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BACKGROUND: Diffuse-type gastric cancer (DGC), for which Helicobacter pylori infection is a causal factor, is associated with poor prognosis among young women, possibly due to female hormones such as estrogen. We aimed to identify the carcinogenesis induced by estrogen and H. pylori in DGC. METHODS: We screened and selected estrogen receptor alpha (ERα)-positive (MKN45) and ERα-negative (SNU5) DGC cell lines. H. pylori strain 60190 and its isogenic mutant strain lacking cytotoxin-associated gene A (60190ΔCagA) were used to infect MKN45 cells. And the cytotoxin-related gene A (CagA) cDNA which was cloned into pSP65-SR-HA (cagA-pSP65SRa) vector was used to transfect MKN45 cells. Tumor samples were used for DGC organoid culture. RESULTS: In MKN45 cells, we found that estradiol promotes epithelial-mesenchymal transition (EMT) and stemness phenotypes via HOTAIR expression. These effects were further enhanced by the addition of CagA secreted by H. pylori but were reversed by co-treatment with fulvestrant (ICI 182,780), a selective ER degrader. We also validated the effect of estrogen on DGC organoids. ERα expression was associated with tumor invasion and HOTAIR expression in DGC patients with overt H. pylori infection. CONCLUSIONS: These findings may explain the rapid DGC progression in young women with physiologically high levels of estrogen and suggest that fulvestrant with ovarian function suppression could serve as a tumor-suppressive agent in premenopausal patients with DGC.
Assuntos
Adenocarcinoma , Antígenos de Bactérias , Proteínas de Bactérias , Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/microbiologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citotoxinas/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Fulvestranto/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologiaRESUMO
CK2α/CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. Considering the role of CSNK2A1 in cancer progression and the phosphorylation of SIRT6 and the role of SIRT6 in chemoresistance through the DNA damage repair pathway, CSNK2A1 and SIRT6 might be involved in resistance to conventional anti-cancer therapies. We evaluated the expression of CSNK2A1 and phosphorylated SIRT6 in the 37 osteosarcoma patients and investigated the effects of CSNK2A1 and the phosphorylation of SIRT6 on Ser338 on resistance to the anti-cancer effects of doxorubicin. Higher expression of CSNK2A1 and phosphorylated SIRT6 was associated with shorter survival in osteosarcoma patients. U2OS and KHOS/NP osteosarcoma cells with induced overexpression of CSNK2A1 were resistant to the cytotoxic effects of doxorubicin, and the knock-down of CSNK2A1 potentiated the cytotoxic effects of doxorubicin. CSNK2A1 overexpression-mediated resistance to doxorubicin was associated with SIRT6 phosphorylation and the induction of the DNA damage repair pathway molecules. CSNK2A1- and SIRT6-mediated resistance to doxorubicin in vivo was attenuated via mutation of SIRT6 at the Ser338 phosphorylation site. Emodin, a CSNK2A1 inhibitor, potentiated the cytotoxic effects of doxorubicin in osteosarcoma cells. This study suggests that blocking the CSNK2A1-SIRT6-DNA damage repair pathway might be a new therapeutic stratagem for osteosarcomas.
Assuntos
Dano ao DNA , Reparo do DNA , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sirtuínas/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Emodina/farmacologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Análise Multivariada , Fosforilação/efeitos dos fármacos , Prognóstico , Modelos de Riscos Proporcionais , Análise de RegressãoRESUMO
BACKGROUND: SIRT6 has diverse roles in cells, and the role of SIRT6 in tumorigenesis is controversial. Considering the role of SIRT6 as an inducer of DNA damage repair, it might be involved in resistance to anti-cancer therapy. METHODS: We evaluated the prognostic significance of SIRT6 in 37 osteosarcomas and investigated the therapeutic efficacy of SIRT6 on the anticancer effects of doxorubicin, olaparib, and ATM inhibitor. RESULTS: Immunohistochemical expression of SIRT6 was significantly associated with shorter overall survival and relapse-free survival of osteosarcoma patients, especially in patients who received adjuvant chemotherapy. In U2OS and KHOS/NP osteosarcoma cells, knock-down of SIRT6 significantly potentiated apoptotic effects of doxorubicin and SIRT6 overexpression induced resistance to doxorubicin. Moreover, SIRT6 induced the DNA damage repair pathway and SIRT6-mediated resistance to doxorubicin was attenuated by blocking the DNA damage repair pathway with olaparib and ATM inhibitor. CONCLUSIONS: This study suggests that suppression of SIRT6 in combination with doxorubicin might be an effective modality in the treatment of osteosarcoma patients, especially for osteosarcomas with shorter survival with high expression of SIRT6.
