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1.
J Periodontal Res ; 47(3): 299-308, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22489671

RESUMO

BACKGROUND AND OBJECTIVE: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. MATERIAL AND METHODS: Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. RESULTS: Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor-κB. CONCLUSION: These results indicate that nicotine-induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Nicotina/toxicidade , Ligamento Periodontal/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/farmacologia , Colágeno Tipo I/efeitos dos fármacos , Elastina/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/efeitos dos fármacos , Matriz Extracelular/enzimologia , Fibronectinas/efeitos dos fármacos , Proteínas de Choque Térmico/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 8 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Nicotina/antagonistas & inibidores , Ligamento Periodontal/citologia , Ligamento Periodontal/enzimologia , Proteínas Quinases/análise , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Fator de Transcrição CHOP/efeitos dos fármacos , Fator de Transcrição CHOP/genética
2.
J Periodontal Res ; 47(6): 719-28, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22571166

RESUMO

BACKGROUND AND OBJECTIVE: Although hypoxia-inducible factor 1α (HIF-1α) is up-regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF-1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF-1α and on the production of its target genes, including cyclooxygenase-2 (COX-2)-derived prostaglandin E(2) (PGE(2) ), MMP-2 and MMP-9 in PDLCs. MATERIAL AND METHODS: The expression of COX-2 and HIF-1α proteins was evaluated using western blotting. The production of PGE(2) and MMPs was evaluated using enzyme immunoassays and zymography, respectively. RESULTS: LPS and nicotine synergistically induced the production of PGE(2) , MMP-2 and MMP-9, and increased the expression of MMP-2, MMP-9, COX-2 and HIF-1α proteins. Inhibition of HIF-1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS- and nicotine-stimulated PGE(2) and MMPs, as well as the expression of COX-2 and HIF-1α. Furthermore, pretreatment with inhibitors of COX-2, p38, extracellular signal-regulated kinase, Jun N-terminal kinase, protein kinase C, phosphatidylinositol 3-kinase and nuclear factor-kappaB decreased the expression of nicotine- and LPS-induced HIF-1α and COX-2, as well as the activity of PGE(2) and MMPs. CONCLUSION: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF-1α is a potential target in periodontal disease associated with smoking and dental plaque.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipopolissacarídeos/farmacologia , Nicotina/farmacologia , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Análise de Variância , Linhagem Celular Transformada , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Fibroblastos/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/química , Estatísticas não Paramétricas , Regulação para Cima
3.
J Oral Rehabil ; 36(10): 748-54, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19702681

RESUMO

This study was designed to radiographically evaluate the effect of surface macro-and microstructures within the coronal portion of the external hex implant at the marginal bone change after loading. The fifty-four patients included in the study were randomly assigned to treatment groups with rough-surface implants (TiUnite, n = 45), a hybrid of smooth and rough surface implants (Restore, n = 45) or rough-surface with microthreads implants (Hexplant, n = 45). Clinical and radiographic examinations were conducted at the time of implant loading (baseline) and at 1-year post-loading. A three-level mixed-effect ancova was used to test the significance of the mean marginal bone change of the three implant groups from baseline to 1-year follow-up. At 1-year, significant differences were noted in marginal bone loss recorded for the three groups (P < 0.0001). The rough surface with microthread implants had a mean crestal bone loss of 0.42 +/- 0.27 mm; the rough surface implants, 0.81 +/- 0.27 mm; and the hybrid surface implants, 0.89 +/- 0.41 mm. Within the limitations of this study, a rough surface with microthreads at the coronal part of implant maintained the marginal bone level against functional loading better than implants without these two features.


Assuntos
Perda do Osso Alveolar/diagnóstico por imagem , Processo Alveolar/diagnóstico por imagem , Mandíbula/diagnóstico por imagem , Maxila/diagnóstico por imagem , Osseointegração/fisiologia , Adulto , Idoso , Perda do Osso Alveolar/cirurgia , Processo Alveolar/cirurgia , Análise de Variância , Materiais Revestidos Biocompatíveis/química , Implantação Dentária Endóssea/métodos , Falha de Restauração Dentária , Feminino , Humanos , Masculino , Mandíbula/cirurgia , Maxila/cirurgia , Pessoa de Meia-Idade , Radiografia , Propriedades de Superfície
4.
J Periodontol ; 66(7): 579-93, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7562350

