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1.
Int J Mol Sci ; 22(19)2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34639022

RESUMO

In this study, ε-polylysine and calcium phosphate precipitation (CPP) methods were employed to induce antibacterial effects and dentin tubule occlusion. Antibacterial effects of ε-polylysine were evaluated with broth dilution assay against P. gingivalis. CPP solution from MCPM, DCPD, and TTCP was prepared. Four concentrations of ε-polylysine(ε-PL) solutions (0.125%, 0.25%, 0.5%, 1%) were prepared. Dentin discs were prepared from recently extracted human third molars. Dentin discs were incubated with P. gingivalis (ATCC 33277) bacterial suspension (ca. 105 bacteria) containing Brain Heart Infusion medium supplemented with 0.1 g/mL Vitamin K, 0.5 mg/mL hemin, 0.4 g/mL L-cysteine in anaerobic jars (37 °C) for 7 days to allow for biofilm formation. P. g-infected dentin specimens were randomly divided into four groups: CPP + 0.125% ε-PL, CPP + 0.25% ε-PL, CPP + 0.5% ε-PL, CPP + 1% ε-PL. On each dentin specimen, CPP solution was applied followed by polylysine solution with microbrush and immersed in artificial saliva. Precipitate formation, antibacterial effects, and occlusion of dentinal tubules were characterized in vitro over up to 72 h using scanning electron microscopy. ε-PL showed 34.97% to 61.19% growth inhibition levels against P. gingivalis (P. g) after 24 h of incubation. On P. g-infected dentin specimens, DCPD + 0.25% ε-PL, and DCPD + 0.5% ε-PL groups showed complete bacterial inhibition and 78.6% and 98.1% dentin tubule occlusion, respectively (p < 0.001). The longitudinal analysis on fractured dentin samples in DCPD and TTCP groups revealed deeply penetrated hydroxyapatite-like crystal formations in dentinal tubules after 72 h of incubation in artificial saliva.


Assuntos
Antibacterianos/farmacologia , Fosfatos de Cálcio/farmacologia , Dentina/química , Polilisina/farmacologia , Antibacterianos/química , Fosfatos de Cálcio/química , Dentina/metabolismo , Sensibilidade da Dentina/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Polilisina/química , Análise Espectral , Propriedades de Superfície
2.
Cells ; 10(9)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34572140

RESUMO

Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules; therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1; however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.


Assuntos
Polpa Dentária/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Odontoblastos/metabolismo , Adolescente , Animais , Diferenciação Celular/genética , Polpa Dentária/fisiologia , Dentina/metabolismo , Dentina/fisiologia , Dentina Secundária/fisiologia , Dentinogênese/genética , Dentinogênese/fisiologia , Feminino , Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Odontoblastos/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/genética , Adulto Jovem
3.
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
4.
Arch Microbiol ; 203(7): 4133-4139, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34059945

RESUMO

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60 s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500 ppm F, as NaF), (4) 0.025 mg/ml CaneCPI-5, and (5) 0.05 mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8 h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60 s) with the solutions for 5 days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.


Assuntos
Biofilmes , Cistatinas , Dentina , Viabilidade Microbiana , Saccharum , Animais , Biofilmes/efeitos dos fármacos , Bovinos , Cistatinas/farmacologia , Dentina/metabolismo , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Saccharum/química , Streptococcus mutans/efeitos dos fármacos
5.
Sci Rep ; 11(1): 10802, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031498

RESUMO

Early childhood caries (ECC) recurrence occurs in approximately 40% of treated cases within one year. The association of Streptococcus mutans and Candida albicans with the onset of ECC is well known. Also, S. mutans strains harboring collagen-binding proteins (Cbps) avidly bind to collagen-rich dentin and are linked to increased caries risk. Here, we investigated the presence of Cbp+ S. mutans and C. albicans in saliva and dental plaque of children with varying caries statuses, and their salivary microbiome. In this cross-sectional study, 143 children who were caries-free (n = 73), treated for ECC with no signs of recurrence after 6 months (n = 45), or treated for ECC and experiencing recurrence within 6 months following treatment (n = 25) were enrolled. Co-infection with C. albicans and S. mutans, especially Cbp+ S. mutans, was strongly associated with caries recurrence. Subjects of the recurrence group infected with Cbp+ S. mutans showed a greater burden of Candida spp. and of Mutans streptococci in dentin than those infected with Cbp- strains. Salivary microbiome analysis revealed that Streptococcus parasanguinis was overrepresented in the caries recurrence group. Our findings indicate that Cbp+ S. mutans and C. albicans are intimately associated with caries recurrence, contributing to the establishment of recalcitrant biofilms.


