization time, and knee flexion loss of patients in TBWCS group were less than those in the TBWKW group (P < 0.05), the starting time of postoperative functional exercise was earlier than that of patients in TBWKW group (P < 0.05), and the incidence rate of secondary operation was lower than that of patients in the TBWKW group (P < 0.05), but there was no statistical difference in the operation time, incision length, and postoperative fracture gap between the two groups. The results of curative effect analysis showed that the knee flexion, Lysholm score, and Bostman score of patients treated with tension band with cannulated screw were higher than those treated with Kirschner wire (P < 0.05), and VAS score was lower. Tension band with cannulated screw had a better curative effect on patella fracture (P < 0.05), lower complication rate (P < 0.05), and higher quality of life of patients (P < 0.05). CONCLUSION: Tension band with cannulated screw has a good curative effect on patella fracture, low incidence of complications, early start of postoperative functional exercise, and high quality of life.

MiR-196-5p involvement in selenium deficiency-induced immune damage via targeting of NFκBIA in the chicken trachea.

Dietary selenium (Se) deficiency can induce multifarious immune injury in tissues, accompanied by inflammation and a decreased expression of selenoproteins. The results of previous studies indicated that these issues are associated with Se-mediated microRNAs involved in immune regulation, although the specific mechanisms associated with these interactions have not been reported in the trachea of chickens. To explore the effects of Se deficiency in the trachea of chickens and the role of miR-196-5p, we established correlational models of tracheal injury in chickens. One hundred broilers were divided into four groups, including a control group (C group), a Se deficient group (L group), a lipopolysaccharide (LPS)-induced control group (C + LPS group) and a LPS-induced Se deficient group (L + LPS group). Light microscopy observations indicated that the infiltration of inflammatory cells was the major histopathological change caused by Se deficiency. Furthermore, ultrastructural observation of the tracheal epithelium and ciliary showed typical inflammatory signs owing to Se deficiency. We determined the targeting relationship between miR-196-5p and NFκBIA by bioinformatics analysis. In the case of Se deficiency, the changes were detected as follows: 19 selenoproteins showed different degrees of decrease (p < 0.05). Significant inhibition of both antimicrobial peptides and immunoglobulin production were observed (p < 0.05). IκB-α (NFκBIA) expression degraded with the increasing miR-196-5p (p < 0.05), and the NF-κB pathway was activated. Thereafter, we can see a significant increase in the mRNA levels of inflammatory cytokines-related genes (tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E (PTGE), interleukin (IL)-1ß, IL-6) and protein expression of NF-κB/iNOS pathway-related genes (NF-κB, iNOS, TNF-α, COX-2) (p < 0.05). The release of IL-2, interferon (IFN)-γ inhibited (p < 0.05) and the secretion of IL-4, IL-6 increased, suggesting the imbalance of Th1/Th2 (Th, helper T cell) cytokines. Compared to the control, the mRNA and protein expression levels of the anti-inflammatory system components with antioxidant activity (PPAR-γ/HO-1) were in an inhibitory state (p < 0.05). Antioxidases (SOD, CAT, GSH-Px) activities were suppressed. The activities of the peroxide markers (MDA, H2O2) were enhanced (p < 0.05). In addition, Se deficiency had a positive effect on the pathological changes of inflammation and the exceptional immunity in LPS-treated groups (p < 0.05). The results confirmed the relationship between miR-196-5p and NFκBIA in chickens, revealing that Se deficiency causes respiratory mucosal immune dysfunction via the miR-196-5p-NFκBIA axis, oxidative stress and inflammation. Moreover, Se deficiency exacerbates the inflammatory damage stimulated by LPS. Our work provides a theoretical basis for the prevention of tracheal injury owing to Se deficiency and can be used as a reference for comparative medicine. Furthermore, the targeted regulation of miR-196-5p and NFκBIA may contribute to the protection of the tracheal mucosa in chickens.

NLRP3 inflammasome mediates M1 macrophage polarization and IL-1ß production in inflammatory root resorption.

AIMS: To explore the involvement of NOD-like receptor protein 3 (NLRP3) inflammasome and M1 macrophage in root resorption (RR). METHODS: A rat RR model was established by excessive orthodontic force. After different force-loading time, the expression levels of NLRP3, caspase-1, and interleukin-1ß (IL-1ß) and distribution of M1 macrophages were analysed by immunohistochemistry and immunofluorescence staining in vivo. Then, the mechanism of NLRP3 activation was further verified by macrophage and human periodontal ligament cell (hPDLC) co-culture system in vitro. The production levels of NLRP3, caspase-1, pro-caspase-1, and IL-1ß in M1 macrophages in the co-culture system were detected by Western blot, and the polarization of CD68+IL-1ß+ M1 macrophages was detected by immunofluorescence staining. RESULTS: In the rat RR model, NLRP3, caspase-1, IL-1ß, and M1 macrophages were expressed in periodontal ligament, mainly concentrated around RR areas. Force-pre-treated hPDLCs promoted M1 macrophage polarization and the production of NLRP3, caspase-1, and IL-1ß in M1 macrophages in co-culture system. When MCC950, an inhibitor of NLRP3 inflammasome, was added, NLRP3 activation and M1 macrophage polarization were inhibited. CONCLUSIONS: In periodontal tissues, hPDLCs stimulated by force promoted M1 macrophage polarization and increased IL-1ß production by activating NLRP3 inflammasome in M1 macrophages, thus initiating the occurrence of RR.

