The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of Staphylococcus aureus and Escherichia coli on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials.

• MeSH
• biofilmy * růst a vývoj   MeSH
• Escherichia coli * fyziologie   MeSH
• glukosa metabolismus   MeSH
• nanovlákna * chemie   MeSH
• nylony chemie   MeSH
• polyestery * chemie   MeSH
• spektrofotometrie metody   MeSH
• Staphylococcus aureus * fyziologie   MeSH
• tetrazoliové soli * metabolismus   chemie   MeSH
• thiazoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: The formation of diabetic ulcers (DU) is a common complication for diabetic patients resulting in serious chronic wounds. There is therefore, an urgent need for complex treatment of this problem. This study examines a bioactive wound dressing of a biodegradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) covered by a thin fibrin layer for sustained delivery of bioactive molecules. METHODS: Electrospun PLCL/PCL nanofibers were coated with fibrin-based coating prepared by a controlled technique and enriched with human platelet lysate (hPL), fibroblast growth factor 2 (FGF), and vascular endothelial growth factor (VEGF). The coating was characterized by scanning electron microscopy and fluorescent microscopy. Protein content and its release rate and the effect on human saphenous vein endothelial cells (HSVEC) were evaluated. RESULTS: The highest protein amount is achieved by the coating of PLCL/PCL with a fibrin mesh containing 20% v/v hPL (NF20). The fibrin coating serves as an excellent scaffold to accumulate bioactive molecules from hPL such as PDGF-BB, fibronectin (Fn), and α-2 antiplasmin. The NF20 coating shows both fast and a sustained release of the attached bioactive molecules (Fn, VEGF, FGF). The dressing significantly increases the viability of human saphenous vein endothelial cells (HSVECs) cultivated on a collagen-based wound model. The exogenous addition of FGF and VEGF during the coating procedure further increases the HSVECs viability. In addition, the presence of α-2 antiplasmin significantly stabilizes the fibrin mesh and prevents its cleavage by plasmin. DISCUSSION: The NF20 coating supplemented with FGF and VEGF provides a promising wound dressing for the complex treatment of DU. The incorporation of various bioactive molecules from hPL and growth factors has great potential to support the healing processes by providing appropriate stimuli in the chronic wound.

• MeSH
• alfa-2-antiplasmin MeSH
• endoteliální buňky MeSH
• hojení ran MeSH
• lidé MeSH
• nanovlákna * MeSH
• obvazy MeSH
• polyestery farmakologie   MeSH
• vaskulární endoteliální růstový faktor A * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Polycaprolactone (PCL) was electrospun with the addition of arginine (Arg), an α-amino acid that accelerates the healing process. The efficient needleless electrospinning technique was used for the fabrication of the nanofibrous layers. The materials produced consisted mainly of fibers with diameters of between 200 and 400 nm. Moreover, both microfibers and beads were present within the layers. Higher bead sizes were observed with the increased addition of arginine. The arginine content within the layers as well as the weight of the resultant electrospun materials were enhanced with the increased addition of arginine to the electrospinning solution (1, 5 and 10 wt%). The PCL + 1% Arg nanofibrous layer contained 5.67 ± 0.04% of arginine, the PCL + 5% Arg layer 22.66 ± 0.24% of arginine and the PCL + 10% Arg layer 37.33 ± 0.39% of arginine according to the results of the elemental analysis. A high burst release within 5 h of soaking was recorded for the PCL + 5% and PCL + 10% nanofibrous layers. However, the release rate of arginine from the PCL + 1% Arg was significantly slower, reaching a maximum level after 72 h of soaking. The resulting materials were hydrophobic. Hemocompatibility testing under static conditions revealed no effect on hemolysis following the addition of arginine and the prolongation of the prothrombin time with the increased addition of arginine, thus exerting an influence on the extrinsic and common pathway of coagulation activation. The results of the dynamic hemocompatibility assessment revealed that the numbers of blood cells and platelets were not affected significantly by the various electrospun samples during incubation. The TAT, β-thromboglobulin and SC5-b9 concentrations were characterized by a moderate increase in the PCL group compared to those of the control group. The presence of arginine resulted in a decrease in the investigated hemocompatibility markers. The PMN elastase levels were comparable with respect to all the groups.

• MeSH
• arginin chemie   MeSH
• biokompatibilní materiály chemie   MeSH
• elektřina MeSH
• hemolýza * MeSH
• hojení ran * MeSH
• lidé MeSH
• nanovlákna chemie   MeSH
• polyestery chemie   MeSH
• protrombinový čas MeSH
• testování materiálů metody   MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH