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The consumption of a high-fat diet (HFD) has been linked to osteoporosis and an increased risk of fragility fractures. However, the specific mechanisms of HFD-induced osteoporosis are not fully understood. Our study shows that exposure to an HFD induces premature senescence in bone marrow mesenchymal stem cells (BMSCs), diminishing their proliferation and osteogenic capability, and thereby contributes to osteoporosis. Transcriptomic and chromatin accessibility analyses revealed the decreased chromatin accessibility of vitamin D receptor (VDR)-binding sequences and decreased VDR signaling in BMSCs from HFD-fed mice, suggesting that VDR is a key regulator of BMSC senescence. Notably, the administration of a VDR activator to HFD-fed mice rescued BMSC senescence and significantly improved osteogenesis, bone mass, and other bone parameters. Mechanistically, VDR activation reduced BMSC senescence by decreasing intracellular reactive oxygen species (ROS) levels and preserving mitochondrial function. Our findings not only elucidate the mechanisms by which an HFD induces BMSC senescence and associated osteoporosis but also offer new insights into treating HFD-induced osteoporosis by targeting the VDR-superoxide dismutase 2 (SOD2)-ROS axis.
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Senescência Celular , Dieta Hiperlipídica , Células-Tronco Mesenquimais , Osteoporose , Espécies Reativas de Oxigênio , Receptores de Calcitriol , Células-Tronco Mesenquimais/metabolismo , Animais , Receptores de Calcitriol/metabolismo , Osteoporose/etiologia , Osteoporose/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Proliferação de Células , Osteogênese/fisiologia , Transdução de Sinais , MultiômicaRESUMO
PURPOSE: To explore the mechanism underlying autophagy disruption in gingival epithelial cells (GECs) in diabetic individuals. METHODS AND MATERIALS: Bone marrow-derived macrophages (BMDMs) and GECs were extracted from C57/bl and db/db mice, the exosomes (Exo) were isolated from BMDMs. qRTâPCR and Western blotting were performed to analyse gene expression. The AnimalTFDB database was used to identify relevant transcription factors, and miRNA sequencing was utilised to identify relevant miRNAs with the aid of the TargetScan/miRDB/miRWalk databases. A dual-luciferase assay was conducted to verify intermolecular targeting relationships. RESULTS: Similar to BMDMs, BMDM-derived Exos disrupted autophagy and exerted proinflammatory effects in GEC cocultures, and ATG7 may play a vital role. AnimalTFDB database analysis and dual-luciferase assays indicated that NR5A2 is the most relevant transcription factor that regulates Atg7 expression. SiRNA-NR5A2 transfection blocked autophagy in GECs and exacerbated inflammation, whereas NR5A2 upregulation restored ATG7 expression and ameliorated ExoDM-mediated inflammation. MiRNA sequencing, with TargetScan/miRDB/miRWalk analyses and dual-luciferase assays, confirmed that miR-381-3p is the most relevant miRNA that targets NR5A2. MiR-381-3p mimic transfection blocked autophagy in GECs and exacerbated inflammation, while miR-381-3p inhibitor transfection restored ATG7 expression and attenuated ExoDM-mediated inflammation. CONCLUSION: BMDM-derived Exos, which carry miR-381-3p, inhibit NR5A2 and disrupt autophagy in GECs, increasing periodontal inflammation in diabetes.
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Autofagia , Células Epiteliais , Exossomos , Gengiva , Macrófagos , Camundongos Endogâmicos C57BL , MicroRNAs , Animais , Masculino , Camundongos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Células Epiteliais/metabolismo , Exossomos/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
The gingiva is a key oral barrier that protects oral tissues from various stimuli. A loss of gingival tissue homeostasis causes periodontitis, one of the most prevalent inflammatory diseases in humans. The human gingiva exists as a complex cell network comprising specialized structures. To understand the tissue-specific pathophysiology of the gingiva, we applied a recently developed spatial enhanced resolution omics-sequencing (Stereo-seq) technique to obtain a spatial transcriptome (ST) atlas of the gingiva in healthy individuals and periodontitis patients. By utilizing Stereo-seq, we identified the major cell types present in the gingiva, which included epithelial cells, fibroblasts, endothelial cells, and immune cells, as well as subgroups of epithelial cells and immune cells. We further observed that inflammation-related signalling pathways, such as the JAK-STAT and NF-κB signalling pathways, were significantly upregulated in the endothelial cells of the gingiva of periodontitis patients compared with those of healthy individuals. Additionally, we characterized the spatial distribution of periodontitis risk genes in the gingiva and found that the expression of IFI16 was significantly increased in endothelial cells of inflamed gingiva. In conclusion, our Stereo-seq findings may facilitate the development of innovative therapeutic strategies for periodontitis by mapping periodontitis-relevant genes and pathways and effector cells.
