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The process of endometrial repair after injury involves the synergistic action of various cells including immune cells and stem cells. In this study, after combing Fibrinogen(Fg) with poly(L-lacticacid)-co-poly(ε-caprolactone)(P(LLA-CL)) by electrospinning, we placed Fg/P(LLA-CL) into the uterine cavity of endometrium-injured rats, and bioinformatic analysis revealed that Fg/P(LLA-CL) may affect inflammatory response and stem cell biological behavior. Therefore, we verified that Fg/P(LLA-CL) could inhibit the lipopolysaccharide (LPS)-stimulated macrophages from switching to the pro-inflammatory M1 phenotype in vitro. Moreover, in the rat model of endometrial injury, Fg/P(LLA-CL) effectively promoted the polarization of macrophages towards the anti-inflammatory M2 phenotype and enhanced the presence of mesenchymal stem cells at the injury site. Overall, Fg/P(LLA-CL) exhibits significant influence on macrophage polarization and stem cell behavior in endometrial injury, justifying further exploration for potential therapeutic applications in endometrial and other tissue injuries.
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Materiales Biocompatibles , Endometrio , Fibrinógeno , Macrófagos , Células Madre Mesenquimatosas , Animales , Femenino , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Endometrio/citología , Endometrio/lesiones , Ratas , Materiales Biocompatibles/química , Fibrinógeno/metabolismo , Ratas Sprague-Dawley , Ratones , Poliésteres/química , Células RAW 264.7 , Inflamación , Lipopolisacáridos , Polaridad Celular/efectos de los fármacosRESUMEN
Early-life stress (ELS) predisposes individuals to psychiatric disorders, including anxiety and depression, and cognitive impairments later in life. However, the underlying molecular mechanisms are not completely understood. Developmental deficits in hippocampal synaptic plasticity are among the primary detrimental alterations in brain function induced by ELS. Impaired synaptic plasticity is usually accompanied by decreased synaptic proteins, such as postsynaptic density 95 (PSD95) and synaptophysin, which are important for synaptic function. The mTOR signaling pathway plays a vital role in regulating protein translation, and mTOR activation is functionally associated with synaptic protein synthesis. In the present study, we observed whether ELS impacts synaptic protein synthesis and mTOR signaling, which is involved in synaptic plasticity. Herein, we established a maternal separation (MS) and chronic restraint stress (CRS) model and evaluated anxiety-like behavior and cognitive function (e.g., learning and memory) in adulthood through behavioral examination and analyzed hippocampal expression levels of PSD95 and synaptophysin. To explore whether the mTOR signaling pathway was associated with ELS, we also examined the activity of mTOR and s6. The behavior tests indicated that maternally separated mice showed increased anxiety-like behavior and cognitive impairments. PSD95 and synaptophysin mRNA and protein expression levels were decreased in the hippocampus, and phosphorylated mTOR and phosphorylated s6 were significantly decreased in maternally separated mice vs. those not exposed to MS. Our data demonstrate that MS impairs synaptic plasticity and inhibits mTOR signaling, specifically via s6. Therefore, we speculate that ELS decreased synaptic plasticity via the inhibition of the mTOR pathway in the hippocampus, which may underlie vulnerability to stress and mental disorders in adulthood.
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Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP-cell derived embryos with two tdTomato-cell derived embryos at the 4-cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP-expressing cells, and this phenomenon was possibly due to the hyper-methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity-related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell.
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The Cre-loxP system is a powerful tool for genetic analysis of distinct cell lineages and tissue-specific gene knockout in animal models. VASA is specifically expressed in reproductive tissues, and is known to play important roles in spermatogenesis and germ-cell growth. In this study, Cre recombinase transgenic pigs under the control of the VASA promoter were generated by somatic cell nuclear transfer. Germ cell-specific expression of Cre recombinase in VASA-Cre transgenic pigs was shown by western blotting and immunohistochemistry. VASA-Cre transgenic pigs will be a useful tool for germ cell-specific gene knockout and a disease model for disorders of the reproductive system.
