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1.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576085

RESUMO

Bitter-taste receptors (T2Rs) have emerged as key players in host-pathogen interactions and important modulators of oral innate immunity. Previously, we reported that T2R14 is expressed in gingival epithelial cells (GECs) and interacts with competence stimulating peptides (CSPs) secreted by the cariogenic Streptococcus mutans. The underlying mechanisms of the innate immune responses and physiological effects of T2R14 on Gram-positive bacteria are not well characterized. In this study, we examined the role of T2R14 in internalization and growth inhibitory effects on Gram-positive bacteria, namely Staphylococcus aureus and S. mutans. We utilized CRISPR-Cas9 T2R14 knockdown (KD) GECs as the study model to address these key physiological mechanisms. Our data reveal that the internalization of S. aureus is significantly decreased, while the internalization of S. mutans remains unaffected upon knockdown of T2R14 in GECs. Surprisingly, GECs primed with S. mutans CSP-1 resulted in an inhibition of growth for S. aureus, but not for S. mutans. The GECs infected with S. aureus induced T2R14-dependent human ß-defensin-2 (hBD-2) secretion; however, S. mutans-infected GECs did not induce hBD-2 secretion, but induced T2R14 dependent IL-8 secretion. Interestingly, our results show that T2R14 KD affects the cytoskeletal reorganization in GECs, thereby inhibiting S. aureus internalization. Our study highlights the distinct mechanisms and a direct role of T2R14 in influencing physiological responses to Gram-positive bacteria in the oral cavity.


Assuntos
Endocitose , Células Epiteliais/metabolismo , Gengiva/citologia , Bactérias Gram-Positivas/metabolismo , Viabilidade Microbiana , Receptores Acoplados a Proteínas G/metabolismo , Paladar , Actinas/metabolismo , Linhagem Celular , Células Epiteliais/ultraestrutura , Humanos , Interleucina-8/metabolismo , Modelos Biológicos , Nitratos/metabolismo , Nitritos/metabolismo , Staphylococcus aureus/metabolismo , Streptococcus mutans/metabolismo , beta-Defensinas/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360848

RESUMO

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonasgingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.


Assuntos
Infecções por Bacteroidaceae/imunologia , Células Epiteliais/imunologia , Gengiva/imunologia , Nanocompostos/efeitos adversos , Peri-Implantite/imunologia , Titânio/efeitos adversos , Linhagem Celular , Citocinas/imunologia , Células Epiteliais/citologia , Gengiva/citologia , Humanos , Lipopolissacarídeos/imunologia , Peri-Implantite/patologia , Porphyromonas gingivalis/imunologia
3.
Int J Mol Sci ; 22(15)2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-34360701

RESUMO

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-ß1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-ß1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-ß receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-ß1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-ß receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.


Assuntos
Proteína Morfogenética Óssea 2/genética , Fibroblastos/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Transdução de Sinais , Adulto , Regeneração Óssea , Células Cultivadas , Feminino , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Gengiva/citologia , Humanos , Masculino , Proteômica , Fator de Crescimento Transformador beta/metabolismo , Adulto Jovem
4.
ACS Appl Mater Interfaces ; 13(30): 35342-35355, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34297530

RESUMO

Growth-factor-free bone regeneration remains a challenge in craniofacial engineering. Here, we engineered an osteogenic niche composed of a commercially modified alginate hydrogel and whitlockite microparticles (WHMPs), which impart tunable physicochemical properties that can direct osteogenesis of human gingival mesenchymal stem cells (GMSCs). Our in vitro studies demonstrate that WHMPs induce osteogenesis of GMSCs more effectively than previously demonstrated hydroxyapatite microparticles (HApMPs). Alginate-WHMP hydrogels showed higher elasticity without any adverse effects on the viability of the encapsulated GMSCs. Moreover, the alginate-WHMP hydrogels upregulate the mitogen-activated protein kinase (MAPK) pathway, which in turn orchestrates several osteogenic markers, such as RUNX2 and OCN, in the encapsulated GMSCs. Concurrent coculture studies with human osteoclasts demonstrate that GMSCs encapsulated in alginate-WHMP hydrogels downregulate osteoclastic activity, potentially due to release of Mg2+ ions from the WHMPs along with secretion of osteoprotegerin from the GMSCs. In vivo studies demonstrated that the GMSCs encapsulated in our osteogenic niche were able to promote bone repair in calvarial defects in murine models. Altogether, our results confirmed the development of a promising treatment modality for craniofacial bone regeneration based on an injectable growth-factor-free hydrogel delivery system.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/uso terapêutico , Hidrogéis/uso terapêutico , Crânio/efeitos dos fármacos , Alginatos/uso terapêutico , Animais , Diferenciação Celular/efeitos dos fármacos , Células Imobilizadas , Gengiva/citologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Engenharia Tecidual/métodos
5.
Sci Rep ; 11(1): 12247, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112817

