RESUMEN
This study aimed to illustrate the biological behavior and changes in cell function during the progression of apical periodontitis in deciduous teeth and to explore the underlying molecular mechanism. Deciduous teeth periodontal ligament stem cells (DePDLSCs) were derived and their identity was confirmed. The viability, inflammation, and osteogenic ability of cells were tested by exposing them to various concentrations of lipopolysaccharide (LPS) (0-100 µg/mL) using the cell counting kit-8 (CCK-8) assay, reverse transcription polymerase chain reaction (real-time PCR), alkaline phosphatase (ALP) staining, and ALP activity assay. In addition, osteogenic-induced cells with and without 10 µg/mL LPS were harvested for high-throughput sequencing. Based on sequencing data, proinflammatory factors and ALP expression were measured after interference with the PI3K-AKT signaling pathway activator, 740Y-P. LPS biphasically affected the proliferation and osteogenesis of DePDLSCs. Low concentrations of LPS showed stimulatory effects, whereas inhibitory effects were observed at high concentrations. Sequencing analysis showed that the PI3K-AKT signaling pathway was significantly downregulated when DePDLSCs were treated with 10 µg/mL LPS. The LPS-induced inflammation and osteogenesis inhibition of DePDLSCs were partially rescued by 740Y-P treatment. In conclusion, LPS affected DePDLSCs proliferation and osteogenesis in a biphasic manner. Moderate activation of PI3K-AKT signaling pathway was beneficial for osteogenic differentiation and anti-inflammatory effect in DePDLSCs. This research may provide etiological probes for apical periodontitis and its treatment.
Asunto(s)
Osteogénesis , Ligamento Periodontal , Células Madre , Diente Primario , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Humanos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Diente Primario/citología , Células Madre/efectos de los fármacos , Lipopolisacáridos/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inflamación , Transducción de Señal/efectos de los fármacos , Células Cultivadas , Reacción en Cadena en Tiempo Real de la Polimerasa , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/análisisRESUMEN
Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-ßI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Osteogénesis , Ligamento Periodontal , Receptor PAR-1 , Células Madre , Ingeniería de Tejidos , Ligamento Periodontal/citología , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Humanos , Diferenciación Celular/efectos de los fármacos , Células Madre/fisiología , Células Madre/efectos de los fármacos , Células Cultivadas , Proliferación Celular/efectos de los fármacos , Ingeniería de Tejidos/métodos , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/metabolismo , Reproducibilidad de los Resultados , Adolescente , Factores de Tiempo , Reacción en Cadena en Tiempo Real de la Polimerasa , Inmunofenotipificación , Análisis de VarianzaRESUMEN
Combretum leprosum Mart. is a plant of the Combretaceae family, widely distributed in the Northeast region of Brazil, popularly used as an anti-inflammatory agent, and rich in triterpenes. This study evaluated in vitro and in silico potential osteogenic of two semisynthetic triterpenes (CL-P2 and CL-P2A) obtained from the pentacyclic triterpene 3ß,6ß,16ß-trihydroxylup-20(29)-ene (CL-1) isolated from C. leprosum. Assays were carried out in cultured murine osteoblasts (OFCOL II), first investigating the possible toxicity of the compounds on these cells through viability assays (MTT). Cell proliferation and activation were investigated by immunohistochemical evaluation of Ki-67, bone alkaline phosphatase (ALP) activity, and mineralization test by Von Kossa. Molecular docking analysis was performed to predict the binding affinity of CL-P2 and CL-P2A to target proteins involved in the regulation of osteogenesis, including: bone morphogenetic protein 2 (BMP-2), proteins related to Wingless-related integration (WNT) pathway (Low-density lipoprotein receptor-related protein 6-LRP6 and sclerostin-SOST), and receptor activator of nuclear factor (NF)-kB-ligand (RANK-L). Next, Western Blot and immunofluorescence investigated BMP-2, WNT, RANK-L, and OPG protein expressions in cultured murine osteoblasts (OFCOL II). None of the CL-P2 and CL-P2A concentrations were toxic to osteoblasts. Increased cell proliferation, ALP activity, and bone mineralization were observed. Molecular docking assays demonstrated interactions with BMP-2, LRP6, SOST, and RANK-L/OPG. There was observed increased expression of BMP-2, WNT, and RANK-L/OPG proteins. These results suggest, for the first time, the osteogenic potential of CL-P2 and CL-P2A.
Asunto(s)
Proteína Morfogenética Ósea 2 , Proliferación Celular , Simulación del Acoplamiento Molecular , Osteoblastos , Osteogénesis , Triterpenos , Animales , Osteogénesis/efectos de los fármacos , Triterpenos/farmacología , Triterpenos/química , Ratones , Proteína Morfogenética Ósea 2/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Proliferación Celular/efectos de los fármacos , Ligando RANK/metabolismo , Simulación por Computador , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfatasa Alcalina/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
The present study aimed to evaluate the effect of photobiomodulation therapy (PBM) on different stages of osteogenesis in vitro. For this, osteoblastic-like cells (Saos-2 cell lineage) were irradiated in two different periods: during the Proliferation phase (PP; from the second to the fourth day) and during the Differentiation phase (DP; from the seventh to the ninth day). The energy density used in the study was 1.5 J/ cm2. The following parameters were evaluated: 1) quantification of collagen type 1 (COL 1), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2); 2) quantification of alkaline phosphatase (ALP) activity; and 3) quantification of extracellular matrix (ECM) mineralization. Non-irradiated cultures were used as controls. The data were analyzed using the Student's t-test or one-way ANOVA, considering a significance level of 5%. The results indicated that COL 1 and BMP-2 quantification was higher in Saos-2 irradiated during the DP in relation to the control group at day 10 (p < 0.05). No differences were observed for other comparisons at this time point (p > 0.05). OPN expression was greater in PP compared with the other experimental groups at day 10 (p < 0.05). Irradiation did not affect ALP activity in Saos-2 regardless of the exposure phase and the time point evaluated (p > 0.05). At day 14, ECM mineralization was higher in Saos-2 cultures irradiated during the DP in relation to the PP (p < 0.05). In conclusion, the results suggested that the effects of PBM on osteoblastic cells may be influenced by the stage of cell differentiation.
Asunto(s)
Fosfatasa Alcalina , Proteína Morfogenética Ósea 2 , Diferenciación Celular , Proliferación Celular , Colágeno Tipo I , Terapia por Luz de Baja Intensidad , Osteoblastos , Osteogénesis , Osteopontina , Osteogénesis/efectos de la radiación , Humanos , Proteína Morfogenética Ósea 2/metabolismo , Fosfatasa Alcalina/metabolismo , Osteopontina/metabolismo , Diferenciación Celular/efectos de la radiación , Colágeno Tipo I/metabolismo , Osteoblastos/efectos de la radiación , Osteoblastos/citología , Osteoblastos/metabolismo , Proliferación Celular/efectos de la radiación , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de la radiaciónRESUMEN
Bioactive and biodegradable scaffolds that mimic the natural extracellular matrix of bone serve as temporary structures to guide new bone tissue growth. In this study, 3D-printed scaffolds composed of poly (lactic acid) (PLA)-tricalcium phosphate (TCP) (90-10 wt.%) were modified with 1%, 5%, and 10 wt.% of ZnO to enhance bone tissue regeneration. A commercial chain extender named Joncryl was incorporated alongside ZnO to ensure the printability of the composites. Filaments were manufactured using a twin-screw extruder and subsequently used to print 3D scaffolds via fused filament fabrication (FFF). The scaffolds exhibited a homogeneous distribution of ZnO and TCP particles, a reproducible structure with 300 µm pores, and mechanical properties suitable for bone tissue engineering, with an elastic modulus around 100 MPa. The addition of ZnO resulted in enhanced surface roughness on the scaffolds, particularly for ZnO microparticles, achieving values up to 241 nm. This rougher topography was responsible for enhancing protein adsorption on the scaffolds, with an increase of up to 85% compared to the PLA-TCP matrix. Biological analyses demonstrated that the presence of ZnO promotes mesenchymal stem cell (MSC) proliferation and differentiation into osteoblasts. Alkaline phosphatase (ALP) activity, an important indicator of early osteogenic differentiation, increased up to 29%. The PLA-TCP composite containing 5% ZnO microparticles exhibited an optimized degradation rate and enhanced bioactivity, indicating its promising potential for bone repair applications.
Asunto(s)
Materiales Biocompatibles , Regeneración Ósea , Fosfatos de Calcio , Diferenciación Celular , Proliferación Celular , Células Madre Mesenquimatosas , Osteoblastos , Poliésteres , Impresión Tridimensional , Ingeniería de Tejidos , Andamios del Tejido , Óxido de Zinc , Andamios del Tejido/química , Fosfatos de Calcio/química , Poliésteres/química , Regeneración Ósea/efectos de los fármacos , Ingeniería de Tejidos/métodos , Células Madre Mesenquimatosas/citología , Óxido de Zinc/química , Materiales Biocompatibles/química , Diferenciación Celular/efectos de los fármacos , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Ensayo de Materiales , Huesos , Regeneración Tisular Dirigida/métodos , Humanos , Animales , Fosfatasa Alcalina/metabolismo , Módulo de Elasticidad , Porosidad , Propiedades de SuperficieRESUMEN
The dental implant surface plays a crucial role in osseointegration. The topography and physicochemical properties will affect the cellular functions. In this research, four distinct titanium surfaces have been studied: machined acting (MACH), acid etched (AE), grit blasting (GBLAST), and a combination of grit blasting and subsequent acid etching (GBLAST + AE). Human amniotic mesenchymal (hAMSCs) and epithelial stem cells (hAECs) isolated from the amniotic membrane have attractive stem-cell properties. They were cultured on titanium surfaces to analyze their impact on biological behavior. The surface roughness, microhardness, wettability, and surface energy were analyzed using interferometric microscopy, Vickers indentation, and drop-sessile techniques. The GBLAST and GBLAST + AE surfaces showed higher roughness, reduced hydrophilicity, and lower surface energy with significant differences. Increased microhardness values for GBLAST and GBLAST + AE implants were attributed to surface compression. Cell viability was higher for hAMSCs, particularly on GBLAST and GBLAST + AE surfaces. Alkaline phosphatase activity enhanced in hAMSCs cultured on GBLAST and GBLAST + AE surfaces, while hAECs showed no mineralization signals. Osteogenic gene expression was upregulated in hAMSCs on GBLAST surfaces. Moreover, α2 and ß1 integrin expression enhanced in hAMSCs, suggesting a surface-integrin interaction. Consequently, hAMSCs would tend toward osteoblastic differentiation on grit-blasted surfaces conducive to osseointegration, a phenomenon not observed in hAECs.
Asunto(s)
Amnios , Implantes Dentales , Propiedades de Superficie , Titanio , Humanos , Titanio/química , Amnios/citología , Amnios/metabolismo , Osteogénesis , Diferenciación Celular , Células Cultivadas , Oseointegración , Células Madre/citología , Células Madre/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Supervivencia Celular , Fosfatasa Alcalina/metabolismoRESUMEN
The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.
Asunto(s)
Aedes , Fosfatasa Alcalina , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Sistemas CRISPR-Cas , Cadherinas , Endotoxinas , Proteínas Hemolisinas , Animales , Aedes/genética , Aedes/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Femenino , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Resistencia a los Insecticidas/genética , Técnicas de Inactivación de Genes , Larva/genética , Larva/crecimiento & desarrollo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Masculino , Insecticidas/farmacologíaRESUMEN
This work presents the effect of the silicocarnotite (SC) and nagelschmidtite (Nagel) phases on in vitro osteogenesis. The known hydroxyapatite of biological origin (BHAp) was used as a standard of osteoconductive characteristics. The evaluation was carried out in conventional and osteogenic media for comparative purposes to assess the osteogenic ability of the bioceramics. First, the effect of the material on cell viability at 24 h, 7 and 14 days of incubation was evaluated. In addition, cell morphology and attachment on dense bioceramic surfaces were observed by fluorescence microscopy. Specifically, alkaline phosphatase (ALP) activity was evaluated as an osteogenic marker of the early stages of bone cell differentiation. Mineralized extracellular matrix was observed by calcium phosphate deposits and extracellular vesicle formation. Furthermore, cell phenotype determination was confirmed by scanning electron microscope. The results provided relevant information on the cell attachment, proliferation, and osteogenic differentiation processes after 7 and 14 days of incubation. Finally, it was demonstrated that SC and Nagel phases promote cell proliferation and differentiation, while the Nagel phase exhibited a superior osteoconductive behavior and could promote MC3T3-E1 cell differentiation to a higher extent than SC and BHAp, which was reflected in a higher number of deposits in a shorter period for both conventional and osteogenic media.
Asunto(s)
Diferenciación Celular , Cerámica , Durapatita , Osteoblastos , Osteogénesis , Silicatos , Animales , Ratones , Durapatita/química , Durapatita/farmacología , Cerámica/química , Cerámica/farmacología , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoblastos/efectos de los fármacos , Silicatos/química , Silicatos/farmacología , Diferenciación Celular/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Materiales Biocompatibles/química , Fosfatasa Alcalina/metabolismo , Compuestos de Calcio/farmacología , Compuestos de Calcio/química , Supervivencia Celular/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Matriz Extracelular/metabolismo , Células 3T3 , Línea CelularRESUMEN
Microbial pigments are considered as one of the main sources of natural types, and the attention to them is increasing in the food and pharmaceutical industries. This study aimed to investigate the effects of pigments extracted from Micrococcus roseus (PEM) on the gene expression of a and b staphylococcal enterotoxins (sea and seb) and their acute toxicity. Real-time PCR was used to study the anti-enterotoxigenic activity of PEM against Staphylococcus aureus at sub-inhibitory concentrations. In addition, the acute toxicity of PEM was evaluated on albino mice through alkaline phosphatase (ALP), aspartate aminotransferas (AST), and alanine aminotransferase (ALT) of liver and its histopathological changes. Based on the results, the expression of sea and seb was decreased in the presence of PEM at sub-inhibitory concentrations. The 2-∆∆CT was measured 0.02 and 0.01 for the expression of sea and seb of S. aureus grown in the MHB containing 16 mg/ml PEM. The results showed that the expression of seb is more sensitive to PEM compared to the expression of sea. After treatment of mice with PEM for two weeks, the condition of mice was normal, and the results of liver enzymatic activities and histopathological changes showed insignificant difference compared to the control sample.
Asunto(s)
Enterotoxinas , Hígado , Pigmentos Biológicos , Staphylococcus aureus , Animales , Ratones , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Hígado/patología , Hígado/efectos de los fármacos , Enterotoxinas/genética , Enterotoxinas/toxicidad , Enterotoxinas/metabolismo , Micrococcus/efectos de los fármacos , Micrococcus/genética , Antibacterianos/farmacología , Antibacterianos/toxicidad , Infecciones Estafilocócicas/microbiología , Masculino , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Pruebas de Sensibilidad Microbiana , Alanina Transaminasa/metabolismo , Alanina Transaminasa/sangreRESUMEN
This study aimed to correlate the values of liver markers with oxidative stress markers in patients with multidrug-resistant tuberculosis in the Brazilian Amazon. A total of 30 patients from the Tuberculosis clinic of a referral hospital were admitted to the study. Whole blood samples were collected for analysis of liver enzyme values and oxidative stress markers by spectrophotometry. The prevalence was male (60%) and the 18-29 age group was the most affected. Patients with multidrug-resistant tuberculosis presented catalase values with a median equal to 6.94 U/gHb and for glutathione, the median was equal to 14.76 µg∕ml. As for the values of liver enzymes (AST, ALT, Gamma-GT and Alkaline phosphatase) the patients had medians equal to 60.50 (U/L); 80 (U/L); 54 (U/L); and 100 (U/L) respectively (p<0.0001). The results suggest a hepatotoxic effect of the drug, which recommends further studies with a larger number of samples in order to investigate the predictors of liver damage in patients with multidrug-resistant tuberculosis.
Asunto(s)
Hígado , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Masculino , Brasil , Estrés Oxidativo , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/metabolismoRESUMEN
Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.
Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.
Asunto(s)
Animales , Osteogénesis , Fosfatasa Alcalina/metabolismo , Rana catesbeiana , Huesos/metabolismo , CinéticaRESUMEN
This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.