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BACKGROUND/AIMS: Parthenogenetic embryos are useful in many applications, such as being an alternative source of embryonic stem cells that would avoid ethical problems. Aberrance in epigenetic reprogramming is considered the major reason for the developmental failure of parthenogenetic embryos. Many histone deacetylase inhibitors have been shown to improve the reprogramming of stem cells and embryos. Here, the relationship between histone modification and parthenogenetic embryonic development was explored. METHODS: Valproic acid (VPA) treatment was applied during the culture of parthenogenetic embryos. The abundance of histone modifiers was examined by immunofluorescence and quantified by Image-pro software. RESULTS: The acH3K9 level in in vitro fertilized embryos was significantly higher than parthenogenetic embryos. VPA treatment improved both the blastocyst formation rate and the acH3K9 level in parthenogenetic embryos. The signal intensities of acH4K5 and H3K4me2 were also enhanced in VPA treated embryos. The H3K27me2 level was decreased in the VPA treated embryos at the 2-cell stage. However, the enhancement in the acH3K9, acH4K5 and H3K4me2 level, or the decrease in the H3K27me2 level disappeared shortly after VPA withdrawal. CONCLUSION: Optimizing histone modifications for a short time following activation was sufficient to enhance the in vitro development of parthenogenetic embryos.
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Desarrollo Embrionario/efectos de los fármacos , Histonas/metabolismo , Ácido Valproico/farmacología , Animales , Ácido Ascórbico/farmacología , Blastocisto/citología , Blastocisto/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Fertilización In Vitro , Microscopía Fluorescente , PorcinosRESUMEN
Although the expression and epigenetic status of imprinted genes have been extensively studied in a number of species, less is known about the genomic imprinting in rabbits. Neuronatin (Nnat) plays significant roles in the brain development and metabolic regulation and has been identified to be imprinted and paternally expressed in humans, mice and pigs; however, it has not yet been investigated in rabbits. In this study, we confirmed the expression of two isoforms of the rabbit Nnat (Nnat-a and Nnat-ß) identified in Genbank and Ensembl by quantitative real-time PCR. In addition, we also determined the methylation profile of the CpG island in the promoter region of the rabbit Nnat using bisulfite sequencing PCR and combined bisulfite restriction analysis. Here, we provide the first evidence that Nnat has two transcripts in rabbit. Additionally, the CpG island located in the promoter region shows oocyte-specific methylation and may be the differentially methylated region of Nnat in rabbits.
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To understand if the genomic imprinting status of the donor cells is altered during the process of SCNT (somatic cell nuclear transfer), cloned pigs were produced by SCNT using PEF (porcine embryonic fibroblast) and P-PEF (parthenogenetic-PEF) cells as donors. Then, the gene expression and methylation patterns of H19, IGF2, NNAT and MEST were compared between PEF vs. C-PEF (cloned-PEF), P-PEF vs. CP-PEF (cloned-P-PEF), respectively. Taken together, the results revealed that there was no significant difference in the expression of imprinted genes and conserved genomic imprints between the donor and cloned cells.
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Clonación de Organismos/veterinaria , Ectogénesis , Desarrollo Embrionario , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Sus scrofa/metabolismo , Animales , Células Cultivadas , China , Células Clonales , Clonación de Organismos/métodos , Metilación de ADN , Transferencia de Embrión/veterinaria , Embrión de Mamíferos/citología , Femenino , Inseminación Artificial/veterinaria , Masculino , Técnicas de Transferencia Nuclear/efectos adversos , Técnicas de Transferencia Nuclear/veterinaria , Partenogénesis , Embarazo , Sus scrofa/genéticaRESUMEN
Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.
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Aborto Espontáneo/genética , Embrión de Mamíferos/metabolismo , Feto/metabolismo , Técnicas de Transferencia Nuclear , ARN Largo no Codificante/genética , Animales , Metilación de ADN/genética , Femenino , Regulación de la Expresión Génica , Genotipo , Masculino , Embarazo , ARN Largo no Codificante/metabolismo , Sus scrofa , Transcripción GenéticaRESUMEN
It is generally believed that aberrant expression of imprinted genes participates in growth retardation of mammalian parthenogenesis. Neuronatin (NNAT), a paternally expressed gene, plays important roles in neuronal growth and metabolic regulation. Here we have compared the gene expression and promoter methylation pattern of NNAT between pig normally fertilized (Con) and parthenogenetic (PA) embryos. The results showed loss of NNAT expression (p<0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partial methylation was observed in Con fetuses, while almost full methylation and unmethylation of NNAT promoter were apparent in Metaphase II (MII) oocytes and mature sperms, respectively, which identified the CpG promoter region as a putative differentially methylated region (DMR) of NNAT. The data demonstrate that promoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis at early stages.
