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BACKGROUND: Vascular endothelial growth factor (VEGF) is an important proangiogenic factor and has been considered as a key target of antiangiogenetic therapy in oral squamous cell carcinoma (OSCC). However, clinical application of bevacizumab, a specific VEGF antibody, didn't improve the survival rate of OSCC patients. One possible explanation is that VEGF gene expresses diverse isoforms, which associate with extracellular vesicles (EVs), and EVs potentially contribute to VEGF resistance to bevacizumab. However, clear solution is lacking in addressing this issue. METHODS: Expression of VEGF isoforms in OSCC cells was confirmed by reverse transcription and polymerase chain reaction (RT-PCR) and western blot. EVs isolated from OSCC cell's conditioned medium (CM) were characterized by western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunogold labeling and western blot were applied to study the VEGF on EVs. Tube formation assay and Matrigel plug angiogenesis assay were used for analyzing the angiogenesis capacity of EV-VEGF. RESULTS: The most popular isoforms expressed by VEGF gene are VEGF121, VEGF165 and VEGF189. In this study, we demonstrated that all three isoforms of mRNA could be detected at varying levels in OSCC cells, while only VEGF165 and VEGF189 proteins were found. CM derived from OSCC cells, both soluble and non-soluble forms of VEGF could be detected. We further confirmed the presence of VGEF189 bound to EVs as a non-soluble form. EV-bound VEGF189 presented angiogenic activity, which could not be neutralized by bevacizumab. It was found that VEGF189 bound to EVs by heparan sulfate proteoglycans (HSPG). In addition, the angiogenic effect of EV-VEGF could be reversed by surfen, a kind of HSPG antagonist both in vitro and in vivo. CONCLUSION: Antagonists targeting HSPG might potentially overcome the resistance of EV-VEGF to bevacizumab and serve as an alternative for anti-VEGF therapy in OSCC.
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BACKGROUND: Small extracellular vesicles derived from milk (Milk-sEVs) have the advantages of easy availability, low cost, low toxicity, and inhibition of inflammation. CD36 mediates inflammation stress in a variety of disease states. The purpose of this study was to investigate the role of Milk-sEVs in inhibiting fibroblast inflammation through CD36 and provide reference data for the treatment of chronic apical periodontitis. RESULTS: The addition of Milk-sEVs resulted in decreased expression of inflammation-related factors in L929 cells, and transcriptome sequencing screened for the DEG CD36 in the Milk-sEV treatment group under inflammation. The mouse model of apical periodontitis was successfully established, and CD36 expression increased with the development of inflammation. Transfection of si-CD36 into L929 cells reduced inflammation by inhibiting activation of the MAPK signaling pathway. CONCLUSIONS: CD36 expression increased with the development of apical periodontitis. In the setting of LPS-mediated inflammation, Milk-sEVs inhibited activation of the MAPK signaling pathway by decreasing the expression of CD36 in L929 cells and thereby reducing inflammation.
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Antígenos CD36 , Vesículas Extracelulares , Sistema de Señalización de MAP Quinasas , Leche , Periodontitis Periapical , Animales , Antígenos CD36/metabolismo , Antígenos CD36/genética , Vesículas Extracelulares/metabolismo , Ratones , Leche/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Periodontitis Periapical/metabolismo , Periodontitis Periapical/patología , Línea Celular , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/patología , MasculinoRESUMEN
The bacteria that invade the periapical tissue of teeth can directly damage tissue cells such as periapical fibroblasts, leading to an inflammatory response in the periapical tissue and ultimately resulting in bone destruction. We investigated the role of fibroblast activation protein α (FAPα) and integrin α5 (ITGA5) in periapical bone destruction. This study found that FAPα and ITGA5 were highly expressed in human tissues from patients with chronic apical periodontitis. Osteoclast differentiation decreased when FAPα or ITGA5 was silenced and inhibited. The results of protein molecular docking showed that FAPα had good binding affinity to ITGA5, and its free energy was -14.5â¯kcal/mol. Immunofluorescence staining and co-immunoprecipitation showed that FAPα and ITGA5 formed protein complexes in the inflammatory microenvironment. In conclusion, this study proved that FAPα and ITGA5 participate in the regulation of osteoclast differentiation by forming protein complexes in the inflammatory microenvironment, which then regulates the occurrence and development of chronic apical periodontitis.
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Proteínas de la Membrana , Periodontitis Periapical , Periodontitis , Humanos , Integrina alfa5/metabolismo , Simulación del Acoplamiento Molecular , EndopeptidasasRESUMEN
Bone regeneration is a dynamic process that involves angiogenesis and the balance of osteogenesis and osteoclastogenesis. In bone tissue engineering, the transplantation of mesenchymal stem cells (MSCs) is a promising approach to restore bone homeostasis. MSCs, particularly their small extracellular vesicles (sEVs), exert therapeutic effects due to their paracrine capability. Increasing evidence indicates that microRNAs (miRNAs) delivered by sEVs from MSCs (MSCs-sEVs) can alter gene expression in recipient cells and enhance bone regeneration. As an ideal delivery vehicle of miRNAs, MSCs-sEVs combine the high bioavailability and stability of sEVs with osteogenic ability of miRNAs, which can effectively overcome the challenge of low delivery efficiency in miRNA therapy. In this review, we focus on the recent advancements in the use of miRNAs delivered by MSCs-sEVs for bone regeneration and disorders. Additionally, we summarize the changes in miRNA expression in osteogenic-related MSCs-sEVs under different microenvironments.
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Chronic apical periodontitis (CAP) is a disease with characteristics of inflammation and bone loss. In this study, our objective was to examine the function of small extracellular vesicles (sEVs) obtained from milk in encouraging osteogenic differentiation and inhibiting inflammation by miR-21 in CAP. The expression of miR-21 was detected using qRT-PCR in human CAP samples. The impact of miR-21 on the process of osteogenic differentiation was investigated using CCK-8, qRT-PCR, immunofluorescence staining, and Western blot analysis. The evaluation of RAW 264.7 cell polarization and the assessment of inflammatory factor expression were conducted through qRT-PCR. The influence of sEVs on MC3T3-E1 cells and RAW 264.7 cells was examined, with a particular emphasis on the involvement of miR-21. In human CAP samples, a decrease in miR-21 expression was observed. MiR-21 increased the expression of osteogenesis-related genes and M2 polarization genes while decreasing the expression of M1 polarization genes and inflammatory cytokines. Treatment with milk-derived sEVs also promoted osteogenesis and M2 polarization while inhibiting M1 polarization and inflammation. Conversely, the addition of miR-21 inhibitors resulted in opposite effects. Our results indicated that sEVs derived from milk had a positive effect on bone formation and activation of anti-inflammatory (M2) macrophages and simultaneously reduced inflammation by regulating miR-21 in CAP.
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Vesículas Extracelulares , MicroARNs , Animales , Diferenciación Celular/genética , Inflamación/genética , MicroARNs/genética , Leche , Osteogénesis/genética , RatonesRESUMEN
Icariin (ICA) played an important role in the treatment of inflammatory bone defects. However, pharmacokinetic studies have shown that its poor bioavailability limited its application. In this context, we isolated bovine milk-derived sEV and prepared sEV-ICA to improve the osteogenic effect of ICA. In this study, we successfully constructed sEV-ICA. sEV-ICA was found to have significantly higher osteogenic efficiency than ICA in cell culture and cranial bone defect models. Mechanistically, bioinformatics analysis predicted that signal transducers and activators of transcription 5 (STAT5a) may bind to the GJA1 promoter, while luciferase activity assays and chromatin immunoprecipitation (ChIP) experiments confirmed that STAT5a directly binded to the GJA1 promoter to promote osteogenesis. We proved that compared with ICA, sEV-ICA showed a better effect of promoting bone repair in vivo and in vitro. In addition, sEV-ICA could promote osteogenesis by promoting the combination of STAT5a and GJA1 promoter. In summary, as a complex drug delivery system, sEV-ICA constituted a rapid and effective method for the treatment of bone defects and could improve the osteogenic activity of ICA.
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Leche , Osteogénesis , Animales , Diferenciación Celular , Flavonoides/farmacología , Flavonoides/uso terapéuticoRESUMEN
BACKGROUND: Phosphoinositide 3-kinase (PI3K) is located within cells, and is involved in regulating cell survival, proliferation, apoptosis and angiogenesis. The purpose of this study was to investigate the role of PI3K in the process of bone destruction in apical periodontitis, and provide reference data for the treatment of this disease. METHODS: The relative mRNA expression of PI3K, Acp5 and NFATc1 in the normal human periodontal ligament and in chronic apical periodontitis were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). A mouse model of apical periodontitis was established by root canal exposure to the oral cavity, and HE staining was used to observe the progress of apical periodontitis. Immunohistochemical staining was used to detect the expression of PI3K and AKT in different stages of apical periodontitis, while enzymatic histochemical staining was used for detection of osteoclasts. An Escherichia coli lipopolysaccharide (LPS)-mediated inflammatory environment was also established at the osteoclast and osteoblast level, and osteoclasts or osteoblasts were treated with the PI3K inhibitor LY294002 to examine the role of PI3K in bone resorption. RESULTS: The expression of PI3K, Acp5 and NFATc1 genes in chronic apical periodontitis sample groups was significantly increased relative to healthy periodontal ligament tissue (P < 0.05). Mouse apical periodontitis was successfully established and bone resorption peaked between 2 and 3 weeks (P < 0.05). The expression of PI3K and Akt increased with the progression of inflammation, and reached a peak at 14 days (P < 0.05). The gene and protein expression of PI3K, TRAP and NFATc1 in osteoclasts were significantly increased (P < 0.05) in the E. coli LPS-mediated inflammatory microenvironment compared to the normal control group. Meanwhile in osteoblasts, the gene and protein expression of PI3K, BMP-2 and Runx2 were significantly reduced (P < 0.05) in the inflammatory microenvironment. With the addition of LY294002, expressions of bone resorption-related factors (TRAP, NFATc1) and bone formation-related factors (BMP-2, Runx2) significantly decreased (P < 0.05). CONCLUSIONS: Under the inflammatory environment induced by LPS, PI3K participates in the occurrence and development of chronic apical periodontitis by regulating the proliferation and differentiation of osteoclasts and osteoblasts.
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Resorción Ósea , Lipopolisacáridos/metabolismo , Periodontitis Periapical , Periodontitis , Fosfatidilinositol 3-Quinasa , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Escherichia coli , Humanos , Ratones , Osteoclastos , Periodontitis/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Small extracellular vesicles (sEVs) are an important component in the paracrine pathway. They can be used as a substitute for seed cells and have shown good application prospects in promoting bone regeneration. Cow's milk could be used as a source of sEVs with good biocompatibility and cost-effectiveness, with easy availability, low cost and low toxicity. This study focused on the role and mechanism of small extracellular vesicles derived from milk in bone repair. In order to explore the mechanism via which Milk-sEVs promote bone repair, we screened the differential gene GJA1 in Milk-sEV-treated osteoblasts through transcriptome chips, and verified the transcript AP3B1 of GJA1 through chromatin immunoprecipitation (CHIP). We have proved by in vivo and in vitro experiments that milk-derived sEVs (Milk-sEVs) increase the repair ability of bone tissue, and promote expression of the osteogenic gene GJA1 through the transcript AP3B1.
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Vesículas Extracelulares , Nanoestructuras , Animales , Bovinos , Vesículas Extracelulares/metabolismo , Femenino , Leche , OsteogénesisRESUMEN
To promote the replacement of nondegradable petrochemical-based polymers with green polylactic acid (PLA) materials, aliphatic chains-grafted cellulose nanocrystals (ECNCs) were prepared and used as nanofillers to overcome the disadvantage of poor toughness of PLA. ECNCs with core-corona structures were obtained by modifying cellulose nanocrystals (CNC) with valeryl chloride, octanoyl chloride, dodecanoyl chloride, and stearoyl chloride. ECNCs consists of a cellulose crystalline core and a soft esterified corona layer with aliphatic chains. Among the diverse ECNCs, the obtained EOCNC by esterification of octanoyl chloride exhibited most efficient enhancement of the toughness of PLA. Specifically, PLA/EOCNC-1% film displayed the best elongation at breakage of 108%, which was 6.4 times that of pure PLA. The esterified outer layer of ECNCs, which improves the interfacial compatibility, is one of the key factors contributing to toughening of PLA. These ECNCs with core-corona structure open up new directions for the application of PLA advanced composites.