RESUMO

Particulate autologous tooth roots are increasingly used for alveolar bone augmentation; however, the proteomic profile of acid dentin lysate and the respective cellular response have not been investigated. Here we show that TGF-ß1 is among the 226 proteins of acid dentin lysate (ADL) prepared from porcine teeth. RNA sequencing identified 231 strongly regulated genes when gingival fibroblasts were exposed to ADL. Out of these genes, about one third required activation of the TGF-ß receptor type I kinase including interleukin 11 (IL11) and NADPH oxidase 4 (NOX4). Reverse transcription-quantitative polymerase chain reaction and immunoassay confirmed the TGF-ß-dependent expression of IL11 and NOX4. The activation of canonical TGF-ß signaling by ADL was further confirmed by the phosphorylation of Smad3 and translocation of Smad2/3, using Western blot and immunofluorescence staining, respectively. Finally, we showed that TGF-ß activity released from dentin by acid lysis adsorbs to titanium and collagen membranes. These findings suggest that dentin particles are a rich source of TGF-ß causing a major response of gingival fibroblasts.


Assuntos
Dentina/metabolismo , Genômica , Proteômica , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Genômica/métodos , Gengiva/citologia , Ligação Proteica , Proteômica/métodos , Transcriptoma
6.
mBio ; 12(3): e0050221, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34182783

RESUMO

Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.


Assuntos
Gengiva/citologia , Inflamação , Queratinócitos/imunologia , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/metabolismo , Ribonucleases/metabolismo , Transdução de Sinais , Animais , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Feminino , Cisteína Endopeptidases Gingipaínas , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/microbiologia , Porphyromonas gingivalis/fisiologia , Ribonucleases/genética , Ribonucleases/imunologia , Organismos Livres de Patógenos Específicos
7.
mBio ; 12(3)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006656

RESUMO

Early childhood caries is a severe oral disease that results in aggressive tooth decay. Particularly, a synergistic association between a fungus, Candida albicans, and a cariogenic bacterium, Streptococcus mutans, promotes the development of hard-to-remove and highly acidic biofilms, exacerbating the virulent damage. These interactions are largely mediated via glucosyltransferases (GtfB) binding to mannans on the cell wall of C. albicans Here, we present an enzymatic approach to target GtfB-mannan interactions in this cross-kingdom consortium using mannan-degrading exo- and endo-enzymes. These exo- and endo-enzymes are highly effective in reducing biofilm biomass without killing microorganisms, as well as alleviating the production of an acidic pH environment conducive to tooth decay. To corroborate these results, we present biophysical evidence using single-molecule atomic force microscopy, biofilm shearing, and enamel surface topography analyses. Data show a drastic decrease in binding forces of GtfB to C. albicans (∼15-fold reduction) following enzyme treatment. Furthermore, enzymatic activity disrupted biofilm mechanical stability and significantly reduced human tooth enamel demineralization without cytotoxic effects on gingival keratinocytes. Our results represent significant progress toward a novel nonbiocidal therapeutic intervention against pathogenic bacterial-fungal biofilms by targeting the interkingdom receptor-ligand binding interactions.IMPORTANCE Biofilm formation is a key virulence factor responsible for various infectious diseases. Particularly, interactions between a fungus, Candida albicans, and a bacterium, Streptococcus mutans, have been known to play important roles in the pathogenesis of dental caries. Although some antimicrobials have been applied to treat fungal-involved biofilm-associated diseases, these often lack targeting polymicrobial interactions. Furthermore, these may not be appropriate for preventive measures because these antimicrobials may disrupt ecological microbiota and/or induce the prevalence of drug resistance over time. By specifically targeting the interaction mechanism whereby mannoproteins on the C. albicans surface mediate the cross-kingdom interaction, we demonstrated that mannoprotein-degrading enzymes can effectively disrupt biofilm interactions without microbiocidal effects or causing cytotoxicity to human cells. This suggests a potential application as a targeted approach for intervening a pathogenic cross-kingdom biofilm associated with a costly and unresolved oral disease.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/metabolismo , Streptococcus mutans/metabolismo , Simbiose , Cárie Dentária/microbiologia , Gengiva/citologia , Humanos , Queratinócitos/microbiologia , Mananas/metabolismo , Microscopia de Força Atômica
8.
Sci Rep ; 11(1): 10770, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031466

RESUMO

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host-pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1ß-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes.


