ABSTRACT
Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
Subject(s)
Adipose Tissue/cytology , Cell Survival , Osteoblasts/cytology , Stem Cells/cytology , Alkaline Phosphatase/metabolism , Biomarkers/metabolism , Cell Differentiation , Cells, Cultured , Collagen Type III/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Gene Expression Regulation , Humans , Membrane Potential, Mitochondrial , Microscopy, Fluorescence , Models, Biological , Osteoblasts/metabolism , Osteopontin/metabolism , RANK Ligand/metabolism , Vimentin/metabolismABSTRACT
Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
ABSTRACT
Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
ABSTRACT
The aim of the present study was to comparatively evaluate DNA damage and cellular death in cells exposed to various commercially available mouthrinses: Listerine Cepacol, Plax alcohol free, Periogard, and Plax Whitening. A total of 75 volunteers were included in the search distributed into five groups containing 15 people each for in vivo study. Exfoliated buccal mucosa cells were collected immediately before mouthrinse exposure and after 2 weeks. Furthermore, blood samples were obtained from three healthy donors for in vitro study. The micronucleus test was used to evaluate mutagenicity and cytotoxicity in vivo. The single-cell gel (comet) assay was used to determine DNA damage in vitro. After 2 weeks exposure, Periogard showed 1.8% of micronucleated cells with significant statistical differences (p < 0.05) compared to before exposure (0.27%). Plax Whitening presented high tail moment value (4.5) when compared to negative control (0.6). The addition of all mouthrinses to cells incubated with methyl methanesulfonate did not alter the number of strand breaks in the genetic material. Listerine was able to reduce genetic damage induced by hydrogen peroxide because a decrease of tail moment was noticed. The results of the present study suggest that Periogard and Plax Whitening can induce genetic damage, whereas Listerine is an antioxidant agent. Since DNA damage is considered to be prime mechanism during chemical carcinogenesis, these data may be relevant in risk assessment for protecting human health and preventing carcinogenesis.
Subject(s)
DNA Damage , Mouth Mucosa/drug effects , Mouthwashes/toxicity , Adult , Cell Death , Cetylpyridinium/toxicity , Chlorhexidine/analogs & derivatives , Chlorhexidine/toxicity , Comet Assay , Ethanol/toxicity , Female , Humans , Lymphocytes/drug effects , Male , Micronucleus Tests , Mouth Mucosa/cytology , Plant Oils/toxicity , Statistics, Nonparametric , Young AdultABSTRACT
Osteoclastogenesis may be regulated via activation of the RANK/RANKL (receptor activator of nuclear factor-kappa B/receptor activator of nuclear factor-kappa B ligand) system, which is mediated by osteoblasts. However, the bone loss mechanism induced by T3 (triiodothyronine) is still controversial. In this study, osteoblastic lineage rat cells (ROS 17/2.8) were treated with T3 (10(-8) M, 10(-9) M, and 10(-10) M), and RANKL mRNA (messenger RNA) expression was measured by semiquantitative RT-PCR. Our results show that T3 concentrations used did not significantly enhance RANKL expression compared to controls without hormone treatment. This data suggests that other mechanisms, unrelated to the RANK/RANKL system, might be to activate osteoclast differentiation in these cells.
Subject(s)
Bone Resorption/drug therapy , Osteoblasts/drug effects , RANK Ligand/metabolism , RNA, Messenger/metabolism , Receptors, Thyroid Hormone/metabolism , Triiodothyronine/pharmacology , Animals , Bone Resorption/metabolism , Cell Differentiation/drug effects , Electrophoresis, Agar Gel , Osteoblasts/cytology , Osteoclasts/metabolism , RANK Ligand/genetics , Rats , Receptors, Thyroid Hormone/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Osteoclastogenesis may be regulated via activation of the RANK/RANKL (receptor activator of nuclear factor-kappa B/ receptor activator of nuclear factor-kappa B ligand) system, which is mediated by osteoblasts. However, the bone loss mechanism induced by T3 (triiodothyronine) is still controversial. In this study, osteoblastic lineage rat cells (ROS 17/2.8) were treated with T3 (10-8 M, 10-9 M, and 10-10 M), and RANKL mRNA (messenger RNA) expression was measured by semiquantitative RT-PCR. Our results show that T3 concentrations used did not significantly enhance RANKL expression compared to controls without hormone treatment. This data suggests that other mechanisms, unrelated to the RANK/RANKL system, might be to activate osteoclast differentiation in these cells.