Assuntos
Proteínas de Bactérias/metabolismo , Candida albicans/patogenicidade , Coinfecção/microbiologia , Cárie Dentária/microbiologia , Streptococcus mutans/patogenicidade , Candida albicans/isolamento & purificação , Candida albicans/metabolismo , Pré-Escolar , Estudos Transversais , Cárie Dentária/metabolismo , Suscetibilidade à Cárie Dentária , Dentina/metabolismo , Feminino , Humanos , Masculino , Recidiva , Saliva/microbiologia , Streptococcus/isolamento & purificação , Streptococcus mutans/isolamento & purificação , Streptococcus mutans/metabolismo
6.
PLoS One ; 16(5): e0250429, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34038418

RESUMO

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.


Assuntos
Dentina/metabolismo , Proteínas da Matriz Extracelular/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Dente/crescimento & desenvolvimento , Animais , Cromossomos Artificiais Bacterianos/genética , Colágeno Tipo II , Dentina/diagnóstico por imagem , Dentina/patologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/metabolismo , Incisivo/metabolismo , Incisivo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Minerais/análise , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/metabolismo , Dente/metabolismo , Microtomografia por Raio-X
7.
J Forensic Sci ; 66(4): 1524-1532, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33942892

RESUMO

It has already been proposed that a combined use of different molecular and morphological markers of aging in multivariate models may result in a greater accuracy of age estimation. However, such an approach can be complex and expensive, and not every combination may be useful. The significance and usefulness of combined analyses of D-aspartic acid in dentine, pentosidine in dentine, DNA methylation in buccal swabs at five genomic regions (PDE4C, RPA2, ELOVL2, DDO, and EDARADD), and third molar mineralization were tested by investigating a sample of 90 oral surgery patients. Machine learning models for age estimation were trained and evaluated, and the contribution of each parameter to multivariate models was tested by assessment of the predictor importance. For models based on D-aspartic acid, pentosidine, and the combination of both, mean absolute errors (MAEs) of 2.93, 3.41, and 2.68 years were calculated, respectively. The additional inclusion of the five DNAm markers did not improve the results. The sole DNAm-based model revealed a MAE of 4.14 years. In individuals under 28 years of age, the combination of the DNAm markers with the third molar mineralization stages reduced the MAE from 3.85 to 2.81 years. Our findings confirm that the combination of parameters in multivariate models may be very useful for age estimation. However, the inclusion of many parameters does not necessarily lead to better results. It is a task for future research to identify the best selection of parameters for the different requirements in forensic practice.


Assuntos
Determinação da Idade pelos Dentes/métodos , Adolescente , Adulto , Idoso , Arginina/análogos & derivados , Arginina/metabolismo , Biomarcadores/metabolismo , Criança , Ilhas de CpG/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , D-Aspartato Oxidase/metabolismo , Ácido D-Aspártico/metabolismo , Metilação de DNA , Dentina/metabolismo , Proteína de Domínio de Morte Associada a Edar/metabolismo , Elongases de Ácidos Graxos/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Aprendizado de Máquina , Pessoa de Meia-Idade , Dente Serotino/crescimento & desenvolvimento , Análise Multivariada , Proteína de Replicação A/metabolismo , Calcificação de Dente , Adulto Jovem
8.
Eur J Oral Sci ; 129(3): e12795, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33905141

RESUMO

FAM20C (family with sequence similarity 20-member C), a kinase that phosphorylates secretory proteins, plays essential roles in various biological processes. In humans, mutations in FAM20C gene cause Raine syndrome, an autosomal recessive hereditary disease manifesting a broad spectrum of developmental defects including skeletal and craniofacial deformities. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets and that high phosphate (hPi) diet significantly improved the development of the skeleton in Fam20c-deficient mice. In this study, we evaluated the effects of hPi diet on the formation of dentin in Fam20c-deficient mice, using plain x-ray radiography, micro-computed tomography (µCT), histology, and immunohistochemistry. Plain x-ray radiography and µCT analyses showed that the hPi diet improved the dentin volume fraction and dentin mineral density of the Fam20c-deficient mice. Histology analyses further demonstrated that the hPi diet dramatically improved the integrity of the mandibular first molars and prevented pulp infection and dental abscesses in Fam20c-deficient mice. Our results support that the hPi diet significantly increased the formation and mineralization of dentin in Fam20c-deficient mice, implying that hypophosphatemia is a significant contributor to the dentin defects in Fam20c-deficient subjects.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas da Matriz Extracelular , Animais , Proteínas de Ligação ao Cálcio/genética , Dentina/metabolismo , Dieta , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Camundongos Knockout , Fosfatos , Microtomografia por Raio-X
9.
Sci Rep ; 11(1): 6083, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727650

RESUMO

This in vitro study evaluated the protective effect of titanium tetrafluoride (TiF4) varnish and silver diamine fluoride (SDF) solution on the radiation-induced dentin caries. Bovine root dentin samples were irradiated (70 Gy) and treated as follows: (6 h): 4% TiF4 varnish; 5.42% NaF varnish; 30% SDF solution; placebo varnish; or untreated (negative control). Microcosm biofilm was produced from human dental biofilm (from patients with head-neck cancer) mixed with McBain saliva for the first 8 h. After 16 h and from day 2 to day 5, McBain saliva (0.2% sucrose) was replaced daily (37 °C, 5% CO2) (biological triplicate). Demineralization was quantified by transverse microradiography (TMR), while biofilm was analyzed by using viability, colony-forming units (CFU) counting and lactic acid production assays. The data were statistically analyzed by ANOVA (p < 0.05). TiF4 and SDF were able to reduce mineral loss compared to placebo and the negative control. TiF4 and SDF significantly reduced the biofilm viability compared to negative control. TiF4 significantly reduced the CFU count of total microorganism, while only SDF affected total streptococci and mutans streptococci counts. The varnishes induced a reduction in lactic acid production compared to the negative control. TiF4 and SDF may be good alternatives to control the development of radiation-induced dentin caries.


Assuntos
Cárie Dentária , Dentina , Compostos de Amônio Quaternário/farmacologia , Lesões Experimentais por Radiação , Protetores contra Radiação/farmacologia , Compostos de Prata/farmacologia , Titânio/farmacologia , Raios X/efeitos adversos , Animais , Bovinos , Cárie Dentária/metabolismo , Cárie Dentária/patologia , Cárie Dentária/prevenção & controle , Dentina/metabolismo , Dentina/patologia , Fluoretos Tópicos/farmacologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/prevenção & controle
10.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33671069

RESUMO

BACKGROUND: Copper-containing biomaterials are increasingly applied for bone regeneration due to their pro-angiogenetic, pro-osteogenetic and antimicrobial properties. Therefore, the effect of Cu2+ on osteoclasts, which play a major role in bone remodeling was studied in detail. METHODS: Human primary osteoclasts, differentiated from human monocytes were differentiated or cultivated in the presence of Cu2+. Osteoclast formation and activity were analyzed by measurement of osteoclast-specific enzyme activities, gene expression analysis and resorption assays. Furthermore, the glutathione levels of the cells were checked to evaluate oxidative stress induced by Cu2+. RESULTS: Up to 8 µM Cu2+ did not induce cytotoxic effects. Activity of tartrate-resistant acid phosphatase (TRAP) was significantly increased, while other osteoclast specific enzyme activities were not affected. However, gene expression of TRAP was not upregulated. Resorptive activity of osteoclasts towards dentin was not changed in the presence of 8 µM Cu2+ but decreased in the presence of extracellular bone matrix. When Cu2+ was added to mature osteoclasts TRAP activity was not increased and resorption decreased only moderately. The glutathione level of both differentiating and mature osteoclasts was significantly decreased in the presence of Cu2+. CONCLUSIONS: Differentiating and mature osteoclasts react differently to Cu2+. High TRAP activities are not necessarily related to high resorption.


Assuntos
Remodelação Óssea/efeitos dos fármacos , Reabsorção Óssea , Cobre/farmacologia , Leucócitos Mononucleares/citologia , Osteoclastos/citologia , Animais , Diferenciação Celular , Células Cultivadas , Dentina/metabolismo , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Suínos , Fosfatase Ácida Resistente a Tartarato/metabolismo
11.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540711

RESUMO

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.