Assuntos
Dano ao DNA/imunologia , Doxorrubicina/uso terapêutico , Osteossarcoma/genética , Sirtuínas/metabolismo , Adulto , Animais , Apoptose , Doxorrubicina/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Osteossarcoma/mortalidade , Osteossarcoma/patologia , Prognóstico , Análise de Sobrevida , TransfecçãoRESUMO
Although we recently demonstrated that static magnetic fields (SMFs) of 3, 15, and 50 mT stimulate osteoblastic differentiation, the effects of SMFs on osteoclastogenesis are still poorly understood. This study focused on the suppressive effects of SMFs on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Direct SMFs inhibit RANKL-induced multinucleated osteoclast formation, tartrate-resistant acid phosphatase activity, and bone resorption in mouse bone marrow-derived macrophage cells. The conditioned medium from osteoblasts treated with SMFs also resulted in the inhibition of osteoclast differentiation as well as resorption. The RANKL-induced expression of osteoclast-specific transcription factors, such as c-Fos and NFATc1, was remarkably downregulated by SMF at 15 mT. In addition, SMF inhibited RANKL-activated Akt, glycogen synthase kinase 3ß (GSK3ß), extracellular signal-regulated kinase, c-jun N-terminal protein kinase, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) formation. These findings indicate that SMF-mediated attenuation of RANKL-induced Akt, GSK3ß, MAPK, and NF-κB pathways could contribute to the direct and indirect inhibition of osteoclast formation and bone resorption. Therefore, SMFs could be developed as a therapeutic agent against periprosthetic or peri-implant osteolysis. Additionally, these could be used against osteolytic diseases such as osteoporosis and rheumatoid arthritis. Bioelectromagnetics. 39:394-404, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Campos Magnéticos , Osteoclastos/fisiologia , Animais , Células da Medula Óssea/citologia , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/metabolismo , Transdução de SinaisRESUMO
FMS-like receptor tyrosine kinase-3 (FLT3) belongs to the family of receptor tyrosine kinase (RTK), and the FLT3 mutation is observed in 1/3 of all acute myeloid leukemia (AML) patients. Potential FLT3 inhibitors have been investigated as potential therapeutic agents of AML. In this study, we identified a potent FLT3 inhibitor LDD1937 containing an indirubin skeleton. The potent inhibitory activity of LDD1937 against FLT3 was shown with an in vitro kinase assay (IC50 = 3 nM). The LDD1937 compound selectively inhibited the growth of MV-4-11 cells (GI50 = 1 nM) and induced apoptotic cell death. LDD1937 caused cell cycle arrest at the G2/M phase and increased the cell population at the sub-G1 phase. Phosphorylation of STAT5, which is the downstream signaling of FLT3, was significantly reduced by LDD1937 in a dose-dependent manner. The pharmacokinetic properties of LDD1937 were investigated in mice. Then, the in vivo anti-tumor effect was investigated using a MV-4-11 xenograft. With the intravenous administration of 5 and 10 mg/kg in nu/nu mice, the tumor volume and weight were significantly reduced compared to the control. LDD1937 is a promising therapeutic candidate to treat AML patients because of its ability to suppress tumor cell growth in vitro and in vivo.