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Metilación de ADN/genética , Feto/metabolismo , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/genética , Porcinos/genética , Animales , Células Cultivadas , Islas de CpG/genética , Expresión Génica/genética , Masculino , Oocitos/metabolismo , Espermatozoides/metabolismoRESUMEN
Bacterial infection is a major challenge in biomaterials development. The adhesion of microorganisms to the material surface is the first step in infectious conditions and this quickly leads to the formation of biofilms on a material surface. A unique attribute of atomic force microscopy (AFM) is that it reveals not only the morphology of cells and the surface roughness of the substrate, but it can also quantify the adhesion force between bacteria and surfaces. We have shown that fluoroalkylsilane (FAS) and octadecyltrichlorosilane (OTS)-coated silicone samples exhibit greater potential for reducing E. coli JM 109 adhesion than heparin- and hyaluronan-modified samples. The force curves obtained from AFM can be used as a primary indicator in predicting bacterial adhesion.
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Adhesión Bacteriana , Escherichia coli/citología , Nanotecnología/métodos , Siliconas/química , Microscopía de Fuerza Atómica , Propiedades de SuperficieRESUMEN
OBJECTIVE: To investigate the expression features of P-glycoprotein (P-gp), glutathione S transferase-pi (GST-pi) and inhibitor of apoptosis proteins like p53, survivin and bcl-2 in lymph node metastases of gastrointestinal carcinomas. METHODS: The expression of P-gp, GST-pi, p53, survivin and bcl-2 were determined by using immunohistochemistry technique in surgical specimens of primary tumor (PT) and lymph node metastases (LNMs) from 54 gastrointestinal cancer patients with metastasis of lymph nodes. The expression difference of 5 multi-drug resistance (MDR)-related factors between LNMs and PT were compared. RESULTS: Significant difference was found in the expression of P-gp and GST-pi between the two groups (both P < 0.05), and expression of p53 and bcl-2 showed positive correlation between LNMs and PT (r = 0.7248, 0.5524; both P < 0.05), respectively. In LNMs, P-gp expression was positively correlated with GST-pi (r = 0.4062, P < 0.05) and survivin (r = 0.6169, P < 0.05), and also GST-pi expression was related positively with survivin (r = 0.4027, P < 0.05). Statistically positive correlations were noted between bcl-2 and P-gp (r = 0.3986, P < 0.05), bcl-2 and survivin (r = 0.2937, P < 0.05), as well as GST-pi and survivin (r = 0.4481, P < 0.01) in PT. Only a positive correlation between GST-pi and survivin expression was simultaneously shown in both LNMs and PT. CONCLUSIONS: There is significant heterogeneity of MDR-related factors expression in LNMs of gastrointestinal carcinomas. Effective adjuvant chemotherapy after operation should target on the metastatic loci of the disease.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Neoplasias del Sistema Digestivo/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Ganglios Linfáticos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Sistema Digestivo/patología , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Survivin , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Bacterial adhesion and colonization are complicated processes that depend on many factors, including surface chemistry, hydrophobicity, and surface roughness. The contribution of each of these factors has not been fully elucidated because most previous studies used different polymeric surfaces to achieve differences in properties. The objective of this study was to modify hydrophobicity and roughness on one polymeric surface, eliminating the confounding contribution of surface chemistry. Mechanically assembled monolayer (MAM) preparation methods (both one- and two-dimensional) were used to impart different degrees of hydrophobicity on fluoroalkylsilane (FAS)-coated silicone. Surface roughness was varied by casting the silicone to templates prepared with different abrasives. Surface hydrophobicity was determined by contact angle measurement, whereas surface roughness was determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Bacterial adhesion and colonization were analyzed using a direct colony-counting method and SEM images. Hydrophobicity increased as a function of stretched length or width (Deltax or Deltay); it reached a maximum at Deltax = 60% with one-dimensional MAM and decreased as Deltax further increased to 80 and 100%. The same trend was observed for the two-dimensional MAM. After 12-h incubation, all the FAS/silicone surfaces had significantly reduced adherence of Staphylococcus epidermidis by 42-89%, compared to untreated silicone, and the degree of which is inversely related to surface hydrophobicity. On the other hand, surface roughness had a significant effect on bacterial adhesion and colonization only when the root-mean-square roughness was higher than 200 nm.