Asunto(s)
Fosfatasa Alcalina , Osteogénesis , Ratas , Animales , Osteocalcina , Diferenciación Celular , Fosfatasa Alcalina/metabolismo , Osteoblastos/metabolismo , Proceso Alveolar/química , Proceso Alveolar/metabolismoRESUMEN
Kisspeptin (Kp-10) is a neuropeptide that binds to GPR54 receptors, exerting several functions mainly in the nervous and reproductive systems of the body. However, its effects and mechanisms of action on the skeletal system remain poorly understood. This study evaluated the effects of different concentrations of Kp-10 on in vitro osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) extracted from the bone marrow (BM) of adult Wistar rats. Two-month-old female rats were euthanized to extract BM from long bones to obtain MSCs. Four experimental groups were established in vitro: a control and Kp-10 at concentrations of 0.01, 0.05 and, 0.1 µg/mL. After induction of osteogenic differentiation, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase activity, collagen synthesis, percentage of area covered by MSCs/field and mineralized nodules/field, and immunocytochemistry of the GPR54 receptor tests. Furthermore, evaluation of gene transcripts for type I collagen, Runx-2, Bmp-2, bone sialoprotein, osteocalcin and osteopontin was performed using real-time RT-qPCR. It was observed that MSCs expressed GPR54 receptor to which Kp-10 binds during osteogenic differentiation, promoting a negative effect on osteogenic differentiation. This effect was observed at all the Kp-10 concentrations in a concentration-dependent manner, characterized by a decrease in the activity of alkaline phosphatase, collagen synthesis, mineralized nodules, and decreased expression of gene transcripts for type I collagen, osteocalcin, osteopontin, and Runx-2. Thus, Kp-10 inhibits in vitro osteogenic differentiation of MSCs extracted from the BM of adult Wistar rats.
Asunto(s)
Kisspeptinas , Células Madre Mesenquimatosas , Osteogénesis , Animales , Femenino , Ratas , Fosfatasa Alcalina/metabolismo , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Kisspeptinas/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteocalcina/genética , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Osteopontina/metabolismo , Osteopontina/farmacología , Ratas WistarRESUMEN
Leukotrienes (LTs) are derived from arachidonic acid metabolism by the 5-lipoxygenase (5-LO) enzyme. The production of LTs is stimulated in the pathogenesis of rheumatoid arthritis (RA), osteoarthritis, and periodontitis, with a relevant contribution to bone resorption. However, its role in bone turnover, particularly the suppression of bone formation by modulating the function of osteoclasts and osteoblasts, remains unclear. We investigated the effects of LTs on bone metabolism and their impact on osteogenic differentiation and osteoclastogenesis using a 5-LO knockout (KO) mouse model. Results from micro-computed tomography (µCT) analysis of femur from 8-week-old 5-LO-deficient mice showed increased cortical bone and medullary region in females and males and decreased trabecular bone in females. In the vertebra, we observed increased marrow area in both females and males 5-LO KO and decreased trabecular bone only in females 5-LO KO. Immunohistochemistry (IHC) analysis showed higher levels of osteogenic markers tissue-nonspecific alkaline phosphatase (TNAP) and osteopontin (OPN) and lower expression of osteoclastogenic marker tartrate-resistant acid phosphatase (TRAP) in the femurs of 5-LO KO mice versus wild-type (WT). Alkaline phosphatase activity and mineralization assay results showed that the 5-LO absence enhances osteoblasts differentiation and mineralization but decreases the proliferation. Alkaline phosphatase (ALP), Bglap, and Sp7 gene expression were higher in 5-LO KO osteoblasts compared to WT cells. Eicosanoids production was higher in 5-LO KO osteoblasts except for thromboxane 2, which was lower in 5-LO-deficient mice. Proteomic analysis identified the downregulation of proteins related to adenosine triphosphate (ATP) metabolism in 5-LO KO osteoblasts, and the upregulation of transcription factors such as the adaptor-related protein complex 1 (AP-1 complex) in long bones from 5-LO KO mice leading to an increased bone formation pattern in 5-LO-deficient mice. We observed enormous differences in the morphology and function of osteoclasts with reduced bone resorption markers and impaired osteoclasts in 5-LO KO compared to WT osteoclasts. Altogether, these results demonstrate that the absence of 5-LO is related to the greater osteogenic profile. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Resorción Ósea , Osteogénesis , Masculino , Femenino , Ratones , Animales , Fosfatasa Alcalina/metabolismo , Microtomografía por Rayos X , Proteómica , Osteoclastos/metabolismo , Osteoblastos/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Ratones Noqueados , Leucotrienos/metabolismo , Leucotrienos/farmacologíaRESUMEN