Assuntos
Infecções por Bacteroidaceae/imunologia , Gengiva/citologia , Interleucina-1beta/genética , Porphyromonas gingivalis/patogenicidade , Telomerase/genética , Fator de Necrose Tumoral alfa/genética , Aderência Bacteriana/efeitos dos fármacos , Infecções por Bacteroidaceae/genética , Células Cultivadas , Metilação de DNA , Dinoprostona/genética , Dinoprostona/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Gengiva/efeitos dos fármacos , Gengiva/imunologia , Gengiva/metabolismo , Humanos , Interleucina-1beta/metabolismo , Lipopeptídeos/farmacologia , Oligopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/agonistas , Fator de Necrose Tumoral alfa/metabolismo
9.
Int J Nanomedicine ; 16: 3201-3216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34007174

RESUMO

Purpose: Polyetheretherketone (PEEK) exhibits high mechanical strengths and outstanding biocompatibility but biological inertness that does not excite the cell responses and stimulate bone formation. The objective of this study was to construct submicro-nano structures on PEEK by femtosecond laser (FSL) for exciting the responses of MC3T3-E1 cells and gingival epithelial (GE) cells, which induce regeneration of bone/gingival tissues for long-term stability of dental implants. Materials and Methods: In this study, submicro-nano structures were created on PEEK surface by FSL with power of 80 mW (80FPK) and 160 mW (160FPK). Results: Compared with PEEK, both 80FPK and 160FPK with submicro-nano structures exhibited elevated surface performances (hydrophilicity, surface energy, roughness and protein absorption). Furthermore, in comparison with 80FPK, 160FPK further enhanced the surface performances. In addition, compared with PEEK, both 80FPK and 160FPK significantly excited not only the responses (adhesion, proliferation, alkaline phosphatase [ALP] activity and osteogenic gene expression) of MC3T3-E1 cells but also responses (adhesion as well as proliferation) of GE cells of human in vitro. Moreover, in comparison with 80FPK, 160FPK further enhanced the responses of MC3T3-E1 cells/GE cells. Conclusion: FSL created submicro-nano structures on PEEK with elevated surface performances, which played crucial roles in exciting the responses of MC3T3-E1 cells/GE cells. Consequently, 160FPK with elevated surface performances and outstanding cytocompatibility would have enormous potential as an implant for dental replacement.


Assuntos
Células Epiteliais/citologia , Células Epiteliais/efeitos da radiação , Gengiva/citologia , Cetonas/química , Lasers , Nanoestruturas/química , Tamanho da Partícula , Polietilenoglicóis/química , Adsorção , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular , Linhagem Celular , Proliferação de Células , Forma Celular , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Microscopia de Força Atômica , Osteogênese/genética , Espectroscopia Fotoeletrônica , Propriedades de Superfície , Água/química
10.
Molecules ; 26(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919066

RESUMO

This study focuses on the role of photosensitizers in photodynamic therapy. The photosensitizers were prepared in combinations of 110/220 µM erythrosine and/or 10/20 µM demethoxy/bisdemethoxy curcumin with/without 10% (w/w) nano-titanium dioxide. Irradiation was performed with a dental blue light in the 395-480 nm wavelength range, with a power density of 3200 mW/cm2 and yield of 72 J/cm2. The production of ROS and hydroxyl radical was investigated using an electron paramagnetic resonance spectrometer for each individual photosensitizer or in photosensitizer combinations. Subsequently, a PrestoBlue® toxicity test of the gingival fibroblast cells was performed at 6 and 24 h on the eight highest ROS-generating photosensitizers containing curcumin derivatives and erythrosine 220 µM. Finally, the antifungal ability of 22 test photosensitizers, Candida albicans (ATCC 10231), were cultured in biofilm form at 37 °C for 48 h, then the colonies were counted in colony-forming units (CFU/mL) via the drop plate technique, and then the log reduction was calculated. The results showed that at 48 h the test photosensitizers could simultaneously produce both ROS types. All test photosensitizers demonstrated no toxicity on the fibroblast cells. In total, 18 test photosensitizers were able to inhibit Candida albicans similarly to nystatin. Conclusively, 20 µM bisdemethoxy curcumin + 220 µM erythrosine + 10% (w/w) nano-titanium dioxide exerted the highest inhibitory effect on Candida albicans.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Curcumina/química , Curcumina/farmacologia , Eritrosina/química , Fotoquimioterapia , Titânio/química , Antioxidantes/química , Antioxidantes/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Fibroblastos/metabolismo , Gengiva/citologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo
11.
Wiad Lek ; 74(3 cz 1): 423-428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33813444