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Gengiva , Periodontite , Humanos , Gengiva/metabolismo , Transcriptoma , Células Endoteliais/metabolismo , Periodontite/genética , Periodontite/metabolismo , Perfilação da Expressão GênicaRESUMO
Periodontitis (PD) is a common chronic oral inflammatory disease that cause alveolar bone loss. Current strategies for bone regeneration achieve limited results in PD. The aberrant host osteoimmunity to pathogenic bacteria is responsible for the destruction of alveolar bone in PD. We aimed to investigate the distinctive activity of immune cells in PD to create more effective and precise therapeutic approaches for treating PD. In this study, we revealed that neutrophils in the inflamed alveolar bone of PD patients expressed higher levels of CXCR1/2 and had a stronger pro-inflammatory capacity and chemotactic ability than that in healthy individuals. Suppressing the recruitment of neutrophils to inflamed sites with the CXCR1/2 inhibitor reparixin reduced alveolar bone loss in PD mice. In this study, we not only revealed that neutrophils exhibit a heterogeneously stronger pro-inflammatory capacity in the inflamed alveolar bone of PD patients but also provided a precise therapeutic treatment for PD involving the suppression of neutrophil recruitment.
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Perda do Osso Alveolar , Periodontite , Humanos , Camundongos , Animais , Perda do Osso Alveolar/patologia , Infiltração de Neutrófilos , Neutrófilos , BactériasRESUMO
Cognitive decline has been linked to periodontitis through an undetermined pathophysiological mechanism. This study aimed to explore the mechanism underlying periodontitis-related cognitive decline and identify therapeutic strategies for this condition. Using single-nucleus RNA sequencing we found that changes in astrocyte number, gene expression, and cellâcell communication were associated with cognitive decline in mice with periodontitis. In addition, activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome was observed to decrease the phagocytic capability of macrophages and reprogram macrophages to a more proinflammatory state in the gingiva, thus aggravating periodontitis. To further investigate this finding, lipid-based nanoparticles carrying NLRP3 siRNA (NPsiNLRP3) were used to inhibit overactivation of the NLRP3 inflammasome in gingival macrophages, restoring the oral microbiome and reducing periodontal inflammation. Furthermore, gingival injection of NPsiNLRP3 reduced the number of Serpina3nhigh astrocytes in the hippocampus and prevented cognitive decline. This study provides a functional basis for the mechanism by which the destruction of periodontal tissues can worsen cognitive decline and identifies nanoparticle-mediated restoration of gingival macrophage function as a novel treatment for periodontitis-related cognitive decline.
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Epithelial-mesenchymal interactions occur during tooth development. The dental epithelium (DE) is regarded as the signal center that regulates tooth morphology. However, the mechanism by which DE regulates the differentiation of mesenchyme-derived dental papilla (DP) into odontoblasts remains unclear. Using miniature pigs as a model, we analyzed the expression profiles of the DE and DP during odontoblast differentiation using high-throughput RNA sequencing. The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is one of the most enriched pathways in both DE and DP. The PI3K/AKT pathway was first activated in the inner enamel epithelium but not in the DP on embryonic day 50. This pathway was then activated in the odontoblast layer on embryonic day 60. We showed that AKT activation promoted odontoblast differentiation of DP cells. We further demonstrated that activation of PI3K/AKT signaling in the DE effectively increased the expression levels of AKT and dentin sialophosphoprotein in DP cells. Additionally, we found that DE cells secreted collagen type IV alpha 6 chain (COL4A6) downstream of epithelial AKT signaling to positively regulate mesenchymal AKT levels. Therefore, our data suggest that PI3K/AKT signaling from the DE to the DP promotes odontoblast differentiation via COL4A6 secretion.
