RESUMEN
The periodontal ligament (PDL) cells contain heterogeneous mesenchymal cell populations, which have the ability to differentiate into cells that produce adjacent mineralized tissues and abundant extracellular matrix (ECM). ECM is essential not only for the homeostasis of the periodontal tissue, but also for controlling the differentiation of the PDL cells. The process of differentiation involves mechanotransduction, which links the ECM to the cytoskeleton. The present study investigated the roles of Rho-associated coiled-coil containing protein kinase (ROCK) signaling, a crucial regulator of the cytoskeleton, during ECM-mediated osteogenic differentiation of PDL cells in vitro. The PDL cells were isolated from human periodontal ligaments of extracted teeth and cultured in osteogenic medium with or without Y-27632, a pharmacological inhibitor of ROCK. ECM-coated plates were used for ECM-mediated differentiation. The osteogenic phenotype was evaluated at different time points by real-time RT-PCR for the gene encoding alkaline phosphatase (ALP) and an ALP activity assay. The effects of ROCK on cytoskeletal changes and ECM synthesis were examined by immunofluorescence analysis. Y-27632 significantly inhibited ALP at the mRNA and protein activity levels in the late stage of differentiation; concomitantly, the actin filament content and the extracellular levels of collagen-I and fibronectin were markedly decreased by Y-27632. Exogenous collagen-I and fibronectin temporally increased ALP activity, with fibronectin showing a more pronounced effect. Importantly, ECM-mediated differentiation was almost completely inhibited by Y-27632. These findings indicated that ECM-mediated differentiation is dependent on ROCK signaling, and ROCK signaling contributes to the establishment of the ECM microenvironment for PDL cell differentiation.
Asunto(s)
Ligamento Periodontal/metabolismo , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/fisiología , Amidas , Diferenciación Celular/fisiología , Células Cultivadas , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Mecanotransducción Celular , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Ligamento Periodontal/citología , Ligamento Periodontal/fisiología , Cultivo Primario de Células/métodos , PiridinasRESUMEN
OBJECTIVE: Gingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-ß (TGF-ß) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-ß, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes. METHODS: Human gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis. RESULTS: By using SB431542, a specific inhibitor of the TGF-ß type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-ß1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, ß4, and ß6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression. CONCLUSION: These findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2.
Asunto(s)
Encía/citología , Proteína Smad2/metabolismo , Animales , Benzamidas/farmacología , Adhesión Celular/efectos de los fármacos , Dioxoles/farmacología , Humanos , Integrinas/metabolismo , Ratones , Ratones Transgénicos , Diente Molar , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transfección , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Aggressive periodontitis is a great challenge to clinicians when providing orthodontic treatment because of the potential for progression of periodontal disease. In this article, we report the successful comprehensive orthodontic treatment of bimaxillary protrusion and severe crowding in an adult with generalized aggressive periodontitis. A woman, aged 22 years 7 months, with a chief complaint of incisal crowding was diagnosed with a skeletal Class I malocclusion associated with severe anterior crowding, possibly worsened by generalized aggressive periodontitis. In addition to a periodontal examination, a blood IgG antibody titer analysis and microbiologic examination for periodontal pathogens were used to diagnose the type of periodontal disease and determine the proper timing to initiate orthodontic treatment. The total active treatment period was 28 months, followed by periodontal prostheses and regeneration therapy. Consequently, satisfactory facial profile, occlusion, and periodontal health were maintained for at least 36 months. These results indicate that efficient screening is important for providing successful orthodontic treatment in patients with advanced periodontal disease. This report also demonstrates the diagnostic importance of blood IgG antibody titer assays and microbiologic examinations to detect periodontal pathogens.
Asunto(s)
Periodontitis Agresiva/terapia , Inmunoglobulina G/sangre , Maloclusión Clase I de Angle/terapia , Grupo de Atención al Paciente , Aggregatibacter actinomycetemcomitans/inmunología , Periodontitis Agresiva/microbiología , Pérdida de Hueso Alveolar/terapia , Anticuerpos Antibacterianos/sangre , Cefalometría/métodos , Femenino , Estudios de Seguimiento , Regeneración Tisular Guiada Periodontal/métodos , Humanos , Maloclusión Clase I de Angle/sangre , Higiene Bucal , Planificación de Atención al Paciente , Prótesis Periodontal , Porphyromonas gingivalis/inmunología , Prevotella intermedia/inmunología , Factores de Tiempo , Técnicas de Movimiento Dental/métodos , Resultado del Tratamiento , Adulto JovenRESUMEN
The periodontal ligament is a multifunctional soft connective tissue, which functions not only as a cushion supporting the teeth against occlusal force, but is also a source of osteogenic cells that can regenerate neighboring hard tissues. Periodontal ligament cells (PDL cells) contain heterogeneous cell populations, including osteogenic cell progenitors. However, the precise mechanism underlying the differentiation process remains elusive. Cell differentiation is regulated by the local biochemical and mechanical microenvironment that can modulate gene expression and cell morphology by altering actin cytoskeletal organization mediated by Rho-associated, coiled-coil containing protein kinase (ROCK). To determine its role in PDL cell differentiation, we examined the effects of ROCK on cytoskeletal changes and kinetics of gene expression during osteogenic differentiation. PDL cells were isolated from human periodontal ligament on extracted teeth and cultured in osteogenic medium for 14 days. Y-27632 was used for ROCK inhibition assay. Osteogenic phenotype was determined by monitoring alkaline phosphatase (ALP) activity and calcium deposition by Alizarin Red staining. ROCK-induced cytoskeletal changes were examined by immunofluorescence analysis of F-actin and myosin light chain 2 (MLC2) expression. Real-time PCR was performed to examine the kinetics of osteogenic gene expression. F-actin and phospho-MLC2 were markedly induced during osteogenic differentiation, which coincided with upregulation of ALP activity and mineralization. Subsequent inhibition assay indicated that Y-27632 significantly inhibited F-actin and phospho-MLC2 expression in a dose-dependent manner with concomitant partial reversal of the PDL cell osteogenic phenotype. PCR array analysis of osteogenic gene expression indicated that extracellular matrix genes, such as fibronectin 1, collagen type I and III, and biglycan, were significantly downregulated by Y27632. These findings indicated crucial effects of ROCK in cytoskeletal reorganization and differentiation of PDL cells toward osteogenic cells. ROCK contributes to induction of osteogenic differentiation by synergistic increases in extracellular matrix gene expression in PDL cells.