RESUMO

OBJECTIVE: The aim of the research was to study the cellular composition of the gums in children of primary school age with normal body weight and overweight for further use of this data in the early diagnostics of periodontal diseases. PATIENTS AND METHODS: Materials and methods: We examined 81 children aged from 6 to 12 years. Cytological examination of gingival cytograms was performed in all examined children. RESULTS: Results: Based on the analysis of the quantitative content of epithelial cells in children with normal body weight, their ratio was established, which is determined by the percentage of 0: 6: 94 (parabasal, intermediate, superficial). The obtained data completely coincide with the percentage of the differentiated ratio of epitheliocytes of multilayered squamous epithelium in children with normal body weight with inflammation and without it in the periodontal tissues. Our cytological examinations of gingival scrape smears in overweight children in contrast to the results of the study of epithelial scrape smears in children with normal body weight have some differences. Thus, in the process of calculation, the degree of differentiation of various epitheliocytes determines their percentage as follows - 3: 7: 90 (parabasal, intermediate, superficial) for children with gingivitis, and 2: 5: 93 (parabasal, intermediate, superficial) for children without inflammation in the periodontal tissues. CONCLUSION: Conclusions: The obtained results allowed us to conclude that in overweight children, in contrast to children with normal body weight, the number of parabasal cells decreases, and the number of superficial and intermediate cells increases.


Assuntos
Peso Corporal , Gengiva , Sobrepeso , Índice de Massa Corporal , Criança , Feminino , Gengiva/citologia , Gengiva/patologia , Humanos , Peso Corporal Ideal , Masculino , Sobrepeso/epidemiologia , Instituições Acadêmicas
12.
Int J Biol Macromol ; 178: 229-239, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33647340

RESUMO

The construction of protein abundance profiles helps to interpret the clinical applications of stem cells. Dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) can be isolated from teeth and used as a highly convenient clinical potential material. Here, we aimed to explore commonalities and differences of DPSCs and GMSCs at the protein level. TMT-based quantitative proteomics and two-dimensional gel electrophoresis technology were used in combination to describe the protein profile of DPSCs and GMSCs extracted from the same donor. A total of 2821 proteins were identified by LC-MS/MS, of which 248 differentially abundant proteins (DAPs) were highly expressed in GMSCs while 782 proteins were highly expressed in DPSCs. The biological functions and molecular pathways of DAPs were annotated with GO enrichment and KEGG analysis. The relationship between molecular abundance and cell characteristics including source, proliferation, angiogenesis and inflammation were connected by WGCNA. Special markers, including Calreticulin (CALR), Annexin A5 (ANXA5) and Rho GDP dissociation inhibitor alpha (GDIR1), were proposed to distinguish DPSCs from GMSCs. Our results provide a molecular basis for in-depth understanding of the protein composition and special functions of dental stem cells, and promote the potential clinical application.


Assuntos
Calreticulina/metabolismo , Polpa Dentária , Gengiva , Células-Tronco Mesenquimais , Biomarcadores , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo
13.
J Mol Histol ; 52(3): 545-553, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33763807

RESUMO

Junctional epithelium (JE) attaching to the enamel surface seals gaps around the teeth, functioning as the first line of gingival defense. Runt-related transcription factor 2 (Runx2) plays a role in epithelial cell fate, and the deficiency of Runx2 in JE causes periodontal destruction, while its effect on the barrier function of JE remains largely unexplored. In the present study, hematoxylin-eosin (H&E) staining revealed the morphological differences of JE between wild-type (WT) and Runx2 conditional knockout (cKO) mice. We speculated that these changes were related to the down-regulation of E-cadherin (E-cad), junctional adhesion molecule 1 (JAM1), and integrin ß6 (ITGB6) in JE. Moreover, immunohistochemistry (IHC) was conducted to assess the expressions of these proteins. To verify the relationship between Runx2 and the three above-mentioned proteins, human gingival epithelial cells (HGEs) were cultured for in vitro experiment. The expression of Runx2 in HEGs was depleted by lentivirus. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis were adopted to analyze the differences in mRNA and protein expressions. Taken together, Runx2 played a crucial role in maintaining the structure and function integrality of JE via regulating the expressions of E-cad and JAM1.