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Odontoblastos , Proteínas Proto-Oncogênicas c-akt , Animais , Suínos , Odontoblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Diferenciação Celular/genética , EpitélioRESUMO
Apical periodontitis (AP) is an infectious disease that causes periapical tissue inflammation and bone destruction. Ferroptosis, a novel type of regulated cell death, is closely associated with inflammatory diseases and the regulation of bone homeostasis. However, the exact involvement of ferroptosis in the bone loss of AP is not fully understood. In this study, human periapical tissues were collected, and a mouse model was established to investigate the role of ferroptosis in AP. Colocalization staining revealed that ferroptosis in macrophages contributes to the inflammatory bone loss associated with AP. A cell model was constructed using RAW 264.7 cells stimulated with LPS to further explore the mechanism underlying ferroptosis in macrophages upon inflammatory conditions, which exhibited ferroptotic characteristics. Moreover, downregulation of NRF2 was observed in ferroptotic macrophages, while overexpression of NRF2 upregulated the level of FSP1, leading to a reduction in reactive oxygen species (ROS) in macrophages. Additionally, ferroptotic macrophages released TNF-α, which activated the p38 MAPK signaling pathway and further increased ROS accumulation in macrophages. In vitro co-culture experiments demonstrated that the osteogenic ability of mouse bone marrow stromal cells (BMSCs) was suppressed with the stimulation of TNF-α from ferroptotic macrophages. These findings suggest that the TNF-α autocrine-paracrine loop in ferroptotic macrophages can inhibit osteogenesis in BMSCs through the NRF2/FSP1/ROS signaling pathway, leading to bone loss in AP. This study highlights the potential therapeutic value of targeting ferroptosis in the treatment of inflammatory bone diseases.
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Ferroptose , Periodontite Periapical , Animais , Humanos , Camundongos , Ferroptose/genética , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Periodontite Periapical/genética , Periodontite Periapical/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Melatonin has been reported to improve nonalcoholic fatty liver disease (NAFLD), and exploring the underlying mechanisms will be beneficial for better treatment of NAFLD. Choline-deficient high-fat diet (CDHFD)- and methionine/choline-deficient diet (MCD)-fed mice with melatonin intervention exhibit significantly decreased liver steatosis, lobular inflammation, and focal liver necrosis. Single-cell RNA sequencing reveals that melatonin selectively inhibits pro-inflammatory CCR3+ monocyte-derived macrophages (MoMFs) and upregulates anti-inflammatory CD206+ MoMFs in NAFLD mice. Liver-infiltrating CCR3+CD14+ MoMFs are also significantly increased in patients with NAFLD. Mechanistically, melatonin receptor-independent BTG2-ATF4 signaling plays a role in the regulation of CCR3+ MoMF endoplasmic reticulum stress, survival, and inflammation. In contrast, melatonin upregulates CD206+ MoMF survival and polarization via MT1/2 receptors. Melatonin stimulation also regulates human CCR3+ MoMF and CD206+ MoMF survival and inflammation in vitro. Furthermore, CCR3 depletion antibody monotherapy inhibits liver inflammation and improves NAFLD in mice. Thus, therapies targeting CCR3+ MoMFs may have potential benefits in NAFLD treatment.
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Proteínas Imediatamente Precoces , Melatonina , Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Colina , Dieta Hiperlipídica/efeitos adversos , Inflamação , Fígado , Metionina , Camundongos Endogâmicos C57BL , Monócitos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores CCR3 , Proteínas Supressoras de TumorRESUMO
The severe pain caused by oral ulcers seriously affects food intake and speech, bringing great inconvenience in daily life. Drug-loaded patches are mostly used to treat oral mucosal diseases such as oral ulcers and oral lichen planus, but their effects are limited because of the influences of saliva and muscle movement. To enhance the adhesion of drug-loaded patches used in the oral cavity, we designed antimicrobial peptides (AMPs)-modified polycaprolactone (PCL)-collagen nanosheets (APCNs). The internal layer is a bioactive and antibacterial collagen layer modified with antimicrobial peptides. The backing layer is a hydrophobic PCL layer with good mechanical strength that can reduce external influences. We have characterized and tested the APCNs. First, the APCNs exhibited continuous and strong adhesion to irregular buccal mucosa surfaces under wet conditions and external force action. Antibacterial experiments showed that the APCNs had high antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria. Moreover, the APCNs showed good biocompatibility and promoted the adhesion of fibroblasts in vitro. Furthermore, APCNs treatment accelerated ulcer healing in a Sprague Dawley rat oral ulcer model. Our study developed antibacterial, wet-adhesive, and healing-promoting PCL-collagen nanosheets and demonstrated that these nanosheets could be promising adhesive therapeutic agents for the treatment of oral mucosal ulcers.
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Úlceras Orais , Ratos , Animais , Úlceras Orais/tratamento farmacológico , Adesivos/farmacologia , Ratos Sprague-Dawley , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antibacterianos/química , Úlcera/tratamento farmacológico , Colágeno/uso terapêuticoRESUMO
BACKGROUND & AIMS: Hepatic inflammation is a hallmark of nonalcoholic fatty liver disease (NAFLD). Double negative T (DNT) cells are a unique subset of T lymphocytes that do not express CD4, CD8, or natural killer cell markers, and studies have suggested that DNT cells play critical and diverse roles in the immune system. However, the role of intrahepatic DNT cells in NAFLD is largely unknown. METHODS: The proportions and RNA transcription profiling of intrahepatic DNT cells were compared between C57BL/6 mice fed with control diet or methionine-choline-deficient diet for 5 weeks. The functions of DNT cells were tested in vitro and in vivo. RESULTS: The proportion of intrahepatic DNT cells was significantly increased in mice with diet-induced NAFLD. In NAFLD mice, the proportion of intrahepatic TCRγδ+ DNT cells was increased along with elevated interleukin (IL) 17A; in contrast, the percentage of TCRαß+ DNT cells was decreased, accompanied by reduced granzyme B (GZMB). TCRγδ+ DNT cell depletion resulted in lowered liver IL17A levels and significantly alleviated NAFLD. Adoptive transfer of intrahepatic TCRαß+ DNT cells from control mice increased intrahepatic CD4 and CD8 T cell apoptosis and inhibited NAFLD progression. Furthermore, we revealed that adrenic acid and arachidonic acid, harmful fatty acids that were enriched in the liver of the mice with NAFLD, could induce apoptosis of TCRαß+ DNT cells and inhibit their immunosuppressive function and nuclear factor kappa B (NF-κB) or AKT signaling pathway activity. However, arachidonic acid facilitated IL17A secretion by TCRγδ+ DNT cells, and the NF-κB signaling pathway was involved. Finally, we also confirmed the variation of intrahepatic TCRαß+ DNT cells and TCRγδ+ DNT cells in humans. CONCLUSIONS: During NAFLD progression, TCRγδ+ DNT cells enhance IL17A secretion and aggravate liver inflammation, whereas TCRαß+ DNT cells decrease GZMB production and lead to weakened immunoregulatory function. Shifting of balance from TCRγδ+ DNT cell response to one that favors TCRαß+ DNT regulation would be beneficial for the prevention and treatment of NAFLD.