MMP9 protects against LPS-induced inflammation in osteoblasts.

The matrix metalloproteinase (MMP) family is widely involved in the destruction of the pulp and apical tissues in the inflammatory process. MMP9 is closely related to oral inflammation. Nevertheless, the specific function of MMP9 during oral inflammation, as well as its mechanism, is not well understood. Our previous studies found that in experimentally induced apical periodontitis, more severe inflammation occurred in MMP9 knockout mice compared with the wild type mice. Moreover, the pathology phenomenon of alveolar bone destruction was even more evident in MMP9 knockout mice compared with the wild type mice. We proposed that MMP9 has "anti-inflammatory" properties. We aimed to study the effects of MMP9 on inflammatory response as well as on bone formation and bone destruction. We found a specific relationship between MMP9 and inflammation. qRT-PCR and Western blot revealed that the production of IL-1ß, TNF-α, RANK, RANKL, TLR2, and TLR4 was reduced by MMP9 in LPS-stimulated MC3T3-E1 cells. Meanwhile, the expressions of OPG and OCN were increased by MMP9 in LPS-stimulated cells. MMP9 plays a protective role in LPS-induced inflammation, thereby providing new clues to the prevention and treatment of apical periodontitis.

[Dens in dente: report of 3 cases and review of the literature].

PURPOSE: To explore the diagnosis and clinical treatment of dens in dente. METHODS: Preventive resin restoration, root canal treatment, apical barrier technique and apexification were used to treat three cases of dens in dente, respectively. The curative effects were assessed by general examinations and imageological examinations during postoperative follow-up visits. RESULTS: Three patients with different type and degree of dens in dente achieved good therapeutic effect and favorable prognosis through different treatment methods. CONCLUSIONS: Dens in dente is complex clinically and the treatment is difficult. Clinicians should improve the understanding of dens in dente. The keys to successful treatment are early diagnosis and early treatment. In addition, it is important to take proper measures according to the type and degree of dens in dente, to preserve the diseased tooth as much as possible.

Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development.

Klf5, a member of the Krüppel-like transcription factor family, has essential roles during embryonic development, cell proliferation, differentiation, migration and apoptosis. This study was to define molecular mechanism of Klf5 during the odontoblastic differentiation. The expression of Klf5, odontoblast-differentiation markers, Dspp and Dmp1 was co-localized in odontoblastic cells at different stages of mouse tooth development and mouse dental papilla mesenchymal cells. Klf5 was able to promote odontoblastic differentiation and enhance mineral formation of mouse dental papilla mesenchymal cells. Furthermore, overexpression of Klf5 could up-regulate Dspp and Dmp1 gene expressions in mouse dental papilla mesenchymal cells. In silico analysis identified that several putative Klf5 binding sites in the promoter and first intron of Dmp1 and Dspp genes that are homologous across species lines. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that Klf5 bound to these motifs in vitro and in intact cells. The responsible regions of Dmp1 gene were located in the promoter region while effect of Klf5 on Dspp activity was in the first intron of Dspp gene. Our results identify Klf5 as an activator of Dmp1 and Dspp gene transcriptions by different mechanisms and demonstrate that Klf5 plays a pivotal role in odontoblast differentiation.

Dentin Sialoprotein is a Novel Substrate of Matrix Metalloproteinase 9 in vitro and in vivo.

Dentin sialoprotein (DSP) is essential for dentinogenesis and processed into fragments in the odontoblast-like cells and the tooth compartments. Matrix metalloproteinase 9 (MMP9) is expressed in teeth from early embryonic to adult stage. Although MMP9 has been reported to be involved in some physiological and pathological conditions through processing substrates, its role in tooth development and whether DSP is a substrate of MMP9 remain unknown. In this study, the function of MMP9 in the tooth development was examined by observation of Mmp9 knockout (Mmp9-/-) mouse phenotype, and whether DSP is a substrate of MMP9 was explored by in vitro and in vivo experiments. The results showed that Mmp9-/- teeth displayed a phenotype similar to dentinogenesis imperfecta, including decreased dentin mineral density, abnormal dentin architecture, widened predentin and irregular predentin-dentin boundary. The distribution of MMP9 and DSP overlapped in the odontoblasts, the predentin, and the mineralized dentin, and MMP9 was able to specifically bind to DSP. MMP9 highly efficiently cleaved DSP into distinct fragments in vitro, and the deletion of Mmp9 caused improper processing of DSP in natural teeth. Therefore, our findings demonstrate that MMP9 is important for tooth development and DSP is a novel target of MMP9 during dentinogenesis.