Assuntos
Caderinas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Epitélio/metabolismo , Moléculas de Adesão Juncional/metabolismo , Dente Molar/metabolismo , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Gengiva/citologia , Humanos , Cadeias beta de Integrinas/metabolismo , Mandíbula/metabolismo , Camundongos Knockout , Periodonto/metabolismo
14.
Int Immunopharmacol ; 95: 107505, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33725636

RESUMO

The purpose of the present study was to investigate the pharmacological effect of Fisetin on experimental periodontitis in rats and explore its potential mechanism. The ligature/LPS method was used to induce periodontitis in rats. LPS was employed to cause inflammation in Human gingival fibroblasts (HGF). The transfections with FGFR1 SiRNA, NLRP3 SiRNA and the selective TLR4 inhibitor TAK242 were used to investigate the mechanism of Fisetin-mediated inflammatory reaction in LPS-induced HGF. As a result, Fisetin reduced the alveolar bone gap, reversed histopathological lesion and inhibited serum inflammatory cytokine concentration in periodontitis rats. Fisetin decreased the inflammatory cytokine contents in the supernatant of LPS-induced HGF. The inhibitory effect of Fisetin might be attributed to FGFR1/TLR4/NLRP3 inflammasome pathway both in vivo and in vitro. The suppressions of FGFR1, TLR4 and NLRP3 proved that FGFR1/TLR4/NLRP3 signaling was involved in the Fisetin-mediated inflammatory response. Fisetin also inhibited NLRP3 priming. The data demonstrated that Fisetin attenuated periodontitis by inhibiting inflammatory reaction via FGFR1/TLR4/NLRP3 inflammasome pathway.


Assuntos
Anti-Inflamatórios/uso terapêutico , Flavonóis/uso terapêutico , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Periodontite/tratamento farmacológico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Flavonóis/farmacologia , Gengiva/citologia , Humanos , Lipopolissacarídeos , Masculino , Periodontite/imunologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
15.
FASEB J ; 35(3): e21375, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33559200

RESUMO

Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown that the bacterial quorum sensing molecules (QSM) can interact with host cell membrane proteins, mainly G protein-coupled receptors (GPCRs), and induce innate immune responses. However, few studies have examined QSM-GPCR interactions and their influence on oral innate immune responses. In this study, we examined the role of bitter taste receptor T2R14 in sensing competence stimulating peptides (CSPs) secreted by cariogenic bacterium Streptococcus mutans and in mediating innate immune responses in gingival epithelial cells (GECs). Transcriptomic and western blot analyses identify T2R14 to be highly expressed in GECs. Our data show that only CSP-1 from S. mutans induces robust intracellular calcium mobilization compared to CSP-2 and CSP-3. By using CRISPR-Cas9, we demonstrate that CSP-1 induced calcium signaling and secretion of cytokines CXCL-8/IL-8, TNF-α, and IL-6 is mediated through T2R14 in GECs. Interestingly, the NF-kB signaling activated by CSP-1 in GECs was independent of T2R14. CSP-1-primed GECs attracted differentiated HL-60 immune cells (dHL-60) and this effect was abolished in T2R14 knock down GECs and also in cells primed with T2R14 antagonist 6-Methoxyflavone (6-MF). Our findings identify S. mutans CSP-1 as a peptide ligand for the T2R family. Our study establishes a novel host-pathogen interaction between cariogenic S. mutans CSP-1 and T2R14 in GECs leading to an innate immune response. Collectively, these findings suggest T2Rs as potential therapeutic targets to modulate innate immune responses upon oral bacterial infections.


Assuntos
Proteínas de Bactérias/fisiologia , Gengiva/imunologia , Interações Hospedeiro-Patógeno , Percepção de Quorum/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Streptococcus mutans/fisiologia , Cálcio/metabolismo , Linhagem Celular , Movimento Celular , Citocinas/biossíntese , Células Epiteliais/imunologia , Gengiva/citologia , Humanos , Imunidade Inata , NF-kappa B/fisiologia , Fosfolipase C beta/fisiologia
16.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33635312

RESUMO

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.


Assuntos
Gengiva/citologia , Gengiva/imunologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/imunologia , Animais , Medula Óssea/metabolismo , Feminino , Hematopoese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , RNA-Seq/métodos , Análise de Célula Única/métodos
17.
Int Immunopharmacol ; 94: 107455, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33582592

RESUMO

OBJECTIVE: The aim of this study was to examine the effect of gingival mesenchymal stem cells derived exosomes (GMSC-Exos) on lipopolysaccharide/interferon-gamma (LPS/INF-γ)-induced inflammatory macrophages in a high-lipid microenvironment. MATERIALS AND METHODS: Exosomes were obtained by culturing gingival mesenchymal stem cells (GMSCs) in alpha-MEM with exosome-free fetal bovine serum for 48 h. The control group was produced in vitro by inducing human acute monocytic leukemia cells (THP-1 cells) into naïve macrophages (M0). Inflammatory macrophages (M1) were made by activating M0 macrophages with LPS/IFN-γ. These M1 macrophages were treated with oxidized low-density lipoprotein (ox-LDL) to create the high-lipid group, of which some macrophages were further treated with GMSC-Exos for 24 h to form the GMSC-Exos group. Supernatants were collected, and total RNA were extracted for downstream analysis. The expression of surface markers in macrophages were analyzed by flow cytometry. The lipid accumulation level was assessed by oil red O staining. RESULTS: Exosomes were successfully isolated from GMSC medium. The GMSC-Exos group showed lower Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß), and cluster of differentiation 86 (CD86) expression levels than the high-lipid group, and the highest levels of Interleukin-10 (IL-10) among all groups. The GMSC-Exos group showed significant reductions in TNF-α levels than the high-lipid group, and significant escalations in IL-10 levels than the other two groups. Oil red o Staining showed that lipid accumulation in macrophages was inhibited in the GMSC-Exos group. CONCLUSIONS: GMSC-Exos reduce the release level and expression of inflammatory factors, inhibit lipid accumulation, and promote the polarization of pro-inflammatory macrophages into anti-inflammatory phenotype in a high-lipid microenvironment.


Assuntos
Exossomos , Macrófagos/imunologia , Células-Tronco Mesenquimais , Adolescente , Adulto , Antígeno B7-2/imunologia , Diferenciação Celular , Gengiva/citologia , Humanos , Inflamação/imunologia , Interleucina-10/imunologia , Lipídeos , Fenótipo , Células THP-1 , Fator de Necrose Tumoral alfa/imunologia , Adulto Jovem
18.
Int Immunopharmacol ; 94: 107456, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33588175

RESUMO

OBJECTIVE: To investigate the effects of hypoxia and Porphyromonas gingivalis- lipopolysaccharide (P. gingivalis-LPS) on activation of the NACHT leucine-rich repeat protein 3 (NLRP3) inflammasome in human gingival fibroblasts (HGFs). DESIGN: Periodontitis was optimally simulated using a hypoxic concentration of 1%. HGFs were stimulated using P. gingivalis-LPS (1.0 µg/ml) in normoxia and hypoxia for 3 h and 6 h, respectively. The expression levels of genes and proteins of hypoxia-inducible factor-1α (HIF-1α), interleukin-1ß, gasdermin D (GSDMD) and the NLRP3 inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, were measured using quantitative real-time polymerase chain reaction and western blot. ELISA was used to detect and determine levels of the inflammatory factor interleukin-1ß in cell supernatants. Lactate dehydrogenase (LDH) release assay, caspase-1 activity assay and Hoechst 33342/Propidium Iodide (PI) staining were performed to further verify the presence of pyroptosis. RESULTS: The NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by individual stimulation using P. gingivalis-LPS or hypoxia. However, the combination of both hypoxia and P. gingivalis-LPS stimulation significantly enhanced inflammasome activation and promoted the expression of interleukin-1ß, gasdermin D and HIF-1α at gene and protein levels; PI positive cells and the release of LDH were also elevated. CONCLUSION: Hypoxia and P. gingivalis-LPS synergistically induced NLRP3 inflammasome activation in HGFs, and subsequently high levels of interleukin-1ß and GSDMD-mediated pyroptosis can cause an HGF inflammatory response, which plays an important role in the pathogenesis of periodontitis.