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Hepatopatia Gordurosa não Alcoólica , Animais , Ácidos Araquidônicos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
Although mesenchymal stem cell-derived exosomes (MSC-exos) have been shown to have therapeutic effects in experimental periodontitis, their drawbacks, such as low yield and limited efficacy, have hampered their clinical application. These drawbacks can be largely reduced by replacing the traditional 2D culture system with a 3D system. However, the potential function of MSC-exos produced by 3D culture (3D-exos) in periodontitis remains elusive. This study showed that compared with MSC-exos generated via 2D culture (2D-exos), 3D-exos showed enhanced anti-inflammatory effects in a ligature-induced model of periodontitis by restoring the reactive T helper 17 (Th17) cell/Treg balance in inflamed periodontal tissues. Mechanistically, 3D-exos exhibited greater enrichment of miR-1246, which can suppress the expression of Nfat5, a key factor that mediates Th17 cell polarization in a sequence-dependent manner. Furthermore, we found that recovery of the Th17 cell/Treg balance in the inflamed periodontium by the local injection of 3D-exos attenuated experimental colitis. Our study not only showed that by restoring the Th17 cell/Treg balance through the miR-1246/Nfat5 axis, the 3D culture system improved the function of MSC-exos in the treatment of periodontitis, but also it provided a basis for treating inflammatory bowel disease (IBD) by restoring immune responses in the inflamed periodontium.
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Colite , Exossomos , Periodontite , Humanos , Periodontite/terapia , Periodonto , Linfócitos T Reguladores , Células Th17RESUMO
Pulpal and periapical diseases account for a large proportion of dental visits, the current treatments for which are root canal therapy (RCT) and pulp revascularisation. Despite the clinical signs of full recovery and histological reconstruction, true regeneration of pulp tissues is still far from being achieved. The goal of regenerative endodontics is to promote normal pulp function recovery in inflamed or necrotic teeth that would result in true regeneration of the pulpodentinal complex. Recently, rapid progress has been made related to tissue engineering-mediated pulp regeneration, which combines stem cells, biomaterials, and growth factors. Since the successful isolation and characterisation of dental pulp stem cells (DPSCs) and other applicable dental mesenchymal stem cells, basic research and preclinical exploration of stem cell-mediated functional pulp regeneration via cell transplantation and cell homing have received considerably more attention. Some of this effort has translated into clinical therapeutic applications, bringing a ground-breaking revolution and a new perspective to the endodontic field. In this article, we retrospectively examined the current treatment status and clinical goals of pulpal and periapical diseases and scrutinized biological studies of functional pulp regeneration with a focus on DPSCs, biomaterials, and growth factors. Then, we reviewed preclinical experiments based on various animal models and research strategies. Finally, we summarised the current challenges encountered in preclinical or clinical regenerative applications and suggested promising solutions to address these challenges to guide tissue engineering-mediated clinical translation in the future.
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Polpa Dentária/metabolismo , Polpa Dentária/fisiologia , Regeneração Tecidual Guiada Periodontal/métodos , Animais , Humanos , Células-Tronco Mesenquimais/metabolismo , Regeneração/fisiologia , Estudos Retrospectivos , Tratamento do Canal Radicular/métodos , Células-Tronco/metabolismo , Engenharia Tecidual/métodosRESUMO
Diabetes-associated periodontitis (DP) aggravates diabetic complications and increases mortality from diabetes. DP is caused by diabetes-enhanced host immune-inflammatory responses to bacterial insult. In this study, we found that persistently elevated CCL2 levels in combination with proinflammatory monocyte infiltration of periodontal tissues were closely related to DP. Moreover, inhibition of CCL2 by oral administration of bindarit reduced alveolar bone loss and increased periodontal epithelial thickness by suppressing periodontal inflammation. Furthermore, bindarit suppressed the infiltration of proinflammatory monocytes and altered the inflammatory properties of macrophages in the diabetic periodontium. This finding provides a basis for the development of an effective therapeutic approach for treating DP.