RESUMO
BACKGROUND: We aimed to determine the effect of fibronectin (FN)-immobilized microgrooved titanium (Ti) on human gingival fibroblast proliferation, gene expression and protein expression. METHODS: Photolithography was used to fabricate the microgrooved Ti, and amine funtionalization (silanization) was used for FN immobilization on titanium surfaces. Cell proliferation, gene expression and protein expression were analyzed, followed by multiple regression analysis for determining the influential factors on cell proliferation. RESULTS: FN-immobilized microgrooved Ti significantly enhanced the fibroblast proliferation in various timelines of culture, among which a burst of fivefold increase is induced at 96 h of culture compared to that on the control smooth Ti. We suggest a presence of the synergistic promotion effect of microgrooves and FN immobilization on fibroblast proliferation. Through a series of analyses on the expression of various genes and proteins involved in cell adhesion and proliferation, cyclin-dependent kinase 6, cyclin D1, integrin α5, oncogene c-Src, osteonectin, paxillin and talin-2 were determined as influential factors on promoting fibroblast proliferation induced by FN-immobilized microgrooved Ti. CONCLUSION: FN-immobilized microgrooved Ti can act as an effective surface for enhancing fibroblast proliferation, and can be used for promoting soft tissue response on the connective tissue attachment zone of biomaterial surfaces.
RESUMO
PURPOSE: This in vitro study evaluated the reliability of a surgical guide with regard to different levels of operator surgical experience and implant site. MATERIALS AND METHODS: A stereolithographic surgical guide for epoxy resin mandibles with three edentulous molar sites was produced using a computer-aided design/computer-assisted manufacture (CAD/CAM) system. Two surgeons with and two surgeons without implant surgery experience placed implants in a model either using or not using the CAD/CAM surgical guide. Four groups were created: inexperienced surgeon without the guide (group 1); experienced surgeon without the guide (group 2); inexperienced surgeon with the guide (group 3); and experienced surgeon with the guide (group 4). Planned implants and placed implants were superimposed using digital software, and deviation parameters were calculated. RESULTS: There were no significant differences in any of the deviation parameters between the groups when using the surgical guide. With respect to the implant sites, there were no significant differences among the groups in any parameter. CONCLUSION: Use of the CAD/CAM surgical guide reduced discrepancies among operators performing implant surgery regardless of their level of experience. Whether or not the guide was used, differences in the anterior-posterior implant site in the molar area did not affect the accuracy of implant placement.
Assuntos
Competência Clínica , Desenho Assistido por Computador , Implantação Dentária Endóssea/métodos , Arcada Parcialmente Edêntula/cirurgia , Mandíbula/cirurgia , Cirurgia Assistida por Computador/métodos , Humanos , Técnicas In Vitro , Planejamento de Assistência ao Paciente , Reprodutibilidade dos TestesRESUMO
We demonstrate that a composite surface of microgroove titanium (Ti) with immobilized fibronectin (FN) or bone sialoprotein II (BSP2) promotes osteoblastic differentiation and osteogenic transcription factor expression in human bone marrow-derived mesenchymal stem cells (MSCs). Comparisons made between smooth microgrooves, microgrooves with silanization and microgrooves with matrix protein (FN or BSP2)-immobilization Ti surfaces revealed a significant promotion of in vitro osteogenic activity and osteoblastic differentiation at various timelines of culture. An even more significant increase was verified on microgrooves with a matrix protein-immobilization Ti surface in 28 d time-dependent gene expression of the main osteogenic transcription factors, such as ARF4, FRA1, RUNX2, and OSX. As a result, a synergestic effect regarding the promotion of osteogenic transcription factor expression and osteoblastic differentiation in the matrix protein-microgroove Ti composite surface was confirmed. From a multiple regression analysis using various timelines of osteogenic culture as independent variables, day 13 was verified as the most prominent influential timeline for the promotion of osteoblastic differentiation induced by the matrix protein-microgroove Ti composite surface. The FN- or BSP2-microgroove Ti composite surface resulting from silanization can strongly induce the promotion of osteoblastic differentiation in human MSCs. The proposed surface is expected to be useful in the development of a variety of osteogenic biomaterial surfaces.