Assuntos
Polpa Dentária/patologia , Periodontite Periapical/patologia , Tecido Periapical/patologia , Pulpite/patologia , Animais , Antígenos de Neoplasias/imunologia , Carcinogênese/imunologia , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/fisiopatologia , Quimiocinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Cárie Dentária/fisiopatologia , Polpa Dentária/microbiologia , Dentina/irrigação sanguínea , Dentina/inervação , Dentina/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neoplasias Bucais/etiologia , Neoplasias Bucais/imunologia , Neoplasias Bucais/fisiopatologia , Rede Nervosa/fisiologia , Neuropeptídeos/metabolismo , Óxido Nítrico/fisiologia , Odontoblastos/fisiologia , Granuloma Periapical/etiologia , Granuloma Periapical/patologia , Tecido Periapical/microbiologia , Cisto Radicular/etiologia , Cisto Radicular/fisiopatologia
12.
Arch Oral Biol ; 125: 105086, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33639479

RESUMO

OBJECTIVES: We aimed to observe the posttranslational role of dentin sialophosphoprotein (DSPP) on postnatal development of mandibular condyle in mice. METHODS: To explore the function of full-length DSPP, four groups of mice were employed: (1) wild type (WT) mice; (2)Dspp knockout (Dspp KO) mice; (3) mice expressing the normal DSPP transgene in the Dspp KO background (Dspp KO/normal Tg); (4) mice expressing the uncleavable full-length DSPP in the Dspp KO background (Dspp KO/D452A Tg). Firstly, Plain X-ray Radiography and Micro-computed Tomography were used to observe the condylar morphology changes of Dspp KO/D452A Tg mice in comparison with the other three groups. Then, Hematoxylin & eosin and toluidine blue staining were applied to uncover the histological changes of mandibular condylar cartilage (MCC) of Dspp KO/D452A Tg mice. To explore the function of the NH2-terminal fragments (i.e. DSP/DSP-PG), three groups of mice were employed: (1) WT mice; (2) Dspp KO mice; (3) mice expressing the NH2-terminal fragments of DSPP in the Dspp-null background (Dspp KO/DSP Tg). The former strategies were utilized to examine the differences of condylar morphology and histological structures changes within three groups of mice. RESULTS: Transgenic full-length DSPP partially maintained mandibular condylar morphology and MCC thickness of Dspp KO mice. Transgenic DSP failed to do so, but led to smaller mandibular condyle and disordered cartilage structure. CONCLUSIONS: Our observations provide insight into the role of posttranslational modification of DSPP in the postnatal development of healthy MCC and maintenance of condylar morphology.


Assuntos
Côndilo Mandibular , Sialoglicoproteínas , Animais , Dentina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Côndilo Mandibular/diagnóstico por imagem , Côndilo Mandibular/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Microtomografia por Raio-X
13.
Biomed Res Int ; 2021: 6652250, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33628801

RESUMO

Objectives: To evaluate the in vitro effectiveness of desensitizing agents in reducing dentine permeability. Methods: The efficacy of desensitizing agents in reducing dentine permeability by occluding dentine tubules was evaluated using a fluid filtration device that conducts at 100 cmH2O (1.4 psi) pressure, and SEM/EDX analyses were evaluated and compared. Forty-two dentine discs (n = 42) of 1 ± 0.2 mm width were obtained from caries-free permanent human molars. Thirty dentine discs (n = 30) were randomly divided into 3 groups (n = 10): Group 1: 2.7% wt. monopotassium-monohydrogen oxalate (Mp-Mh oxalate), Group 2: RMGI XT VAR, and Group 3: LIQ SiO2. Dentine permeability was measured following treatment application after 10 minutes, storage in artificial saliva after 10 minutes and 7 days, and citric acid challenge for 3 minutes. Data were analysed with a repeated measures ANOVA test. Dentine discs (n = 12) were used for SEM/EDX analyses to acquire data on morphological changes on dentine surface and its mineral content after different stages of treatment. Results: Desensitizing agents' application on the demineralized dentine discs exhibited significant reduction of permeability compared to its maximum acid permeability values. Mp-Mh oxalate showed a significant reduction in dentine permeability (p < 0.05) when compared to RMGI XT VAR and LIQ SiO2. On SEM/EDX analysis, all the agents formed mineral precipitates that occluded the dentine tubules. Conclusions: 2.7% wt. monopotassium-monohydrogen oxalate was significantly effective in reducing dentine permeability compared to RMGI XT VAR and LIQ SiO2.


