Inspect s50b

A total of 4.0 g of bone grafting nanomaterial was weighed and placed in six Petri dishes with a diameter of 6 cm. In order to control the speed of blood clotting, six additional empty Petri dishes were prepared. Whole venous blood (60 mL) was collected from a male volunteer who provided consent for the study. Prior to conducting the study, the Ethics Committee on Medical Research of the Medical Institute of Sumy State University approved the protocol. Next, 5 mL of blood was immediately added to each dish containing the bone grafting nanomaterial. The samples were gently stirred with a glass rod to ensure even distribution of the blood. A timer was started as soon as the blood was added, and it was stopped once a clot had formed. For scanning electron microscopy (SEM) analysis, bone grafting nanomaterial with coagulated blood weighing approximately 0.5 g was fixed in a 2.5% glutaraldehyde solution and then dehydrated in alcohols of increasing concentration for 24 h. Once dry, the samples were covered with a 30–50 nm layer of silver using a vacuum set-up VUP-5M (SELMI, Sumy, Ukraine). The SEM images of the blood clot on the hydroxyapatite were captured using an FEI Inspect S50B (FEI, Brno, Czech Republic) with an Everhart–Thornley secondary electron detector.

The cell morphology and arrangement of ESKAPE pathogens in biofilms were assessed using SEM. Small glass slides (0.5 × 0.5 cm) were placed in 24-well plate wells containing 2 mL Mueller-Hinton broth with bacterial cells at 5 × 10⁵ CFU/mL concentration and incubated for 48 h at 37 °C. Then, samples were divided into three groups. The first and second groups were added with AgNPs-1 and AgNPs-2 diluted in Muller-Hinton broth at 20 µg/mL concentration. The third group was added to Muller-Hinton broth. After that, all samples were incubated for 24 h. Then, the media were discarded. Samples were washed three times with PBS, fixed with glutaraldehyde 2.5% in 0.1 M phosphate buffer (pH 7.2) for 60 min, dehydrated in ethanol-water mixture with increasing ethanol concentrations (65%, 75%, 85%, 95%, and 100%), and air-dried overnight. Dehydrated specimens were coated with a thin film of silver in a sputter coater. Morphological analysis was performed by the examination of the SEM (Inspect S50-B, FEI, Brno, Czech Republic; accelerating voltage −15 kV) images.

The morphology analysis of the obtained coatings was conducted by scanning electron microscopy (SEM, Phenom ProX, Phenom-World BV, Eindhoven, the Netherlands, accelerating voltage = 25 kV). The pore size and pore distribution were described by the Trial Version Image-Pro 10.0.7 software. The elemental composition of the oxide coatings was characterized by an energy-dispersive X-ray spectrometer attached to the SEM.
The oxide layers’ cross-sections were determined to study the morphology and thickness of the PEO coatings. PEO modified samples were embedded in epoxy resin (Eposir F 740 + Ipox EH 2260) and then placed in a vacuum unit for 3 min before drying. Then, all samples were air-dried for 24 h and ground with a grinding machine (Einhell TH-US 400, 1440 rpm) using abrasive papers (Hermes BW114) with 400, 1000, 1200 and 1500 granulation. The morphology and chemical composition of the oxide layer structures were studied using scanning electron microscopy (SEO-SEM Inspect S50-B, FEI, Brno, Czech Republic; accelerating voltage-15 kV) paired with an energy-dispersive X-ray spectrometer (AZtecOne with X-MaxN20, Oxford Instruments plc, Abingdon, UK).