Hypophosphatasia (HPP) is an inherited disease caused by a low activity of tissue-nonspecific alkaline phosphatase, a hydrolase that removes phosphate groups from many molecules. Decreased alkaline phosphatase activity leads to the accumulation of three main metabolites, i.e., pyridoxal 5´-phosphate (PLP), inorganic pyrophosphate (PPi), and phosphoethanolamine. Impairment in PLP dephosphorylation induces seizures, while PPi accumulation inhibits bone mineralization. Clinically, HPP has a wide spectrum of presentations, ranging from neonatal death to an apparent lack of symptoms. This disease is classified into six subtypes according to the age at onset of first signs or symptoms. The clinical manifestations of the disease include rickets-like bone changes, bone demineralization, fragility fractures, reduced muscular strength, chest deformity, pulmonary hypoplasia, nephrolithiasis, nephrocalcinosis, and chondrocalcinosis. Treatment of HPP consists of enzyme replacement therapy. Before this therapy was approved, treatment was palliative and associated with high morbidity and mortality. Asfotase alfa has changed the prognosis of the disease by reducing bone deformity and improving bone mineralization, lung function, and muscle weakness, among other benefits. In adults, teriparatide and anti-sclerostin antibody have been used off-label in selected cases, demonstrating benefit in accelerating fracture healing and in concomitant treatment of osteoporosis. This review summarizes the main aspects of HPP and identifies the particularities of the disease in adult patients.
Asunto(s)
Hipofosfatasia , Osteoporosis , Adulto , Recién Nacido , Humanos , Fosfatasa Alcalina/metabolismo , Hipofosfatasia/terapia , Hipofosfatasia/tratamiento farmacológico , Osteoporosis/tratamiento farmacológico , Terapia de Reemplazo EnzimáticoRESUMEN
The increase in life expectancy has led to a higher incidence of osteoporosis, characterized by an imbalance in bone remodeling. Several drugs are used for its treatment, but most promote undesirable side effects. The present investigation evaluated the effects of two low concentrations of grape seed extract (GSE) rich in proanthocyanidins on MC3T3-E1 osteoblastic cells. The cells were cultured in an osteogenic medium and divided into control (C), 0.1 µg/mL GSE (GSE0.1), and 1.0 µg/mL GSE (GSE1.0) groups to evaluate cell morphology, adhesion, and proliferation, in situ alkaline phosphatase (ALP) detection, mineralization and immunolocalization of osteopontin (OPN). The data obtained were analyzed by statistical tests for a significance of 5%. Cell morphology was maintained with both GSE concentrations, whereas cell adhesion significantly increased within three days in all groups. Cell proliferation increased significantly at seven days of culture, followed by a significant decrease in all experimental periods, with no statistical difference among them. In situ detection of ALP and mineralization increased with time, but within each period, no statistical differences among groups were observed. The expression of osteopontin was distributed regularly with more intensity after 24 hours in the GSE0.1 group. After three days, OPN expression was more intense in the control group, followed by GSE0.1 and GSE1.0 groups. Data obtained suggest that low concentrations of GSE do not affect the morphology and may stimulate the functional activity of osteoblastic cells.
Asunto(s)
Extracto de Semillas de Uva , Extracto de Semillas de Uva/farmacología , Osteopontina/metabolismo , Adhesión Celular , Osteogénesis , Proliferación Celular , Osteoblastos , Diferenciación Celular , Fosfatasa Alcalina/metabolismoRESUMEN
Degenerative diseases, such as osteoporosis, could be treated by stem cells. The aim of this study was to identify the gene expression of bone marrow mesenchymal stem cells (BM-MSC) derived from Sprague Dawley rats and to assess the effect of Cissus quadrangularis Salisb. extract on their maturation into bone cells. The BM-MSC were divided into three groups: (a) BM-MSCs + osteoblast cell growth basal medium as the positive control; (b) BM-MSCs + Dulbecco's modified eagle's medium (DMEM) + 0.3 mg/mL methanol extract of C. quadrangularis as methanol group; and (c) BM-MSC + DMEM + 0.3 mg/mL ethyl acetate extract of C. quadrangularis as ethyl acetate group. A relative quantification approach using was used to analyze the expression of the alp (alkaline phosphatase) gene, with the beta-actin gene was used to normalize the expression of the alp gene. The intra-assay variation was calculated to validate the RT-qPCR data. Our study found that the intra-assay variation value was acceptable, with most of the coefficients of variability (CV) value <5. Ethyl acetate solvent outperformed methanol solvent in extracting the active compound C. quadrangularis. In the ethyl acetate extract group, the expression of the alp gene increased three times compared to the positive control. In methanol extract group, the expression of alp gene was lower six times compared to positive control. This study suggests that C. quadrangularis extracts using ethyl acetate could induce the maturation of BM-MSCs. However, further studies are warrant to confirm this effect using different indicators.