RESUMO

Periodontal regeneration requires formation of periodontal tissues lost due to periodontal disease. To better understand the formation of new periodontal tissues during periodontal repair and regeneration, immunohistochemical expression of extracellular matrix components of normal as well as healing periodontal tissues was evaluated and compared using the avidin-biotin complex immunohistochemical technique. For this purpose, horizontal furcation defects were created around mandibular P2 and P4 of 6 dogs after extraction of P1 and P3. The root surfaces were conditioned with citric acid and expanded polytetrafluoroethylene (ePTFE) membranes were placed and retained 0.5 mm above the cemento-enamel junction. The mucoperiosteal flaps were sutured in a coronal position. Two animals were sacrificed at 2, 4, and 8 weeks, and mesio-distal tissue slices containing normal or healing periodontal tissues were demineralized, dehydrated, and embedded in paraffin. Immunohistochemical localization of type I collagen (CI), fibronectin (FN), secreted protein, acidic and rich in cysteine (SPARC), vitronectin (VN), and bone sialoprotein (BSP) was performed on 6 microns thick sections. Morphological results demonstrated that at 2 weeks after defect creation, lesions were filled primarily with granulation tissue which was gradually replaced by newly-formed fibrous connective tissue, periodontal ligament (PDL), cementum, and bone between 4 and 8 weeks. The results of immunohistochemical study revealed that at 2 weeks the granulation tissue, especially in the intercellular spaces of inflammatory cells, was intensively stained for FN and VN. At 4 and 8 weeks, staining for CI, FN, and VN was found in fibrous connective tissue, the newly-formed PDL, cementum, and osteoid. Further the attachment zone of the PDL collagen fibers to cementum showed intense staining for FN. Immunostaining for SPARC was positive in the new PDL, cementum, and bone, while staining for BSP was restricted to the new cementum and bone. Interestingly, the PDL, especially in areas adjacent to active bone formation, demonstrated intense staining for BSP. However, fibrous connective tissue and PDL proper were unstained for BSP. These results indicate that FN and VN are involved in the early stages of periodontal repair, and periodontal regeneration is achieved through formation of periodontal tissues that are composed of different matrix components specific to different types of periodontal tissues.


Assuntos
Proteínas da Matriz Extracelular/análise , Periodonto/metabolismo , Condicionamento Ácido do Dente , Animais , Dente Pré-Molar , Citratos/administração & dosagem , Ácido Cítrico , Colágeno/análise , Colágeno/genética , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Cisteína/análise , Cisteína/genética , Cemento Dentário/metabolismo , Cemento Dentário/patologia , Cães , Proteínas da Matriz Extracelular/genética , Fibronectinas/análise , Fibronectinas/genética , Defeitos da Furca/metabolismo , Defeitos da Furca/patologia , Defeitos da Furca/cirurgia , Expressão Gênica , Tecido de Granulação/metabolismo , Tecido de Granulação/patologia , Regeneração Tecidual Guiada Periodontal , Imuno-Histoquímica , Sialoproteína de Ligação à Integrina , Masculino , Membranas Artificiais , Ligamento Periodontal/metabolismo , Ligamento Periodontal/patologia , Periodonto/patologia , Periodonto/cirurgia , Politetrafluoretileno , Regeneração , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Vitronectina/análise , Vitronectina/genética , Cicatrização
5.
J Periodontol ; 66(8): 716-30, 1995 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7473015

RESUMO

The origin of fibroblasts, their proliferative activity and roles in the early stages of periodontal repair were investigated in order to better understand the periodontal healing process in furcation defects of the beagle dog after guided tissue regenerative therapy. Newly divided cells were identified by immunolocalization of bromodeoxyuridine (BrdU) injected 1 hour prior to sacrificing the animals. At 1 and 2 weeks after creation of the defects, the lesions were occupied primarily by granulation tissue. Under this condition, periodontal ligaments (PDL) fibroblasts in a coronal portion of the remaining PDL close to wounds proliferated actively, migrated along the root surface and formed fibrous connective tissue on the surface. Similarly, the fibroblasts adjacent to the bone surface also showed proliferative activity and engaged in active formation of fibrous connective tissue on the bone surface. The majority of labeled cells in both areas were located in the extravascular area. At 3 and 4 weeks, the defects were filled with an increased amount of new connective tissue and bone. The labeled fibroblasts were preferentially found in the most coronal portion of connective tissue formed on the root surface that was in direct contact with inflamed tissue, and the collagen fibers projected into granulation tissue. In areas of active bone formation, numerous labeled fibroblasts were located in connective tissue adjacent to the newly-formed bone. However, fibroblasts in the endosteum of new bone were rarely labeled These results indicate that fibroblasts involved in periodontal repair originate primarily from both the remaining PDL and alveolar bone, and actively engage in fibrous connective tissue formation in the early stages of periodontal repair The ability of PDL fibroblasts to proliferate, migrate, and form connective tissue on the root surfaces in the early repair stages appears to play a crucial role in the formation of the PDL and cementum, and consequently, in periodontal regeneration in the absence of root resorption and ankylosis. As the formation of new connective tissue and bone continues, the precursor cells for fibroblasts and osteoblasts are supplied locally through the continued divisions of the fibroblastic cells in association with the newly-formed connective tissue. Paravascular and endosteal cells appear to be minor contributors to new cell population during furcation defect repair in the beagle dog.


Assuntos
Fibroblastos/patologia , Fibroblastos/fisiologia , Defeitos da Furca/patologia , Defeitos da Furca/cirurgia , Processo Alveolar/patologia , Animais , Antimetabólitos , Bromodesoxiuridina , Divisão Celular , Movimento Celular , Colágeno , Tecido Conjuntivo/patologia , Tecido Conjuntivo/fisiopatologia , Cemento Dentário/patologia , Cemento Dentário/fisiopatologia , Cães , Defeitos da Furca/fisiopatologia , Tecido de Granulação/patologia , Tecido de Granulação/fisiopatologia , Regeneração Tecidual Guiada Periodontal , Masculino , Osteoblastos/patologia , Osteoblastos/fisiologia , Osteogênese , Ligamento Periodontal/patologia , Ligamento Periodontal/fisiopatologia , Periodontite/patologia , Periodontite/fisiopatologia , Regeneração , Raiz Dentária/patologia , Cicatrização
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