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Quimiocina CCL2/antagonistas & inibidores , Diabetes Mellitus , Periodontite , Humanos , Indazóis/farmacologia , Macrófagos , Monócitos , Periodontite/complicações , Periodontite/tratamento farmacológico , Propionatos/farmacologiaRESUMO
Periodontitis is caused by host immune-inflammatory response to bacterial insult. A high proportion of pro-inflammatory macrophages to anti-inflammatory macrophages leads to the pathogenesis of periodontitis. As stem cell-derived exosomes can modulate macrophage phenotype, dental pulp stem cell-derived exosomes (DPSC-Exo) can effectively treat periodontitis. In this study, we demonstrated that DPSC-Exo-incorporated chitosan hydrogel (DPSC-Exo/CS) can accelerate the healing of alveolar bone and the periodontal epithelium in mice with periodontitis. Gene Ontology (GO) term enrichment analysis showed that treatment with DPSC-Exo/CS ameliorated periodontal lesion by suppressing periodontal inflammation and modulating the immune response. Specifically, DPSC-Exo/CS facilitated macrophages to convert from a pro-inflammatory phenotype to an anti-inflammatory phenotype in the periodontium of mice with periodontitis, the mechanism of which could be associated with miR-1246 in DPSC-Exo. These results not only shed light on the therapeutic mechanism of DPSC-Exo/CS but also provide the basis for developing an effective therapeutic approach for periodontitis.
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INTRODUCTION: Dental pulp is a major composition in the pulp-dentin complex, which serves as protective system against dental trauma/infection. Functional dental pulp regeneration is highly desirable after pulpitis or pulp necrosis. However, endodontic regeneration has remained challenging for decades because of the deconstructive microenvironment and the lack of functional cells within the root canal system. The present study developed a decellularized matrix hydrogel derived from human dental pulp (hDDPM-G), which might serve as a growth-permissive microenvironment for dental pulp regeneration. METHODS: Human dental pulps extracted from healthy wisdom teeth were decellularized and digested and then underwent sol-gel transition to form hDDPM-G. The protein compositions were identified by proteomic analysis. Human dental pulp stem cells (hDPSCs) were seeded on hDDPM-G-coated surfaces and evaluated by immunofluorescence staining, transwell migration, and Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) assays. Induced hDPSC differentiation was examined in vitro and characterized by immunostaining, Western blotting, and reverse transcription polymerase chain reaction. RESULTS: Complete decellularization was implemented. Protein contents found in the human decellularized dental pulp matrix were identified to contribute in promoting cell proliferation, migration, and regulation of stem cell differentiation. The hDDPM-G-coated surfaces promoted hDPSC adhesion, migration, and proliferation. Furthermore, hDDPM-G coatings facilitated odontoblastlike, neural-like, and angiogenic differentiation of the seeded hDPSCs after being cultured in induction media for 14 days. CONCLUSIONS: This study showed that hDDPM-G effectively contributed in promoting hDPSC proliferation and migration and induced multidirectional differentiation. Considering the injectability and gelation at body temperature, hDDPM-G may hold translational potential for endodontic regeneration.
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Polpa Dentária , Hidrogéis , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , ProteômicaRESUMO
OBJECTIVE: This study aims to investigate the expression pattern of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 6 (NLRP6) in human dental pulp tissues and cells, and roughly explore the role of NLRP6 in dental pulp immunity. METHODS: Immunohistochemistry and immunofluorescence double staining were performed to determine the expression and localization of NLRP6 in healthy and inflamed pulp tissues. The expression of NLRP6 in human dental pulp cells (HDPCs) was investigated by immunocytofluorescence. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) and western blot were used to evaluate the impact of lipopolysaccharide (LPS) stimulation on NLRP6 expression in HDPCs. Last, NLRP6 gene was silenced by lentiviral short hairpin RNA to explore the impact of NLRP6 on LPS-induced interleukin (IL)-1ß. RESULTS: NLRP6 was predominantly expressed in odontoblasts layer and blood vessels of healthy dental pulp, as well as infiltrated immune cells and fibroblasts of inflamed pulp. Further immunofluorescence double staining showed that pericytes and endothelial cells in the dental pulp blood vessels, macrophages and T cells as well as fibroblasts in the inflamed pulp expressed NLRP6. NLRP6 was also basically expressed in cultured HDPCs and upregulated by LPS stimulation. Knockdown of NLRP6 in HDPCs significantly inhibited the LPS-induced IL-1ß expression. CONCLUSIONS: Our study revealed the expression and distribution of NLRP6 in human dental pulp tissues. Furthermore, NLRP6 was also basically expressed in cultured HDPCs, which could be upregulated by LPS stimulation, indicating the involvement of NLRP6 in dental pulp immune response.