Assuntos
Dessensibilizantes Dentinários , Permeabilidade da Dentina/efeitos dos fármacos , Sensibilidade da Dentina , Dentina/metabolismo , Dente Molar/metabolismo , Dessensibilizantes Dentinários/química , Dessensibilizantes Dentinários/farmacologia , Sensibilidade da Dentina/tratamento farmacológico , Sensibilidade da Dentina/metabolismo , Humanos
14.
FASEB J ; 35(2): e21325, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33508145

RESUMO

The objectives of our study were to investigate the roles of mTORC1 in odontoblast proliferation and mineralization and to determine the mechanism by which mTORC1 regulates odontoblast mineralization. In vitro, MDPC23 cells were treated with rapamycin (10 nmol/L) and transfected with a lentivirus for short hairpin (shRNA)-mediated silencing of the tuberous sclerosis complex (shTSC1) to inhibit and activate mTORC1, respectively. CCK8 assays, flow cytometry, Alizarin red S staining, ALP staining, qRT-PCR, and western blot analysis were performed. TSC1-conditional knockout (DMP1-Cre+ ; TSC1f/f , hereafter CKO) mice and littermate control (DMP1-Cre- ; TSC1f/f , hereafter WT) mice were generated. H&E staining, immunofluorescence, and micro-CT analysis were performed. Transcriptome sequencing analysis was used to screen the mechanism of this process. mTORC1 inactivation decreased the cell proliferation. The qRT-PCR and western blot results showed that mineralization-related genes and proteins were downregulated in mTORC1-inactivated cells. Moreover, mTORC1 overactivation promoted cell proliferation and mineralization-related gene and protein expression. In vivo, the micro-CT results showed that DV/TV and dentin thickness were higher in CKO mice than in controls and H&E staining showed the same results. Mineralization-related proteins expression was upregulated. Transcriptome sequencing analysis revealed that p53 pathway-associated genes were differentially expressed in TSC1-deficient cells. By inhibiting p53 alone or both mTORC1 and p53 with rapamycin and a p53 inhibitor, we elucidated that p53 acts downstream of mTORC1 and that mTORC1 thereby promotes odontoblast mineralization. Taken together, our findings demonstrate that the role of mTORC1 in odontoblast proliferation and mineralization, and confirm that mTORC1 upregulates odontoblast mineralization via the p53 pathway.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Odontoblastos/metabolismo , Calcificação de Dente , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Dentina/citologia , Dentina/metabolismo , Camundongos , Odontoblastos/fisiologia , Transcriptoma , Proteína 1 do Complexo Esclerose Tuberosa/genética
15.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477897

RESUMO

Hydroxyapatite (HA) reinforced collagen fibrils serve as the basic building blocks of natural bone and dentin. Mineralization of collagen fibrils play an essential role in ensuring the structural and mechanical functionalities of hard tissues such as bone and dentin. Biomineralization of collagen can be divided into intrafibrillar and extrafibrillar mineralization in terms of HA distribution relative to collagen fibrils. Intrafibrillar mineralization is termed when HA minerals are incorporated within the gap zone of collagen fibrils, while extrafibrillar mineralization refers to the minerals that are formed on the surface of collagen fibrils. However, the mechanisms resulting in these two types of mineralization still remain debatable. In this review, the evolution of both classical and non-classical biomineralization theories is summarized. Different intrafibrillar mineralization mechanisms, including polymer induced liquid precursor (PILP), capillary action, electrostatic attraction, size exclusion, Gibbs-Donnan equilibrium, and interfacial energy guided theories, are discussed. Exemplary strategies to induce biomimetic intrafibrillar mineralization using non-collagenous proteins (NCPs), polymer analogs, small molecules, and fluidic shear stress are discussed, and recent applications of mineralized collagen fibers for bone regeneration and dentin repair are included. Finally, conclusions are drawn on these proposed mechanisms, and the future trend of collagen-based materials for bone regeneration and tooth repair is speculated.