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Icariin is a class IV drug of low solubility, permeability, and poor bioavailability. Synthetic nanomaterials have developed rapidly. However, some literatures point out that synthetic nanomaterials such as liposomes, aptamers, metal nanoparticles, and nanogels have high toxicity and are affected by the reticuloendothelial system or mononuclear phagocyte system. It is known that exosomes could be used as an ideal clinical drug delivery vehicle to avoid the above-mentioned problems to a certain extent. Studies have shown that drugs can be loaded into exosomes by passive and active loading. We used Fetal bovine serum (FBS) exosomes to carry Icariin for the first time in this experiment, FBS exosomes-Icariin (FBS EXO-ICA) more effectively promoted the proliferation of osteoblasts and bone regeneration than Icariin alone. FBS EXO-ICA could become a new nano scale drug formulation for treating diseases associated with bone loss.
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BACKGROUND/PURPOSE: Composite resin is currently the most widely used dental restoration material. Previous studies have demonstrated that the application of Chlorhexidine (CHX) on the dentin surface after acid etching can result in an improvement in the integrity and stability of tooth restoration through time. In order to better understand whether CHX can help improve the stability of the resin-dentin bond strength, in this study, a comprehensive review of the effect of adding CHX to the adhesive system on the stability of immediate and long-term resin-dentin bond strength was conducted. MATERIALS AND METHODS: This article was written in accordance with the PRISMA Statement and is registered on the International Prospective Register of Systematic Reviews (registration number CRD42018084962). Six electronic databases including PubMed, Embase, Cochrane library, ISI Web of Science, ClinicalTrials.gov and China National Knowledge Infrastructure (CNKI) were searched up to October, 2018. Ten articles were selected from 340 possible eligible articles for meta-analysis, and 41 sets of data were analyzed in the meta-analysis. RESULTS: The results indicated that the use of 0.1% and 0.2% CHX does not adversely affect the immediate bond strength (pâ¯>â¯0.05), but both 0.1% and 0.2% CHX increased bond strength compared with the control group over 12 months (pâ¯<â¯0.05). However, this trend does not represent a longer period of aging. CONCLUSION: In these in vitro clinical trials, CHX incorporated into the bonding systems maintained the stability of bond strength.
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The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. In this study, we isolated primary carcinoma-associated fibroblasts (CAFs) from four human oral squamous cell carcinoma (OSCC) specimens. Compared with other non-extracellular vesicle components, CAF-derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in human umbilical vein endothelial cells (HUVEC) was dependent on the association between sEVs and VEGF. In addition, sEV-bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab-resistant manner. VEGF was found to interact with heparan sulfate proteoglycans on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab. These findings suggest that the combined use of heparinase and bevacizumab might inhibit angiogenesis in patients with high levels of sEV-bound VEGF.
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Bevacizumab/uso terapéutico , Fibroblastos Asociados al Cáncer/fisiología , Vesículas Extracelulares/fisiología , Neoplasias de la Boca/irrigación sanguínea , Neovascularización Patológica/etiología , Carcinoma de Células Escamosas de Cabeza y Cuello/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/fisiología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Liasa de Heparina/farmacología , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Refractory periapical periodontitis, which is a persistent infection after root canal treatment, still has no effective treatment. Its most common pathogen is Enterococcus faecalis. Here, the precursor of phytosteroids, dioscin, is introduced to fight against the inflammation induced by Enterococcus faecalis. The findings suggest that dioscin inhibits the nuclear transport of NF-κB and the expression of reactive oxygen species (ROS) induced by lipoteichoic acid from the Enterococcus faecalis. The decrease in mRNA and protein levels of NLRP3, Caspase-1, and IL-1ß is observed in dioscin treated mouse macrophages. In the MC3T3-E1 cells, dioscin also promotes the expression of osteogenic-related factors, ALP, Runx2, and OCN. The increased formation of mineralized nodules after the application of dioscin further indicates that dioscin has the potential to promote osteogenesis. The above results suggest dioscin can be a potential root canal irrigation or root canal sealant for the treatment of refractory apical periodontitis.