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Polpa Dentária/metabolismo , Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Odontoblastos/metabolismo , Células Cultivadas , Polpa Dentária/imunologia , Humanos , Lipopolissacarídeos/farmacologiaRESUMO
Enhanced odontoblast differentiation of human dental pulp cells (hDPCs) is considered a keystone in dentin-pulp complex formation. We have revealed lncRNA DANCR was implicated in this differentiation program, however, its mechanism in odontoblast differentiation of hDPCs remains further explored. In this study, by employing loss-of-function approach, we identified downregulation of DANCR drived odontoblast differentiaion of hDPCs. Bioinformatics analysis was utilized to show that DANCR contained binding site for miR-216a and an inverse correlation between DANCR and miR-216a was obtained. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) were applied to further confirm that DANCR conferred its functions by directly binding to miR-216a. Notably, miR-216a was able to bind to the 3'-UTR of c-Cbl and repressed its expression. In addition, the protein level of c-CBL was significantly downregulated during hDPCs differentiation, while c-Cbl overexpression inhibited odontoblast differentiation of hDPCs. Moreover, downregulation of miR-216a efficiently reversed the suppression of c-Cbl level and odontoblast differentiation induced by knockdown of DANCR. Taken together, these analyses indicated that DANCR positively regulated the expression of c-Cbl, through sponging miR-216a, and inhibited odontoblast differentiation of hDPCs. Our results will extend the field of clinical application for cell-based therapy in regenerative medicine.
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Diferenciação Celular/genética , MicroRNAs/genética , Odontoblastos/fisiologia , Proteínas Proto-Oncogênicas c-cbl/genética , RNA Longo não Codificante/genética , Regulação para Cima/genética , Adolescente , Adulto , Linhagem Celular , Regulação para Baixo/genética , Humanos , Adulto JovemRESUMO
BACKGROUND: Hyperglycemic micro-environment induced by diabetes could regulate the response of periodontal tissues to pathogenic microorganisms in which disruption of autophagy lysosomal pathway (ALP) may participate. This study aimed to explore the mechanisms underlying how high glucose (HG) regulates ALP in gingival epithelial cells (GECs). METHODS: Human gingival tissues in healthy group (C), periodontitis group (P), diabetes group (DM), and diabetes + periodontitis group (DP) were collected and were applied to observe the fusion of autophagy and lysosome. Diabetic mouse model with periodontitis was established and RNA-seq was applied to investigate the expression of ALP-associated genes in gingival epithelium. To explore the key role of ATPase transmembrane v0 domain, subunit c (ATP6V0C) in the disruption of ALP by HG, human gingival epithelial cells (HGECs) were cultured in 5.5 mM/25 mM glucose medium for 48 hours and followed by Porphyromonas gingivalis stimulation for 0, 6, and 12 hours. HBLV-h-ATP6V0C was transfected in HGECs that were stimulated by 25 mM HG condition. RESULTS: Immunofluorescence double staining exhibited the disruption of ALP in human gingival epithelium in diabetes groups and HGECs under 25 mM glucose condition, accompanied with significantly downregulated lysosomal acidity. RNA-seq of mouse gingival epithelium screened out Atp6v0c. Compared with HGECs in normal culture medium, ATP6V0C expression and LC3-II/I expression ratio were significantly downregulated, with an upregulated expression of P62, IL-1ß in HGECs under HG condition. Over-expression of ATP6V0C rescued HG-induced disruption of ALP in HBLV-h-ATP6V0C transfected HGECs, with significantly upregulation of LC3-II/I and downregulation of P62, IL-1ß. CONCLUSION: ATP6V0C mediates HG-induced ALP disruption in HGECs, eventually exacerbates periodontal inflammation.