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Adhesión Bacteriana/fisiología , Siliconas/química , Staphylococcus epidermidis/metabolismo , Materiales Biocompatibles/química , Biopelículas , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Polímeros/química , Propiedades de SuperficieRESUMEN
A new approach of chemically immobilizing antibody within a pattern based on thin-film cracking is presented. An adjustable pattern width is achieved with resolutions varied from nano- to microscale by using loading stress on thin-film coated elastomer substrate in both one and two dimensions. By introduction of solution or chemical vapor deposition approaches, antibodies were covalently immobilized in the channels. To demonstrate the bioactivity, specificity, and response rate of antibody patterned structure, scanning electron microscopy was used to enumerating bacteria. The chemically coupled antibody is found to retain its specificity when incubated with different bacteria solutions. Trichloro(1H,1H,2H,2H-perfluoroctyl)silane coating on nonsensing regions exhibits a distinguished bacteria-resistant function that is beneficial for providing a low intrinsic background signal in detection. This technique shows a great potential for applications in the fields of sensing and tissue engineering.
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Anticuerpos Monoclonales/química , Técnicas Biosensibles/métodos , Elastómeros de Silicona/química , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos de Superficie/inmunología , Toxinas Bacterianas/inmunología , Escherichia coli/citología , Escherichia coli/inmunología , Cinética , Mecánica , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Propiedades de Superficie , Ingeniería de Tejidos/métodosRESUMEN
The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to AlN/sapphire surfaces which may allow the antibody to freely reorient and potentially improving the antigen capture efficiency. Spacer density was one of the key parameters to be optimized in studying its effect on the immobilization of antibody. The spacer density was controlled by functionalizing AlN/sapphire surfaces with a mixed (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and trichloro(1H,1H,2H,2H-perfluoroctyl)silane (FAS) self-assembled monolayer (SAM) through a step-wise method. Contact angle measurement and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface coverage of GPTMS and surface chemical composition. Compared to spacer fully covered samples, the capture efficiency was improved by approximately 28% with optimal Jeffamine ED 600 spacer density, which depends on the spacer properties such as the number of monomer units and its size.
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Anticuerpos Antibacterianos/inmunología , Escherichia coli/inmunología , Fimbrias Bacterianas/inmunología , Sitios de Unión de Anticuerpos , Microscopía Electrónica de Rastreo , Análisis Espectral/métodos , Propiedades de Superficie , Rayos XRESUMEN
Providing a long period of sustained antibiotic release is one of the important challenges in the development of clinical shunts for long-term implantation. A cast-molding approach was used to load rifampicin into the silicone precursor before curing. Sustained in vitro release from rifampicin-loaded silicone for upwards of 110 days at a level of approximately 2-4 microg/cm2 per day was achieved. Quantitative comparisons of Staphylococcus epidermidis adhesion on untreated and rifampicin-loaded silicone surfaces were carried out to demonstrate the effect of rifampicin to discourage the bacterial adhesion. It was shown that the fresh 8.3% rifampicin-loaded silicone decreased bacterial adherence by 99.8%. Bacterial adherence on rifampicin-loaded silicone surfaces after 90 days release (eluted silicone) was decreased by 94.8%. A new approach was used to tune the initial burst effect and prolong long lasting release by introducing self-assembled perfluorodecyltrichlorosilane (FAS) and octadecyltrichlorosilane (OTS) layers. FAS layered structures can tune the burst effect and prolong the subsequent continuous release to achieve the long-term delivery. Significant decreases in initial burst effect (70.3% for multi-FAS layers and 39.7% for FAS monolayer) and enhanced long-term release (approx. 10 microg/cm2 per day for FAS monolayer for 110 days and approx. 15 microg/cm2 per day for multi-FAS layers for 60 days) were observed.