Assuntos
Biomineralização/genética , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Colágeno/genética , Dentina/metabolismo , Biomimética , Colágeno/química , Colágeno/metabolismo , Dentina/crescimento & desenvolvimento , Durapatita/farmacologia , Matriz Extracelular/efeitos dos fármacos , Humanos , Polímeros/química , Polímeros/farmacologia , Engenharia Tecidual , Cicatrização/efeitos dos fármacos , Difração de Raios X
16.
J Mol Histol ; 52(1): 63-75, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33141361

RESUMO

Much information is currently available for molecules in early odontogenesis, but there is limited knowledge regarding terminal cytodifferentiation of ameloblasts and odontoblasts for the determination of normal crown morphology. The present differential display PCR (DD-PCR) revealed that insulin-like growth factor-binding protein 5 (IGFBP5) was differentially expressed in molar tooth germs between the cap (before crown mineralization) and root formation (after crown mineralization) stages. Real-time PCR confirmed that the expression levels of IGFBP1-4 were not significantly changed but those of IGFBP5-7 were upregulated in a time-dependent manner. Immunoreactivities for IGFBP5-7 were hardly seen in molar germs at the cap/early bell stage and protective-stage ameloblasts at the root formation stage. However, the reactivity was strong in odontoblasts and maturation-stage ameloblasts, which are morphologically and functionally characterized by wide intercellular space and active enamel matrix mineralization. The localization of each IGFBP was temporospatial. IGFBP5 was localized in the nuclei of fully differentiated odontoblasts and ameloblasts, while IGFBP6 was localized in the apical cytoplasm of ameloblasts and odontoblasts with dentinal tubules, and IGFBP7 was mainly found in the whole cytoplasm of odontoblasts and the intercellular space of ameloblasts. IGFBP silencing using specific siRNAs upregulated representative genes for dentinogenesis and amelogenesis, such as DMP1 and amelogenin, respectively, and augmented the differentiation media-induced mineralization, which was confirmed by alizarin red s and alkaline phosphatase staining. These results suggest that IGFBP5-7 may play independent and redundant regulatory roles in late-stage odontogenesis by modulating the functional differentiation of ameloblasts and odontoblasts.


Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Odontogênese , Calcificação de Dente , Amelogênese/genética , Animais , Esmalte Dentário/metabolismo , Dentina/metabolismo , Regulação da Expressão Gênica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Dente Molar/metabolismo , Odontoblastos/metabolismo , Odontogênese/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Calcificação de Dente/genética , Germe de Dente/metabolismo , Regulação para Cima/genética
17.
Biochem Biophys Res Commun ; 534: 837-842, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33168184

RESUMO

Functional peptides derived from the active domains of odontogenesis-related proteins have been reported to promote dental hard tissue regeneration. The purpose of this study was to evaluate the effects of an artificially synthesized peptide, TVH-19, on odontoblast differentiation and tertiary dentin formation in indirect pulp capping (IPC) using in vitro and in vivo experiments. TVH-19 did not exhibit any effect on the proliferation of human dental pulp cells (hDPCs) but significantly promoted cell migration, compared with the control (p < 0.05). TVH-19-treated hDPCs showed significantly higher alkaline phosphatase (ALP) activity and stronger alizarin red staining (ARS) reactivity than the control group (p < 0.05). TVH-19 also upregulated the mRNA and protein expression levels of odontogenic genes. After generating IPC in rats, the samples of teeth were studied using micro-computed tomography (Micro-CT), hematoxylin & eosin (HE) staining, and immunohistochemical staining to investigate the functions of TVH-19. The in vivo results showed that TVH-19 induced the formation of tertiary dentin, and reduced inflammation and apoptosis, as evident from the downregulated expression of interleukin 6 (IL-6) and cleaved-Caspase-3 (CL-CASP3). Overall, the results of our study suggest that TVH-19 induces differentiation of hDPCs, promotes tertiary dentin formation, relieves inflammation, and reduces apoptosis, indicating the potential applications of TVH-19 in IPC.


Assuntos
Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Dentina/metabolismo , Peptídeos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/metabolismo , Humanos , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Peptídeos/química , Calcificação de Dente/efeitos dos fármacos
18.
J Cell Physiol ; 236(1): 480-488, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32537777