Assuntos
Fibronectinas/metabolismo , Sialoproteína de Ligação à Integrina/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Titânio , Animais , Diferenciação Celular , Células Cultivadas , Humanos , Camundongos , Osteocalcina/genética , Osteogênese , Propriedades de Superfície , Fatores de Transcrição/metabolismoRESUMO
Magnetism has recently been implicated to play significant roles in the regulation of cell responses. Allowing cells to experience a magnetic field applied externally or scaffolding them in a material with intrinsic magnetic properties has been a possible way of utilizing magnetism. Here we aim to investigate the combined effects of the external static magnetic field (SMF) with magnetic nanocomposite scaffold made of polycaprolactone/magnetic nanoparticles on the osteoblastic functions and bone formation. The SMF synergized with the magnetic scaffolds in the osteoblastic differentiation of primary mouse calvarium osteoblasts, including the expression of bone-associated genes (Runx2 and Osterix) and alkaline phosphatase activity. The synergism was demonstrated in the activation of integrin signaling pathways, such as focal adhesion kinase, paxillin, RhoA, mitogen-activated protein kinase, and nuclear factor-kappaB, as well as in the up-regulation of bone morphogenetic protein-2 and phosphorylation of Smad1/5/8. Furthermore, the SMF/magnetic scaffold-stimulated osteoblasts promoted the angiogenic responses of endothelial cells, including the expression of vascular endothelial growth factor and angiogenin-1 genes and the formation of capillary tubes. When the magnetic scaffolds were implanted in mouse calvarium defects, the application of SMF significantly enhanced the new bone formation at 6 weeks, as revealed by the histological and micro-computed tomographic analyses. Current findings suggest that the combinatory application of external (SMF) and internal (scaffold) magnetism can be a promising tool to regenerative engineering of bone.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Campos Magnéticos , Nanocompostos/química , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Alicerces Teciduais/química , Animais , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Integrinas/genética , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoblastos/citologia , Paxilina/genética , Paxilina/metabolismo , Poliésteres/química , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transdução de Sinais , Engenharia Tecidual , Regulação para Cima , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
The aim of this study is to compare the characteristics of stem cells derived from human exfoliated deciduous teeth (SHED) from cryopreserved intact deciduous teeth with those of fresh SHED. In total, 20 exfoliated deciduous teeth were randomly divided into a fresh group (f-SHED; n = 11) and cryopreserved group (c-SHED; n = 9; stored for 1-8 months). Following thawing and separation of the pulp, the SHED cells were cultured, and the characteristics as mesenchymal stem cells were investigated using proliferation assays, cell-cycle analysis, colony-forming unit-fibroblast (CFU-F) assays, and flow cytometry analyses. Furthermore, differentiation into adipogenic and osteogenic lineages was investigated in vitro as well as in vivo via transplantation in mice. We found no significant differences between the two groups in the proliferation analyses, in the expression of mesenchymal stem cell markers, or in the adipogenic and osteogenic differentiation in vitro (p < 0.05). Furthermore, the in vivo transplantation results showed no significant differences in the quantity of bone tissue that formed or in histochemistry performance (p < 0.05). In conclusion, cryopreservation of intact exfoliated deciduous teeth appears to be a useful method for preserving SHED.
Assuntos
Criopreservação/métodos , Células-Tronco/citologia , Dente Decíduo/citologia , Animais , Diferenciação Celular , Citometria de Fluxo , Humanos , CamundongosRESUMO
This study aimed to explore effects of static magnetic fields (SMFs) of moderate intensity (3-50 mT) as biophysical stimulators of proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). MSCs were exposed to SMFs of three intensities: 3, 15, and 50 mT. Proliferation was assessed by cell counting and bromodeoxyuridine incorporation, and differentiation by measuring alkaline phosphatase (ALP) activity, calcium content, mineralized nodule formation, and transcripts of osteogenic markers. Moderate intensity SMFs increased cell proliferation, ALP activity, calcium release, and mineralized nodule formation in a dose- and time-dependent manner, which peaked at 15 mT. In the same manner, they upregulated expression of osteogenic marker genes such as ALP, bone sialoprotein 2 (BSP2), collagen1a1 (COL1a1), osteocalcin (OCN), osteonectin (ON), osteopontin (OPN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) with peak at 15 mT after 14 or 21 days of exposure. Results demonstrate that moderate intensity SMFs promote proliferation and osteoblastic differentiation of MSCs. This effect could help to improve MSC responses during osseointegration between a dental implant and surrounding bone.