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Antibióticos Antituberculosos/química , Materiales Biocompatibles Revestidos , Infecciones Relacionadas con Prótesis/prevención & control , Rifampin/química , Siliconas/química , Adhesión Bacteriana , Catéteres de Permanencia/microbiología , Preparaciones de Acción Retardada/farmacología , Hidrocefalia/terapia , Silanos/química , Siliconas/farmacología , Staphylococcus epidermidis/efectos de los fármacos , Factores de TiempoRESUMEN
The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Poly(ethylene glycol) (PEG) and Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to silicon wafer surfaces for the purpose of improving the orientation of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a variety of linear polymers were used to covalently attach the antibodies to silicon surfaces. Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical composition at each reaction step. The effect of spacer length in improving the specificity of immobilized antibody was investigated by attaching E. coli on the end of an AFM tip. The distribution of unbinding force and rupture distance from the force-distance curves obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the incidence of interactions by up to 90%. J600 proved to be the most effective spacer overcoming the steric hindrance seen with direct immobilization of antibody. In addition, the force spectroscopy reveals the elementary force quantum of E. coli-antibody to be 0.3 nN.
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Anticuerpos Antibacterianos/química , Reacciones Antígeno-Anticuerpo , Escherichia coli , Proteínas Fimbrias/química , Tamaño de la Partícula , Polietilenglicoles/química , Biotecnología/métodos , Células Cultivadas , Escherichia coli/inmunología , Microscopía de Fuerza Atómica/métodos , Estructura Molecular , Silicio/química , Propiedades de Superficie , Tensoactivos/químicaRESUMEN
Cerebrospinal fluid (CSF) shunts for the treatment of hydrocephalus are generally made of silicone rubber. The growth of bacterial colonies on the silicone surface leads to frequent CSF shunt complications. A systematic study of the effect of the surface modification of silicone on Staphylococcus epidermidis adhesion and colonization was performed for different incubation times by means of colony counting and scanning electron microscopy (SEM). Silicone was modified with different biopolymers and silanes, including heparin, hyaluronan, octadecyltrichlorosilane (OTS), and fluoroalkylsilane (FAS) to provide a stable and biocompatible surface with different surface functional groups and degrees of hydrophobicity. The modified silicone surfaces were studied by using contact angle measurements, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After 4 and 8 h of incubation, the FAS- and OTS-coated silicone and the hyaluronan coated OTS/silicone surfaces showed significantly reduced bacterial adhesion and colonization compared to blank silicone by both quantification methods. However, the heparin coated OTS/silicone showed significantly increased bacterial adhesion. These results indicate that the nature of the surface functional group and surface roughness determine the extent of bacterial adhesion and colonization. However, the degree of hydrophobicity of the surface did not appear to play a determining role in bacterial adhesion and colonization.
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Adhesión Bacteriana , Materiales Biocompatibles Revestidos , Contaminación de Equipos , Elastómeros de Silicona , Infecciones Estafilocócicas/prevención & control , Staphylococcus epidermidis , Derivaciones del Líquido Cefalorraquídeo , Contaminación de Equipos/prevención & control , Hidrocefalia/terapia , Microscopía de Fuerza Atómica , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus epidermidis/ultraestructura , Propiedades de SuperficieRESUMEN
A self-assembled monolayer (SAM) of fluoroalkyl silane (FAS) (CF(3)(CF(2))(5)(CH(2))(2)SiCl(3)) was deposited on the surface of silicon wafers, aiming to enhance its stability and to reduce the inflammatory response in the central nervous system. Following implantation of the FAS SAM coated silicon in rat brains, the FAS SAM coating failed to reduce the inflammatory response of silicon, because it could not prevent the corrosion of the underlying silicon. The corrosion was hindered for the initial 10 days by the FAS SAM coating, but there was nearly no difference when compared to the uncoated silicon when the implantation periods were extended to 28 and 90 days. The FAS SAM coating was completely removed within 28 and 90 days. Meanwhile, on all the extracted uncoated and FAS SAM coated silicon wafers, there were proteinaceous substances deposited on the surfaces, and the amount of the deposits increased with exposure time.