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Diosgenina/análogos & derivados , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Osteogénesis/efectos de los fármacos , Animales , Línea Celular , Diosgenina/farmacología , Humanos , Lipopolisacáridos/farmacología , Ratones , Ácidos Teicoicos/farmacologíaRESUMEN
Although the role of cathepsin C (Cat C) in inflammation is gradually being elucidated, its function in periapical periodontitis, which is one of the most common infectious diseases worldwide, has not been studied. This study evaluated a surgically-induced model of periapical periodontitis in cathepsin C (Cat C) knock-down (KD) mice, which was constructed with a tetracycline operator, to evaluate the role of Cat C in the pathogenesis and progression of periapical periodontitis. Our results showed, for the first time, that there was a statistically significant increase in the expression of Cat C as periapical periodontitis progressed; this increase started from 1 week after surgery and reached a peak at 3 weeks after surgery, before gradually decreasing. The volume of periapical bone resorption in Cat C KD mice was significantly smaller than that in wild-type mice at 3 and 4 weeks after surgery (P<0.05). Inflammatory cell infiltration into the apical tissues of wild-type mice was also significantly higher than that of Cat C KD mice. The expression of receptor activator of nuclear factor-κB ligand (RANKL) in wild-type mice was also higher than that in Cat C KD mice. The difference in the number of osteoclasts in the apical area between the two groups was statistically significant after 2 weeks. Correlation analysis showed that there was a significant correlation between Cat C and RANKL expression (r= 0.835). Therefore, our data indicated that Cat C promoted the apical inflammation and bone destruction in mice.
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OBJECTIVE: To observe the effect of Bruton's tyrosine kinase (BTK) on the proliferation and differentiation of osteoclasts and to explore the mechanism of BTK on bone destruction in periapical periodontitis. METHODS: After RAW264.7 cells induced with 100 ng·L⻹ receptor activator for nuclear factor-κB ligand (RANKL) for 5 days, osteoclast induction was confirmed by light microscopy, tartrate-resistant acid phosphatase (TRAP) staining, and quantitative real-time PCR (RT-qPCR). Then, BTK-small interfering RNA (BTK-siRNA) was transfected into cells induced for 5 days. After 24 h, the expression of TRAP mRNA was measured using RT-qPCR, and the proliferation and differentiation of osteoclasts were detected using CCK-8 and TRAP activity assay. Statistical analysis was performed. RESULTS: After RAW264.7 was induced with RANKL for 5 days, a large number of round, ellipse, irregularly protuberant, and TRAP-positive macrophages were observed under light microscopy. The expression of TRAP mRNA significantly reduced after 24 h of BTK-siRNA transfection (P<0.05). The detection of CCK-8 and TRAP activities showed that the proliferation and differentiation of osteoclasts significantly decreased (P<0.05). CONCLUSIONS: Silencing of BTK can inhibit the proliferation and differentiation of osteoclasts. BTK can be used as a new target for the inhibition of osteoclasts.
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Agammaglobulinemia Tirosina Quinasa , Osteoclastos , Diferenciación Celular , Proliferación Celular , Macrófagos , Ligando RANKRESUMEN
OBJECTIVES: This study aimed to investigate the role of JAK2-STAT3 (Janus kinase 2/signal transducer and activator of transcription 3) in periapical disease caused by Enterococcus faecalis, as well as the correlation between lipoteichoic acid (LTA) in E. faecalis and the activity of the JAK2-STAT3 signaling pathway and osteoclast formation. MATERIALS AND METHODS: A rat model of periapical periodontitis induced by E. faecalis was established. Periapical bone resorption was confirmed by HE staining. The expression of JAK2, p-JAK2, STAT3, and p-STAT3 was assessed with immunohistochemical staining. Osteoclasts were observed through enzyme histochemical staining. LTA acted on mouse osteoclast precursor cells (RAW264.7 cells); a JAK2 inhibitor (AG490) was used to inhibit the JAK2-STAT3 pathway in RAW264.7 cells. The expression of proteins in the JAK2-STAT3 pathway and TRAP (tartrate resistant acid phosphatase) in RAW264.7 cells was also detected. RESULTS: Rat periapical periodontitis was successfully established and bone resorption peaked at day 21. The expression of critical components in the JAK2-STAT3 pathway increased with the progression of inflammation. LTA promoted the differentiation of RAW264.7 cells into osteoclasts. NFATc1 was highly expressed and was inhibited by AG490. CONCLUSIONS: JAK2-STAT3 signaling pathway plays an important role in the process of periapical bone resorption and osteoclastogenesis.
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Resorción Ósea , Enterococcus faecalis/fisiología , Janus Quinasa 2/metabolismo , Osteoclastos/fisiología , Osteogénesis , Periodontitis Periapical/fisiopatología , Factor de Transcripción STAT3/metabolismo , Animales , Regulación de la Expresión Génica , Janus Quinasa 2/genética , Ratones , Osteoclastos/microbiología , Periodontitis Periapical/etiología , Ratas , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
Chronic apical periodontitis is characterized by alveolar bone absorption in the apical region and is the result of the participation of various inflammatory mediators. Studies have shown that the Bruton tyrosine kinase- (Btk-) phospholipase Cγ2 (PLCγ2) signaling pathway plays an important role in bone absorption, but it is unknown whether it plays a role in apical periodontitis bone destruction. Therefore, this study verified the role of Btk and PLCγ2 in bone resorption of apical periodontitis by in vivo and in vitro experiments. In the in vivo experiment, a mice model of apical periodontitis was established; apical bone resorption was confirmed by the numbers of osteoclasts and HE staining. Btk, PLCγ2, and nuclear factor of activated T-cells 1 (NFATc-1) were detected by immunohistochemical staining. In the in vitro experiment, lipopolysaccharides (LPS) were used to stimulate osteoclast precursor cell RAW264.7 to establish an inflammatory microenvironment and detect osteoclast differentiation. By silencing Btk, the expression of Btk, PLCγ2, and NFATc-1 was detected by real-time qPCR and Western blot, and osteoclastogenesis was detected by enzyme histochemical staining to further confirm the role of Btk in bone resorption. It was found that the expression of Btk, PLCγ2, and NFATc-1 changed significantly with the progression of inflammation and bone destruction, indicating that Btk and PLCγ2 may be involved in the progression of inflammation in apical periodontitis and bone absorption. In vitro experiments confirmed that the differentiation of osteoclasts and the expression of PLCγ2 and NFATc-1 were significantly inhibited after silencing Btk expression, but osteoclast precursor cells could be differentiated due to the proinflammatory factor lipopolysaccharide. This study demonstrates that Btk and PLCγ2 are key factors involved in the apical inflammatory response and bone destruction.
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Agammaglobulinemia Tirosina Quinasa/metabolismo , Periodontitis Periapical/metabolismo , Fosfolipasa C gamma/metabolismo , Animales , Resorción Ósea/metabolismo , Diferenciación Celular/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Osteoclastos/fisiología , Células RAW 264.7 , Transducción de Señal/fisiologíaRESUMEN
To determine the expression of Bruton's tyrosine kinase (BTK) in refractory periapical periodontitis and analyze the relationship between BTK and bone resorption in refractory periapical periodontitis. The mechanism of bone resorption is also discussed. The OneArray Plus expression microarray was used to screen for genes related to refractory periapical periodontitis. Real-time PCR was used to detect the expression of BTK in refractory periapical periodontitis tissues. A model of periapical periodontitis was established by sealing E.faecalis into the pulp of rats. To establish a model of E.faecalis LTA infection of osteoclasts, the relationship between BTK and bone destruction during refractory periapical periodontitis was analyzed. OneArray Plus expression microarray results showed that we found that the expression of 1787 genes in the two samples was different. After validating these samples, we found that BTK was closely related to refractory periapical periodontitis. The results showed that the expression of BTK in refractory periapical periodontitis tissues was higher than that in normal tissues. Immunohistochemistry, enzyme histochemistry and real-time PCR showed that the BTK expression curve in the experimental model resembled a reverse V shape from week 1 to week 4. Osteoclasts were cultured in vitro and treated with E. faecalis LTA. The expression of BTK in the E. faecalis model was greater than that in the control group. BTK played an important role in the progression of refractory periapical periodontitis.
