RESUMO
Abstract This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.
Resumo O objetivo deste estudo foi investigar a influência do modelo de cultura celular tridimensional e das partículas de vidro bioativo (BG) sobre a expressão fenotípica de culturas de células osteogênicas da calvária de ratos. As células foram mantidas em culturas sobre superfícies colágenas bi-dimensionais (2D) e em géis de colágeno tridimensional (3D) com e sem partículas de BG até 14 dias. Foram avaliadas: viabilidade celular, atividade de fosfatase alcalina (ALP), morfologia celular e imunomarcação de proteínas da matriz não-colágena do osso através de epifluorescência e microscopia confocal. As expressões de marcadores osteogênicos foram analisadas utilizando RT-PCR. A formação de nódulos mineralizados foi visualizada através de microscopia e o conteúdo de cálcio foi avaliado quantitativamente pelo Alizarina Red. As culturas experimentais produziram uma taxa crescente de viabilidade até 14 dias. Embora a atividade ALP em 7 dias tenha sido maior em culturas com BG, as células em 3D e 3D+BG apresentaram uma diminuição da atividade ALP aos 14 dias. As condições tridimensionais favoreceram a imunomarcação para OPN e BSP e a expressão de mRNAs para ALP e COL I. As partículas de BG influenciaram positivamente a expressão do mRNAs para OPN e OC e a formação de nódulos calcificados in vitro. Os resultados indicaram que as culturas em 3D e partículas BG contribuíram para a expressão do fenótipo osteoblástico e para a diferenciação e formação de matriz mineralizada.
Assuntos
Animais , Materiais Biocompatíveis , Vidro , Osteoblastos/citologia , Osteogênese , Crânio/citologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Sialoproteína de Ligação à Integrina/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Osteopontina/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/genética , Crânio/enzimologia , Crânio/metabolismo , Alicerces TeciduaisRESUMO
This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.
Assuntos
Materiais Biocompatíveis , Vidro , Osteoblastos/citologia , Osteogênese , Crânio/citologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Cálcio/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Sialoproteína de Ligação à Integrina/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Osteopontina/metabolismo , RNA Mensageiro/genética , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Crânio/enzimologia , Crânio/metabolismo , Alicerces TeciduaisRESUMO
AIM: The aim of this randomized controlled clinical study was to compare the use of an acellular dermal matrix graft (ADMG) with or without the enamel matrix derivative (EMD) in smokers to evaluate which procedure would provide better root coverage. MATERIAL AND METHODS: Nineteen smokers with bilateral Miller Class I or II gingival recessions ≥3 mm were selected. The test group was treated with an association of ADMG and EMD, and the control group with ADMG alone. Probing depth, relative clinical attachment level, gingival recession height, gingival recession width, keratinized tissue width and keratinized tissue thickness were evaluated before the surgeries and after 6 months. Wilcoxon test was used for the statistical analysis at significance level of 5%. RESULTS: No significant differences were found between groups in all parameters at baseline. The mean gain recession height between baseline and 6 months and the complete root coverage favored the test group (p = 0.042, p = 0.019 respectively). CONCLUSION: Smoking may negatively affect the results achieved through periodontal plastic procedures; however, the association of ADMG and EMD is beneficial in the root coverage of gingival recessions in smokers, 6 months after the surgery.
