Antibakteriální nanovlákenné vrstvy pro prevenci infekcí z důvodu kolorektálního leaku
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Za účelem prevence infekcí z důvodu kolorektálního leaku byl vyvinut a následně také otestován nanovlákenný materiál, do něhož bylo inkorporováno antibiotikum Vankomycin hydrochlorid (VCH) o hmotnostních koncentracích 0%; 0,5%; 1% a 1,5%. Antibiotikum bylo inkorporováno do roztoku poly--kaprolaktonu (PCL) a roztok byl následně zvlákněn na bezjehlovém elektrostatickém zařízení NanospiderTM. Morfologie připravených vrstev byla sledována pomocí skenovací elektronové mikroskopie, ze získaných snímků byly měřeny průměry vláken pro ověření homogenity. Smáčivost vlákenných vrstev byla hodnocena pomocí kontaktního úhlu, byla sledována interakce s destilovanou vodou a simulovanou střevní tekutinou, která byla zvolena vzhledem k finální aplikaci. Obsah antibiotik byl otestován pomocí elementární analýzy a vysoce-účinné kapalinové chromatografie (HPLC). Dále byly provedeny testy antibakteriální, jež potvrdily přítomnost, uvolňování a také účinnost antibiotika Vankomycin hydrochlorid v nanovlákenných materiálech. Pomocí testování in vitro, při kterém byly materiály osazeny buněčnou myší linií 3T3 myších fibroblastů, byla hodnocena cytotoxicita extraktů materiálů i samotného antibiotika VCH v médiu. Materiály byly vyhodnoceny jako cytokompatibilní a byla nalezena hraniční toxická koncentrace VCH v médiu. Pomocí testování in vitro byla také ověřena buněčná adheze a proliferace po osazení na materiál. Toho bylo dosaženo za použití metabolického CCK-8 testu, fluorescenční mikroskopie (morfologie buněk a jejich počet), a skenovací elektronové mikroskopie. Test byl vyhodnocován 4.; 7.; 14. a 21. den. Po celou dobu trvání experimentu bylo také pozorováno, jaký má na vyrobené nanovlákenné materiály vliv sterilizace ethylenoxidem, která je vybranou sterilizační metodou před aplikací materiálu in vivo. Žádné významné změny pozorovány nebyly.
In order to prevent colorectal leak infections, a nanofibrous material was developed and subsequently tested, into which the antibiotic Vancomycin hydrochloride (VCH) by weight was incorporated at 0%; 0,5%; 1% and 1,5% concentration. The antibiotic was incorporated into the poly--caprolactone (PCL) solution and the solution was subsequently spun on a NanospiderTM needleless electrostatic device. The morphology of the prepared layers was monitored by scanning electron microscopy, and the fiber diameters were measured from the obtained images to verify homogeneity. The wettability of the fibrous layers was evaluated by means of a contact angle, the interaction with distilled water and simulated intestinal fluid was monitored, which was chosen with respect to the final application. The antibiotic content was tested by elemental analysis and high performance liquid chromatography (HPLC). In addition, antibacterial tests were performed to confirm the presence, release and also the effectiveness of the antibiotic Vancomycin hydrochloride in nanofibrous materials. The cytotoxicity of the material extracts and the VCH antibiotic alone in the medium was evaluated by in vitro testing, in which the materials were seeded with the mouse fibroblast 3T3 cell line. The materials were evaluated as cytocompatible and the borderline toxic concentration of VCH in the medium was found. Cell adhesion and proliferation after attachment to the material were also verified by in vitro testing. This was achieved using a metabolic CCK-8 assay, fluorescence microscopy (cell morphology and number), and scanning electron microscopy. The test was evaluated 4th; 7th; 14th and 21st day. Throughout the experiment, the effect of sterilization with ethylene oxide, which is the chosen sterilization method before the application of the material in vivo, was also observed on the produced nanofibrous materials. No significant changes were observed.
In order to prevent colorectal leak infections, a nanofibrous material was developed and subsequently tested, into which the antibiotic Vancomycin hydrochloride (VCH) by weight was incorporated at 0%; 0,5%; 1% and 1,5% concentration. The antibiotic was incorporated into the poly--caprolactone (PCL) solution and the solution was subsequently spun on a NanospiderTM needleless electrostatic device. The morphology of the prepared layers was monitored by scanning electron microscopy, and the fiber diameters were measured from the obtained images to verify homogeneity. The wettability of the fibrous layers was evaluated by means of a contact angle, the interaction with distilled water and simulated intestinal fluid was monitored, which was chosen with respect to the final application. The antibiotic content was tested by elemental analysis and high performance liquid chromatography (HPLC). In addition, antibacterial tests were performed to confirm the presence, release and also the effectiveness of the antibiotic Vancomycin hydrochloride in nanofibrous materials. The cytotoxicity of the material extracts and the VCH antibiotic alone in the medium was evaluated by in vitro testing, in which the materials were seeded with the mouse fibroblast 3T3 cell line. The materials were evaluated as cytocompatible and the borderline toxic concentration of VCH in the medium was found. Cell adhesion and proliferation after attachment to the material were also verified by in vitro testing. This was achieved using a metabolic CCK-8 assay, fluorescence microscopy (cell morphology and number), and scanning electron microscopy. The test was evaluated 4th; 7th; 14th and 21st day. Throughout the experiment, the effect of sterilization with ethylene oxide, which is the chosen sterilization method before the application of the material in vivo, was also observed on the produced nanofibrous materials. No significant changes were observed.
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tkáňový nosič, nanovlákna, elektrostatické zvlákňování, střevní anastomóza, antibiotika, scaffold, nanofibers, electrospinning, intestinal anastomosis, antibiotics