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Agammaglobulinemia Tirosina Quinasa/biosíntesis , Periodontitis Periapical/enzimología , Periodontitis Periapical/patología , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Osteoclastos/enzimología , Osteoclastos/patología , Periodontitis Periapical/microbiología , Células RAW 264.7 , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: Periapical periodontitis is caused by bacterial infection and results in both one destruction and tooth loss. Osteopontin (OPN) is a secreted phosphorylated glycoprotein that participates in bone metabolism. METHODS: Thirty-three patients with chronic periapical periodontitis and 10 patients who had undergone the orthodontic removal of healthy tooth tissue (control) at the periodontal ligament were investigated, and an animal model of mouse periapical periodontitis was established for an in vivo analysis. The relationship between OPN and bone destruction during periapical periodontitis was analyzed. Osteoblasts and osteoclasts were cultured in vitro and treated with lipopolysaccharide. An inhibitor of NF-κB was used to pretreat the transfected cells. RESULTS: OPN increased osteoclast proliferation and differentiation, but reduced osteoblasts proliferation and differentiation. OPN activated the NF-κB pathway during periapical periodontitis and accelerated the transfer and phosphorylation of P65 from the cytoplasm to the nucleus. CONCLUSION: This study demonstrated that OPN played important roles in the progression of periapical periodontitis, and a dual role in bone metabolism during periapical periodontitis, linking osteoclasts and osteoblasts. The underlying mechanism may be related to the NF-κB pathway.
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FN-kappa B/metabolismo , Osteopontina/metabolismo , Periodontitis Periapical/patología , Transducción de Señal , Animales , Catepsina K/genética , Catepsina K/metabolismo , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Masculino , Mandíbula/diagnóstico por imagen , Ratones , Ratones Endogámicos C57BL , Osteopontina/antagonistas & inhibidores , Osteopontina/genética , Periodontitis Periapical/diagnóstico por imagen , Periodontitis Periapical/metabolismo , Tejido Periapical/diagnóstico por imagen , Tejido Periapical/metabolismo , Ligamento Periodontal/metabolismo , Células RAW 264.7 , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Orthodontic treatments often include dental displacement using a fixed retainer such as braces, which may result in the accumulation of plaque that provides a suitable environment for microorganisms to cause oral infection. So, this study was designed to investigate the microbial diversity among orthodontic patients and healthy individuals. METHODS: Fifty individuals i.e. 30 orthodontic patients and 20 normal individuals were included in this study. Samples were collected during the midterm of orthodontic treatment (10-12 months). Saliva samples were collected and total DNA was isolated. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) with universal primers targeting the V3 region of 16s rRNA was used to study the bacterial diversity among different orthodontic and control groups. After DGGE profile analysis, the predominant product bands from the gel were excised, cloned, and sequenced to confirm the taxonomic identity followed by its quantification by using real-time PCR with gene-specific primers. RESULTS: Both orthodontic treatment and control groups formed two distinct clustering profiles, but the Shannon-Weaver index (H') indicated greater microbial diversity in the orthodontic group (Pâ¯=â¯0.08). Sequence analysis and real-time PCR revealed a greater number of Pseudomonas spp. in the orthodontic group, while there was no significant difference in Streptococcal spp. CONCLUSION: This study suggested alterations in the oral microbiota following orthodontic treatment would provide diagnostic tools to identify prevalent microbes associated with oral infections that may prove useful for developing future therapies.