A osteoclastogênese pode ser regulada via ativação do sistema RANK/RANKL (receptor ativador do fator nuclear kapa B/ ligante do receptor do fator nuclear kapa B), que é mediado pelos osteoblastos. Entretanto, o mecanismo de perda óssea induzido pelo T3 (triiodotironina) ainda é controverso. Neste estudo, a linhagem osteoblástica de células de rato ROS 17/2.8 foi tratada com T3 (10-8 M, 10-9 M e 10-10 M), e a expressão do mRNA do RANKL foi medida por RT-PCR semiquantitativo. Nossos resultados mostraram que as concentrações de T3 utilizadas não induziram significativamente a expressão do RANKL, comparado ao controle (sem tratamento hormonal). Estes dados sugerem que outros mecanismos, não relacionados ao sistema RANK/RANKL, são usados para ativar a diferenciação osteoclástica nestas células.
Subject(s)
Animals , Rats , Bone Resorption/drug therapy , Osteoblasts/drug effects , RANK Ligand/metabolism , RNA, Messenger/metabolism , Receptors, Thyroid Hormone/metabolism , Triiodothyronine/pharmacology , Bone Resorption/metabolism , Cell Differentiation/drug effects , Electrophoresis, Agar Gel , Osteoblasts/cytology , Osteoclasts/metabolism , RANK Ligand/genetics , Reverse Transcriptase Polymerase Chain Reaction , Receptors, Thyroid Hormone/geneticsABSTRACT
Devido a crescente busca por métodos de diagnóstico mais eficientes, por meio de técnicas não invasivas e que não sejam prejudiciais aos pacientes, este estudo objetivou-se avaliar in vitro a utilização da fluorescência a laser no diagnóstico do cálculo dental. Utilizaram-se 21 dentes humanos extraídos e posteriormente armazenados em formol a 10 por cento, os quais foram submetidos a uma avaliação clínica pré-operatória, determinando dois sítios em cada dente (sítio controle e sítio teste). Em seqüencia, os dentes foram examinados utilizando o aparelho KaVo DIAGNOdent, e os valores obtidos foram anotados. Após o exame dos 21 dentes, estes foram submetidos ao processo de raspagem e alisamento radiculares, utilizando curetas de Gracey até a obtenção de superfícies radiculares limpas, lisas, duras e sem cálculo à inpeção visual. Ao término do procedimento de raspagem e alisamento radicular, os sítios controle e teste de cada dente foram reexaminados, e os valores obtidos foram registrados. Os resultados aparesentaram uma concordância de 100 por cento para todas as mediadas obtidas pelo aparelho de fluorescência a laser. Considerando as condições experimentais concluiu-se que a fluorescência a laser é eficaz no diagnóstico de cálculo dental.
Subject(s)
Humans , Periodontics , Lasers , Dental CalculusABSTRACT
A proposta deste estudo foi apresentar através da literatura as diferentes substâncias que podem ser utilizadas para o tratamento químico da superfície radicular e avaliar qual vem sendo a mais utilizada por seus efeitos favoráveis. Os estudos apresentaram que nas diversas modalidades de terapia peiodontal é importante o tratamento da superfície radicular para sua descontaminação e biomodificação, favorecendo a cicatrização por nova inserção. A utilização do tratamento mecânico da superfície radicular com o intuito de reduzir a concentração subgengival de endotoxinas livres, pode resultar em efeitos negativos, como a raspagem excessiva da raiz sem preservação do cemento para estimular a osteogenese e reinserção. Além disso, os procedimentos mecânicos nem sempre são suficientes para reduzir ou eliminar as endotoxinas. Por esta razão tem sido utilizadas substâncias associadas à terapia mecânica. Ao longo dos anos, vários estudos mostraram que esta associação remove endotoxinas localizadas e tem ação desmineralizante prevenindo a migração apical do epitélio juncional. O ácido cítrico vem sendo o mais estudado por seus resultados favoráveis e por não haver comprovação de efeitos deletérios aos tecidos adjacentes.