Assuntos
Hipóxia Celular/imunologia , Fibroblastos/imunologia , Inflamassomos/imunologia , Lipopolissacarídeos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Porphyromonas gingivalis , Adolescente , Adulto , Feminino , Gengiva/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/imunologia , Adulto Jovem
19.
Int J Nanomedicine ; 16: 725-740, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33542627

RESUMO

Purpose: As a dental material, polyetheretherketone (PEEK) is bioinert that does not induce cellular response and bone/gingival tissues regeneration. This study was to develop bioactive coating on PEEK and investigate the effects of coating on cellular response. Materials and Methods: Tantalum pentoxide (TP) coating was fabricated on PEEK surface by vacuum evaporation and responses of rat bone marrow mesenchymal stem (RBMS) cells/human gingival epithelial (HGE) were studied. Results: A dense coating (around 400 nm in thickness) of TP was closely combined with PEEK (PKTP). Moreover, the coating was non-crystalline TP, which contained many small humps (around 10 nm in size), exhibiting a nanostructured surface. In addition, the roughness, hydrophilicity, surface energy, and protein adsorption of PKTP were remarkably higher than that of PEEK. Furthermore, the responses (adhesion, proliferation, and osteogenic gene expression) of RBMS cells, and responses (adhesion and proliferation) of HGE cells to PKTP were remarkably improved in comparison with PEEK. It could be suggested that the nanostructured coating of TP on PEEK played crucial roles in inducing the responses of RBMS/HGE cells. Conclusion: PKTP with elevated surface performances and outstanding cytocompatibility might have enormous potential for dental implant application.


Assuntos
Células Epiteliais/citologia , Gengiva/citologia , Cetonas/farmacologia , Células-Tronco Mesenquimais/citologia , Nanoestruturas/química , Óxidos/farmacologia , Polietilenoglicóis/farmacologia , Tantálio/farmacologia , Adsorção , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Nanoestruturas/ultraestrutura , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Difração de Raios X
20.
Molecules ; 26(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477984

RESUMO

Casein and whey being food supplements have been considered to be used in oral health care products. However, the response of oral cells to micellar casein and whey powder remains unclear. Considering that milk contains the growth factor TGF-ß, and lactoperoxidase was recently reported to decrease the expression of inhibitor of DNA-binding (ID) proteins, there is a rationale to assume that casein and whey can also provoke these responses in oral cells. To examine the TGF-ß activity, gingival fibroblasts were exposed to reconstituted casein and whey powder from food supplement before the expression of TGF-ß target genes were analyzed by reverse transcription-quantitative polymerase chain reaction. Immunoassays were performed for interleukin11 (IL11) in the cell culture supernatant and for TGF-ß in the reconstituted casein and whey. We blocked TGF-ß by neutralizing the antibody and the TGF-ß receptor type I kinase with the inhibitor SB431542. We also showed smad3 phosphorylation and smad2/3 nuclear translocation by Western blot and immunostaining, respectively. Moreover, with reconstituted casein and whey powder, ID1 and ID3 expression analysis was evaluated in HSC2 human oral squamous carcinoma cells. We report here that casein and whey powder caused a robust increase of TGF-ß target genes interleukin11 (IL11), NADPH oxidase 4 (NOX4) and proteoglycan4 (PRG4) in gingival fibroblasts that was blocked by SB431542 and the neutralizing antibody. Moreover, casein and whey powder increased the phosphorylation of smad3 and nuclear translocation of smad2/3. No changes of proliferation markers Ki67 and cyclinD1 were observed. Furthermore, reconstituted casein and whey powder decreased ID1 and ID3 expression in the HSC2 oral squamous carcinoma cells. These findings suggest that the processing of milk into casein and whey powder maintains the TGF-ß activity and its capacity to regulate ID1 and ID3 genes in oral fibroblasts and oral squamous carcinoma cells, respectively. These data increase the scientific knowledge on the biological activity of casein and whey with a special emphasis on oral health.


Assuntos
Caseínas/química , Caseínas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Inibidoras de Diferenciação/genética , Micelas , Fator de Crescimento Transformador beta/metabolismo , Soro do Leite/química , Animais , Linhagem Celular , Fibroblastos/metabolismo , Gengiva/citologia , Pós
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