RESUMO

Tooth root development occurs through the interaction of multiple growth factors and transcription factors expressed in Hertwig's epithelial root sheath (HERS) and dental mesenchyme. Previously, we demonstrated that bobby sox homolog (Bbx) regulates odontoblast differentiation of human dental pulp stem cells. Here, we generated Bbx knockout (Bbx-/- ) mice to address the functional role of Bbx in tooth formation. During tooth development, Bbx was expressed in both dental epithelium and mesenchyme. However, molar and incisor morphology in Bbx-/- mice at postnatal Day 0 (P0) exhibited no prominent abnormalities compared with their wild-type (Bbx+/+ ) littermates. Until P28, the crown morphology in Bbx-/- mice was not distinctively different from Bbx+/+ littermates. Meanwhile, the length of the mandibular base in Bbx-/- mice was notably less at P28. Compared with Bbx+/+ mice, the mesial and distal root lengths of the first molar were reduced by 21.33% and 16.28% at P14 and 16.28% and 16.24% at P28, respectively, in Bbx-/- mice. The second molar of Bbx-/- mice also showed 10.16% and 6.4% reductions at P28 in the mesial and distal lengths, compared with Bbx+/+ mice, respectively. The gene expression analysis during early tooth root formation (P13) showed that the expression of dentin sialophosphoprotein (Dspp) was significantly decreased in Bbx-/- mice. Collectively, our data suggest that Bbx participates in tooth root formation and might be associated with the regulation of Dspp expression.


Assuntos
Dentina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Dente Molar/metabolismo , Odontogênese/fisiologia , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Raiz Dentária/crescimento & desenvolvimento , Raiz Dentária/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Epitélio/metabolismo , Feminino , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Dente Molar/crescimento & desenvolvimento , Odontoblastos/metabolismo , Fatores de Transcrição/metabolismo
19.
Methods Mol Biol ; 2206: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754821

RESUMO

Exiting developments in tissue engineering and new insights in stem cell biology have led to new possible strategies for the regeneration of damaged tissues in the oral cavity. The regeneration of the pulp-dentin complex regeneration in particular, has drawn the attention of many researchers because of the high clinical needs. While it is still important to perform in vitro research using a wide variety of cells, scaffolds and growth factors, it is also critical to have a reliable animal model for preclinical trials. In this chapter, we describe a mouse model in which a scaffold resembling a tooth containing dental pulp cells is implanted subcutaneously. We also describe which histological stainings could be used to examine blood vessel formation and the regeneration of the pulp-dentin complex.


Assuntos
Polpa Dentária/citologia , Regeneração/fisiologia , Pele/citologia , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Polpa Dentária/metabolismo , Dentina/metabolismo , Camundongos , Camundongos Nus , Camundongos SCID , Modelos Animais , Pele/metabolismo , Células-Tronco/citologia , Engenharia Tecidual/métodos , Tecidos Suporte
20.
Int J Mol Sci ; 21(23)2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33276685

RESUMO

Particulate autogenous tooth roots are used for alveolar bone augmentation surgery; however, dental plaque may provoke an inflammatory response that may counteract the desired graft consolidation process. Traditional mechanical cleaning of extracted teeth may be of support to lower a possible inflammatory response of the autograft. To test this assumption, extracted porcine teeth were left either uncleaned or underwent mechanical cleaning with a toothbrush and toothpaste before being fragmented and subjected to acid lysis, termed as unclean acid dentine lysate (ucADL) and clean acid dentine lysate (cADL), respectively. The inflammatory responses of murine macrophage RAW 264.7 cells being exposed to the respective acid dentine lysates were evaluated at the level of inflammatory gene expression and IL6 immunoassays. We report here that acid lysates obtained from uncleaned teeth provoked a robust increase in IL1ß, IL6, and COX2 in RAW 264.7 cells. The mechanical removal of dental plaque significantly reduced the inflammatory response. Consistently, Limulus tests revealed that tooth cleaning lowers the presence of endotoxins in dentine lysates. To further prove the involvement of endotoxins, a toll-like receptor 4 (TLR4) inhibitor TAK242 was introduced. TAK242 abolished the inflammatory response provoked by acid lysates obtained from uncleaned teeth in RAW 264.7 cells. Moreover, nuclear translocation and phosphorylation of the TLR4 downstream NFκB-p65 were attenuated at the presence of cleaned versus uncleaned dentine lysates. Taken together, our data support the importance of dental plaque removal of teeth being extracted for alveolar bone augmentation surgery.


Assuntos
Dentina/metabolismo , Concentração de Íons de Hidrogênio , Macrófagos/metabolismo , Higiene Bucal , Animais , Biomarcadores , Assistência Odontológica , Placa Dentária , Endotoxinas/efeitos adversos , Endotoxinas/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos , Fosforilação , Células RAW 264.7 , Receptor 4 Toll-Like/metabolismo , Raiz Dentária , Escovação Dentária
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