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Campos Magnéticos , Células-Tronco Mesenquimais/citologia , Fosfatase Alcalina/metabolismo , Calcificação Fisiológica , Cálcio/metabolismo , Proliferação de Células , Espaço Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , OsteogêneseRESUMO
In our previous studies, we confirmed that low-level laser therapy (LLLT) with a 980-nm gallium-aluminum-arsenide diode laser was beneficial for the healing of the alveolar bone in rats with systemic disease. However, many factors can affect the biostimulatory effects of LLLT. Thus, we attempted to investigate the effects of irradiation time on the healing of extraction sockets by evaluating the expressions of genes and proteins related to bone healing. The left and right first maxillary molars of 24 rats were extracted. Rats were randomly divided into four groups in which extraction sockets were irradiated for 0, 1, 2, or 5 min each day for 3 or 7 days. Specimens containing the sockets were examined using quantitative real-time reverse transcription polymerase chain reaction and western blotting. LLLT increased the expressions of all tested genes, Runx2, collagen type 1, osteocalcin, platelet-derived growth factor-B, and vascular endothelial growth factor, in a time-dependent manner. The highest levels of gene expressions were in the 5-min group after 7 days. Five minutes of irradiation caused prominent increases of the expression of all tested proteins after both 3 and 7 days. The expression level of each protein in group 4 was higher by almost twofold compared with group 1 after 7 days. Laser irradiation for 5 min caused the highest expressions of genes and proteins related to bone healing. In conclusion, LLLT had positive effects on the early stages of bone healing of extraction sockets in rats, which were irradiation time-dependent.
Assuntos
Lasers Semicondutores , Terapia com Luz de Baixa Intensidade , Alvéolo Dental/patologia , Alvéolo Dental/efeitos da radiação , Cicatrização/efeitos da radiação , Animais , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Masculino , Osteocalcina/genética , Osteocalcina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo , Extração Dentária , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/genéticaRESUMO
BACKGROUND: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)-α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus. METHODS: Cultured PDL cells were stimulated with TNF-α and analyzed with an MMP antibody array. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP-3, -10, and -13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP-3, -10, and -13 was observed after blocking the TNFR with an antagonist. Results from real-time PCR, ELISA, and western blot were analyzed by paired t test. RESULTS: The antibody array showed that the protein most strongly upregulated by TNF-α stimulation was MMP-3, followed by MMP-13 and MMP-10. The TNF-α receptor blocker specifically inhibited expression of MMP-3 and -13. In addition, TNF-α increased levels of MMP mRNAs in MMP-3, -13, and -10 (in decreasing order). However, ELISAs showed that MMP-13 was the most upregulated protein, followed by MMP-10 and MMP-3. Western blotting indicated that TNF-α increased MMP-3 and -13 levels but had no significant effect on the level of MMP-10, and zymography showed that TNF-α increased the activities of all forms of MMP-3 and -13, but MMP-10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1. CONCLUSIONS: TNF-α (10 ng/mL) upregulates levels of MMP-3, -10, and -13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.