The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor.

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin ß6. The 36 amino acid residues of DSP are sufficient to bind to integrin ß6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP (aa183-219) stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin ß6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP (aa183-219) in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin ß6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Photoluminescence properties of a new orange-red-emitting Sm(3+)-La3SbO7 phosphor.

The antimonate compound La3SbO7 has high chemical stability, lattice stiffness and thermal stability. Orange-red-emitting antimonate-based phosphors La3SbO7:xSm(3+) (x = 0.02, 0.05, 0.08, 0.10, 0.15, 0.20 and 0.25) were synthesized. The phase structure and photoluminescence properties of these phosphors were investigated. The emission spectrum obtained on excitation at 407 nm contained exclusively the characteristic emissions of Sm(3+) at 568, 608, 654 and 716 nm, which correspond to the transitions from (4)G5/2 to (6)H5/2, (6)H7/2, (6)H9/2 and (6)H11/2 of Sm(3+), respectively. The strongest emission was located at 608 nm due to the (4)G5/2→(6)H7/2 transition of Sm(3+), generating bright orange-red light. The critical quenching concentration of Sm(3+) in La3SbO7:Sm(3+) phosphor was determined as 10% and the energy transfer between Sm(3+) was found to be through an exchange interaction. The International Commission on Illumination chromaticity coordinates of the La3SbO7:0.10Sm(3+) phosphors are located in the orange-red region. The La3SbO7:Sm(3+) phosphors may be potentially used as red phosphors for white light-emitting diodes.

Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.

Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes.

Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription.

Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages.

Autophagy in SDF-1α-mediated DPSC migration and pulp regeneration.

Critical morphological requirements for pulp regeneration are tissues replete with vascularisation, neuron formation, and dentin deposition. Autophagy was recently shown to be related to angiogenesis, neural differentiation, and osteogenesis. The present study aimed to investigate the involvement of autophagy in stromal cell-derived factor-1α (SDF-1α)-mediated dental pulp stem cell (DPSC) migration and pulp regeneration, and identify its presence during pulp revascularisation of pulpectomised dog teeth with complete apical closure. In vitro studies showed that SDF-1α enhanced DPSCs migration and optimised focal adhesion formation and stress fibre assembly, which were accompanied by autophagy. Moreover, autophagy inhibitors significantly suppressed, whereas autophagy activator substantially augmented SDF-1α-stimulated DPSCs migration. Furthermore, after ectopic transplantation of tooth fragment/silk fibroin scaffold with DPSCs into nude mice, pulp-like tissues with vascularity, well-organised fibrous matrix formation, and new dentin deposition along the dentinal wall were generated in SDF-1α-loaded samples accompanied by autophagy. More importantly, in a pulp revascularisation model in situ, SDF-1α-loaded silk fibroin scaffolds improved the de novo ingrowth of pulp-like tissues in pulpectomised mature dog teeth, which correlated with the punctuated LC3 and Atg5 expressions, indicating autophagy. Our findings provide novel insights into the pulp regeneration mechanism, and SDF-1α shows promise for future clinical application in pulp revascularisation.

MMP9 deficiency increased the size of experimentally induced apical periodontitis.

INTRODUCTION: Apical periodontitis is an inflammation and destruction of periapical tissues. Matrix metalloproteinase-9 (MMP-9) is thought to be involved in periapical lesion formation and progression. The aim of this study was to evaluate the lesion progression in MMP-9 knockout (KO) mice compared with that in control mice (wild type [WT]). METHODS: The pulps of mouse mandibular first molars were exposed; animals were killed at 0, 7, 14, 21, and 28 days after surgery. Hematoxylin-eosin-stained sections were observed for the description of pulpal, apical, periapical features, and the periapical lesion size. The periapical lesion size was further measured with micro-computed tomographic imaging. The number of osteoclasts was also counted by tartrate-resistant acid phosphatase histoenzymology. Real-time polymerase chain reaction and immunohistochemistry were used to analyze the expression levels of receptor activator of NF-κB (RANK), receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), MMP-2, and MMP-8. RESULTS: There was a significant difference (P < .05) between the 2 types of animals regarding the periapical lesion size, which was larger in MMP-9 KO animals. No significant differences (P > .05) were found between WT and MMP-9 KO mice related to the osteoclast number as well as the pulpal, apical, and periapical features. More neutrophil cells were observed in MMP-9 KO animals than WT mice (P < .05). The expression levels of RANK, RANKL, OPG, IL-1ß, TNF-α, MMP-2, and MMP-8 were found up-regulated in MMP-9 KO mice (P < .05). CONCLUSIONS: MMP-9 KO animals developed larger periapical lesions with greater inflammatory response, indicating an important role of MMP-9 in the host's immune and inflammatory response to root canal and periradicular infection.