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Materiales Biocompatibles/química , Encéfalo/cirugía , Silanos/química , Animales , Materiales Biocompatibles/toxicidad , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Materiales Biocompatibles Revestidos/química , Corrosión , Estabilidad de Medicamentos , Proteína Ácida Fibrilar de la Glía/metabolismo , Inflamación/etiología , Inflamación/patología , Ensayo de Materiales , Degeneración Nerviosa/etiología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Prótesis e Implantes , Ratas , Ratas Sprague-Dawley , Silanos/toxicidad , Propiedades de SuperficieRESUMEN
Shunt infections are one of the most serious complications in shunt implant surgery. Previous studies have suggested that cerebrospinal fluid (CSF) proteins could affect bacterial adhesion and subsequent shunt infection. A systematic study using immobilized protein on the surface of silane-modified silicone was conducted to determine how these modifications influenced Staphylococcus epidermidis adhesion and colonization. A comparison was also made with silicone having physically adsorbed protein. A colony-counting adhesion assay and scanning electron microscopy (SEM) were used to provide quantitative analysis of bacterial adhesion and semi-quantitative analysis of bacterial colonization, respectively. In order to determine the appropriate silanization process for effective protein immobilization, the effect of bovine serum albumin (BSA) immobilized on n-3-(trimethoxysilyl)propyl-ethylenediamine (AEAPS)/silicone, aminopropyltriethoxysilane (APTMS)/silicone, 3-(glycidyloxypropyl)trimethoxysilane (GPTMS)/silicone, and octadecyltrichlorosilane (OTS)/silicone on bacterial adhesion was investigated. Upon identifying that OTS is the most effective silane, different types of proteins, including: BSA, human serum albumin (HSA), gamma-globulin, and fibrinogen were immobilized on OTS/silicone by a photo-immobilization method. Immobilized protein on modified silicone surfaces was found to be stable in saline for 30 days, while physically adsorbed protein showed instability within hours as determined by contact angle measurements and X-ray photoelectron spectroscopy (XPS). For HSA/OTS/silicone, BSA/OTS/silicone, gamma-globulin/OTS/silicone, fibrinogen/OTS/silicon, and physically absorbed BSA on silicone, the contact angles were 78.5 degrees, 80.7 degrees, 78.9 degrees, 81.3 degrees, and 96.5 degrees; and the amount of nitrogen content was found to be 4.6%, 5.0%, 5.6%, 7.2%, and 3.2%, respectively. All protein immobilized on OTS/silicone surfaces significantly reduced bacterial adhesion by around 75% compared to untreated silicone, while physically adsorbed BSA on silicone reduced by only 29.4%, as determined by colony-counting adhesion assay. However, there was no significant difference on bacterial adhesion among the different types of proteins immobilized on OTS/silicone. Minimizing bacterial adhesion and colonization can be attributed to the increased concentration of -NH2 group, and stability and more hydrophilic nature of the protein/OTS/silicone surfaces.
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Adhesión Bacteriana/fisiología , Proteínas de la Membrana/química , Siliconas/química , Staphylococcus epidermidis/fisiología , Animales , Adhesión Bacteriana/genética , Bovinos , Microanálisis por Sonda Electrónica , Fibrinógeno/química , Fibrinógeno/fisiología , Humanos , Proteínas de la Membrana/fisiología , Albúmina Sérica/química , Albúmina Sérica/fisiología , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/fisiología , Siliconas/farmacología , Staphylococcus epidermidis/citología , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus epidermidis/ultraestructura , gammaglobulinas/química , gammaglobulinas/fisiologíaRESUMEN
Bacterial adhesion on biomaterial surfaces is the initial step in establishing infections and leads to the formation of biofilms. In this study, silicone was modified with different biopolymers and silanes, including: heparin, hyaluronan, and self-assembled octadecyltrichlorosilane (OTS), and fluoroalkylsilane (FAS). The aim was to provide a stable and bacteria-resistant surface by varying the degree of hydrophobicity and the surface structure. The adhesion of Escherichia coli (JM 109) on different modified silicone surfaces was investigated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Mica, an ideal hydrophilic and smooth surface, was employed as a control specimen to study the effect of hydrophobicity and surfaces roughness on bacterial adhesion. AFM probes were coated with E. coli and the force measurements between the bacteria-immobilized tip and various materials surfaces were obtained while approaching to and retracting from the surfaces. A short-range repulsive force was observed between the FAS coated silicone and bacteria. The pull-off force of bacteria to FAS was the smallest among coated surfaces. On the other hand, heparin exhibited a long-range attractive force during approach and required a higher pull-off force in retraction. Both AFM and SEM results indicated that FAS reduced bacterial adhesion whereas heparin enhanced the adhesion compared to pure silicone. The work demonstrates that hydrophobicity cannot be used as a criterion to predict bacterial adhesion. Rather, both the native properties of the individual strain of bacteria and the specific functional structure of the surfaces determine the strength of force interaction, and thus the extent of adhesion.