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Metaloproteinase 10 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Inibidores de Metaloproteinases de Matriz/farmacologia , Ligamento Periodontal/citologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Fator de Necrose Tumoral/análise , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral/análise , Regulação para CimaRESUMO
INTRODUCTION: The purpose of this study was to investigate whether in vitro stimulation of pulp cells leads to increased secretion of matrix metalloproteinases (MMPs) and, if so, to identify which MMPs are affected. METHODS: Cells cultured from dental pulp were stimulated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, and lysates were analyzed with an antibody array (Bio-Rad Laboratories, Hercules, CA). The mRNA and protein levels of MMP-3, -10, and -13 were measured by real-time polymerase chain reaction (real-time PCR), enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and zymography. In addition, tumor necrosis factor receptors in the pulp cells were assayed by flow cytometry. The ELISA and real-time PCR results were analyzed by paired t tests. RESULTS: The expression of MMP-3, -10, and -13 was up-regulated in the pulp cells after 24 hours of stimulation with TNF-α (10 ng/mL) as seen in the antibody array, real-time PCR, and ELISA results, but MMP-10 was not detected by Western blotting or casein zymography. Flow cytometry analysis showed that the majority of the pulp cells expressed tumor necrosis factor receptor 1. CONCLUSIONS: In regions of inflammation, TNF-α may initiate the degradation of dental connective tissue by activating MMP-3 and MMP-13. These proteins may play an important pathologic role in the inflammation of dental pulp.
Assuntos
Polpa Dentária/enzimologia , Metaloproteinases da Matriz/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Humanos , Metaloproteinase 10 da Matriz/análise , Metaloproteinase 10 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/análise , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/análise , Receptores Tipo I de Fatores de Necrose Tumoral/análise , Fatores de Tempo , Regulação para CimaRESUMO
OBJECTIVE: This study aimed to identify the optimal micro- and submicroscale topographies of titanium (Ti) substrata that would most significantly influence adhesion, proliferation, and other activities of these cells. DESIGN: Truncated V-shaped microgrooves in 60 µm-wide and 10 µm-deep cross-sections with 0°, 30°, 60°, or 90° angles between the microgrooves and ridge-top submicroscale texture were created on the Ti substrata (designated NE60/10-0°, NE60/10-30°, NE60/10-60° and NE60/10-90°, respectively). Ground titanium with submicroscale texture but with no microgrooves was used as the control substratum, NE0. Scanning electron microscopic observation and the assays determining the cell adhesion, cell proliferation and osteoblast differentiation were performed. RESULTS: Cells more actively migrated into the microgrooves on NE60/10-30° than into the microgrooves on any other substrata tested, suggesting that the cells utilise the increased surface area of the substrata at the microscale level. NE60/10-0° and NE60/10-30° substrata generally enhanced adhesion, proliferation, alkaline phosphatase activity, and osteoblast differentiation of human primary cells when compared to other Ti substrata, and significant correlations were observed between these cellular activities. CONCLUSIONS: Here, we show that the contact guidance of human primary cells grown on Ti substrata can be controlled more by specific submicroscale textures on ridge tops than by the dimensions of surface microgrooves only. Also, the degree of angles created between the submicroscale textures and microgrooves on Ti substrata significantly affect cell adhesion, proliferation and differentiation in human primary cells.
Assuntos
Diferenciação Celular , Proliferação de Células , Fibroblastos/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Ligamento Periodontal/citologia , Titânio/química , Fosfatase Alcalina/metabolismo , Análise de Variância , Adesão Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Gengiva/citologia , Gengiva/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Ligamento Periodontal/metabolismo , Propriedades de SuperfícieRESUMO
This study was designed to investigate the histomorphometric and biomechanical comparison of small-diameter implants with different designs. These implants can be placed surgically in narrow bone spaces, such as the lower incisor region, that have low occlusal loading. Specimens of screw-shaped pin implants were designed for the study. These specimen implants were divided into 6 groups: group 1, machined implants; group 2, resorbable blast media (RBM)-treated implants; group 3, machined implants with a long vertical groove; group 4, RBM-treated implants with a long vertical groove; group 5, RBM-treated implants with a vertical groove on the upper thread; and group 6, RBM-treated implants with a vertical groove on the lower trunk. The specimen implants were placed surgically on the medial side of the rabbit tibia. Animals were sacrificed 2, 4, and 8 weeks after surgery. The removal torque was measured and tissues were prepared for histologic and histomorphometric analysis. The bone-to-implant contact and the percentage of the bone area inside the threads were measured. RBM-treated implants with vertical groove groups showed significantly higher values of removal torque, bone-implant contact, and bone area rate than the ones of machined surface groups.