2.5. Platelet Adhesion Test

Platelet adhesion testing was carried out according to ISO 10993-4 standards. Platelet adhesion, aggregation, and activation response of native, decellularized, and composite scaffolds were investigated in thrombogenicity studies. Briefly, anticoagulated sheep blood obtained from the slaughterhouse was quickly delivered to the laboratory in blood tubes mixed with 3.8% (w/v) sodium citrate solution at a ratio of 9:1. Anticoagulated blood was centrifuged at 1700 rpm for 10 min to obtain platelet-rich plasma (PRP). Platelet concentration in the plasma was determined to be approximately 2 × 10⁸/mL. 500 μL of platelet-rich plasma was added to the samples placed in a 24-well plate and incubated at 37 °C, 100 rpm in an orbital shaker for 1 h. The samples were washed with PBS twice to remove nonadherent platelets followed by the samples being fixed with 2.5% (v/v) glutaraldehyde/PBS solution at 4 °C. After the samples were rinsed with PBS, they were dehydrated with a series of graded alcohol solutions (0%, 25%, 50%, 75%, and 100% (v/v)) for 10 min. Finally, the samples were lyophilized, coated with gold–palladium (Au–Pd), and examined by SEM at various magnifications (Tescan, Czechia).

In order to confirm the SEM images and quantify the amount of cell adhesion and viability more accurately on the surfaces of native, decellularized, and composite pericardium that interact with platelet-rich plasma, Calcein-AM staining was also performed. In this staining, living cells are stained green due to intracellular esterase activity and fluorescence. 500 μL of platelet-rich plasma was added to the 1 × 1 cm² samples placed in a 24-well plate and incubated for 1 h at 100 rpm in an orbital shaker at 37 °C. At the end of the incubation, samples were washed with PBS twice to remove nonadherent platelets followed by treatment with 500 μL of Calcein-AM dye (2 μM) and incubation for 30 min. Afterward, the materials were washed twice with PBS and examined under a green filter with a fluorescence microscope (Leica, Germany).