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Autofagia , ATPases Vacuolares Próton-Translocadoras , Animais , Células Epiteliais , Gengiva , Glucose , Humanos , Lisossomos , Camundongos , Porphyromonas gingivalisRESUMO
OBJECTIVES: To compare the sealing ability and biocompatibility of Biodentine with mineral trioxide aggregate (MTA) when used as root-end filling materials. METHODOLOGY: The Cell Counting Kit-8 (CCK-8) assay was used to compare the cytotoxicity of MTA and Biodentine. Twenty-one extracted teeth with a single canal were immersed in an acidic silver nitrate solution after root-end filling. Then, the volume and depth of silver nitrate that infiltrated the apical portion of the teeth were analyzed using micro-computed tomography (micro-CT). Seventy-two roots from 3 female beagle dogs were randomly distributed into 3 groups and apical surgery was performed. After six months, the volume of the bone defect surrounding these roots was analyzed using micro-CT. RESULTS: Based on the results of the CCK-8 assay, MTA and Biodentine did not show statistically significant differences in cytotoxicity (P>0.05). The volume and the depth of the infiltrated nitrate solution were greater in the MTA group than in the Biodentine group (P<0.05). The volume of the bone defect was larger in the MTA group than in the Biodentine group. However, the difference was not significant (P>0.05). The volumes of the bone defects in the MTA and Biodentine groups were smaller than the group without any filling materials (P<0.05). CONCLUSIONS: MTA and Biodentine exhibited comparable cellular biocompatibility. Biodentine showed a superior sealing ability to MTA in root-end filling. Both Biodentine and MTA promoted periradicular bone healing in beagle dog periradicular surgery models.
Assuntos
Compostos de Alumínio/farmacologia , Compostos de Cálcio/farmacologia , Óxidos/farmacologia , Tecido Periapical/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Materiais Restauradores do Canal Radicular/farmacologia , Tratamento do Canal Radicular/métodos , Silicatos/farmacologia , Cicatrização/efeitos dos fármacos , Adolescente , Animais , Regeneração Óssea/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Cães , Combinação de Medicamentos , Humanos , Masculino , Teste de Materiais , Osteogênese/efeitos dos fármacos , Tecido Periapical/citologia , Tecido Periapical/diagnóstico por imagem , Ligamento Periodontal/diagnóstico por imagem , Reprodutibilidade dos Testes , Fatores de Tempo , Raiz Dentária/diagnóstico por imagem , Raiz Dentária/efeitos dos fármacos , Raiz Dentária/cirurgia , Resultado do Tratamento , Microtomografia por Raio-X , Adulto JovemRESUMO
Exosomes are important mediators of intercellular communication and have a vital part in the diagnosis and treatment of various diseases in humans. Here, we investigated the benefits and underlying mechanism of exosomes secreted via stem cells from human exfoliated deciduous teeth (SHED-derived exosomes) in promoting alveolar bone regeneration, thus providing new insights into exosome-based therapy for periodontitis. SHED-derived exosomes were isolated by ultracentrifugation. The impacts of SHED-derived exosomes on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) and the osteogenic capability of rat bone marrow mesenchymal stem cells (BMSCs) were evaluated in vitro. Compound C, a pharmacological blocker of adenosine monophosphate-activated protein kinase (AMPK), was used to examine the role of the AMPK signaling cascade in these processes. Periodontal defect rat models were established and treated with PBS, ß-tricalcium phosphate (ß-TCP), or a grouping of exosomes/ß-TCP. Microcomputed tomography (micro-CT) scanning, hematoxylin and eosin (HE) staining, Masson staining, and immunofluorescence staining were done to inspect the impacts of the exosomes/ß-TCP combination on periodontal bone regeneration. Our outcomes indicated that the expression of angiogenesis-related genes (KDR, SDF-1, and FGF2), osteogenesis-related genes (COL1, RUNX2, and OPN), and phosphorylated (p)-AMPK were upregulated after treatment with exosomes, while the positive impacts of SHED-derived exosomes on HUVECs and BMSCs were partially reversed by compound C. Micro-CT analysis demonstrated that the exosomes/ß-TCP group exhibited better bone regeneration than either the ß-TCP group or the control group. Additionally, the results of HE and Masson staining as well as immunofluorescence staining showed neovascularization and new bone formation in the exosomes/ß-TCP group but only limited new bone formation in the other two groups. Thus, SHED-derived exosomes contribute to periodontal bone regeneration by promoting neovascularization and new bone formation, possibly through the AMPK signaling pathway.