Asunto(s)
Cissus , Células Madre Mesenquimatosas , Ratas , Animales , Cissus/metabolismo , Extractos Vegetales/farmacología , Ratas Sprague-Dawley , Metanol , Fosfatasa Alcalina/metabolismo , Solventes , Células Madre Mesenquimatosas/metabolismoRESUMEN
This study aimed to evaluate the pre-clinical behavior of niobium-containing bioactive glasses (BAGNb) by their ability to promote bone repair and regulate alkaline phosphatase (ALP) levels in an animal model. BAGNbs were produced as powders and as scaffolds and surgically implanted in the femur of male rats (Wistar lineage n = 10). Glasses without Nb (BAG) were produced and implanted as well. The Autogenous Bone (AB) was used as a control. After 15, 30, and 60 days of surgical implantation, blood serum samples were collected to quantify ALP activity, and femurs were removed to assess bone repair. Bone samples were histologically processed and stained with H&E to quantify the % new bone into defects. No postoperative complications were identified. Early-stage repair (15 days) resulted in increased ALP activity for all groups, with increased values ââfor powdered BAGNb. The maturation of the new bone led to a reduction in serum ALP levels. Histological sections showed the formation of immature bone tissue and vascularization with the progression of bone deposition to mature and functional tissue over time. BAG powder showed less new bone formation in 15 days, while the analysis at 30 and 60 days showed no difference between groups (p > .05). Niobium-containing bioactive glasses safely and successfully induced bone repair in vivo. The modulation of ALP activity may be a pathway to describe the ability of niobium-containing materials to contribute to new bone formation.
Asunto(s)
Fosfatasa Alcalina , Niobio , Ratas , Masculino , Animales , Niobio/farmacología , Fosfatasa Alcalina/metabolismo , Ratas Wistar , Huesos/metabolismo , Fémur/metabolismo , Osteogénesis , Regeneración ÓseaRESUMEN
The development of nanoscale biomaterials associated with polymers has been growing over the years, due to their important structural characteristics for applications in biological systems. The present study aimed to produce and test polymeric scaffolds composed of polylactic acid (PLA) fibers associated with a 58S bioglass doped with therapeutic ions for use in tissue engineering. Three 58S Bioglass was obtained by the sol-gel route, pure and doped with 5% strontium and cobalt ions. Solutions of 7% PLA was used as control and added the three different bioglass, 4% of 58S bioglass (PLA-BG), 4% bioglass-doped strontium (PLA-BGSr) and 4% bioglass-doped cobalt (PLA-BGCo). Scaffolds were produced through electrospinning process, and was characterized chemical and morphologically. The in vitro tests were performed using mesenchymal cells cultures from femurs of nine rats, grown in osteogenic supplemented total culture medium. After osteoblastic differentiation induction cell viability, alkaline phosphatase activity, total protein content quantification, and visualization of mineralization nodule tests were performed. Analysis of normal distribution used the Shapiro-Wilk test (nanofibers diameter and biological assay). Data were compared using the Kruskal-Wallis nonparametric test (p = 0.05). The bioglasses produced proved to be free of nitrate, chlorinated and nano-sized, with effective incorporation of therapeutic ions in their structure. All materials showed cell viability (>70%), total protein production, and alkaline phosphatase activity. It was possible to develop polylactic acid scaffolds associated with 58S bioglass doped with therapeutic ions without cytotoxicity. Scaffolds characteristics appear to sustain its application in bone tissue engineering.
Asunto(s)
Estroncio , Ingeniería de Tejidos , Ratas , Animales , Estroncio/farmacología , Andamios del Tejido/química , Fosfatasa Alcalina/metabolismo , Cobalto/farmacología , Poliésteres/química , Osteogénesis , IonesRESUMEN
Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.