ABC | Volume 113, Nº1, July 2019

Original Article Wollmann et al Characterization of decellularized pericardium Arq Bras Cardiol. 2019; 113(1):11-17 bind the GAG. The GAG-dye complex was then collected by centrifugation. Subsequently, the supernatant was removed and the tube drained, and the dissociation reagent was added. Then solution was transferred into a 96-well plate. Absorbance against the background control was obtained at a wavelength of 656 nm on a Versamax spectrophotometer (Molecular Devices, Sunnyvale, USA). The sGAG amount was calculated based on a standard curve obtained with the standard sGAG supplied with the kit. Collagen Collagen contents in the pericardium were determined using the Sircol collagen assay (Biocolor, Newtownabbey, UK) according to the manufacturer’s instructions. The pericardia were incubated for 48 h at 48°C in 0.5 M acetic acid containing 0.1 mg/ml pepsin. The samples were added to Sircol dye reagent, and collagen-dye complexes formed and precipitated out from the soluble unbound dye. After centrifugation, the pellet was washed once with Acid-Salt Wash Reagent and suspended in alkali reagent. The solution was transferred to a 96-well plate and the absorbance was read at 550 nm on a Versamax spectrophotometer (Molecular Devices, Sunnyvale, USA). The amount of collagen was calculated based on a standard curve obtained with standard bovine type I collagen supplied with the kit. Cytotoxicity Assay Cytotoxicity of decellularized pericardiumwas performed in accordance with ISO 10993/5. L929 fibroblasts (ATCC cell line CCL 1, NCTC clone 929) were seeded in 6-well plates (Nest) at a density of 1x10 5 cells/well. Each well, with inner diameter of 35 mm, received 4 mL of DMEM + 2mM Glutamine + 10% Fetal Bovine Serum. The plates were incubated for 48 h at 37ºC in an atmosphere of 5% ± 0.5% CO 2 in the air, until a monolayer, with greater than 80% confluence. The medium was removed from the wells and replaced with agar 1.8% added to neutral red dye 0.01%. Samples in triplicate, positive, negative and blank controls on the agar overlay featured 6-well plates. After an incubation period of 24 h at 37 ºC in a humidified atmosphere of 5%± 0.5% CO 2 in air, the inhibition zone around the samples was measured, as neutral red is free from dead cells. The biological reactivity of the samples was scored from 0 to 4 in accordance with the following criteria: 0 = absent cytotoxicity (absence of a zone of lysis underneath the sample); 1= slight (zones of cell lysis underneath the sample only); 2 = mild (zones of cell lysis ≤5 mm from the sample); 3 = moderate (zones of cell lysis>5 mm and ≤10 mm from the sample); 4 = severe (zones of cell lysis >10 mm, but not involving the entire well). In addition, the wells were examined with an inverted microscope (Nikon Eclipse TS 100; Nikon) to observe cell changes. Biomechanical Assay The biomechanical assay of human pericardium was performed with tensile test to compare the decellularization effects on tissue properties of tensile strength, elongation and elastic modulus. An EMIC DL 500 universal testing machine was used to perform the tests. The sample quantity of fresh and decellularized tissue used was 8 and 6 respectively, and collagen fibers traction direction was random. The dumbbell‑shaped sample was based on the ASTMD1708-13 standard because of the small-size tissue available to the tests. To obtain the samples, it was necessary to manufacture a steel rule die according to standard specifications, which was used to cut the pericardium. Before each test, sample thickness, width and initial length values were provided to the equipment. Thickness and width are necessary to calculate the cross-sectional area and consequently the stress (equation 1). Initial length is necessary to calculate the strain and consequently the elongation percentage (equation 2). Thickness was measured with a thickness gauge at three points of the tissue. Pre-load of 0.1 N and 5 mm/min velocity were applied to the test. Equation 1. Stress calculation. σ = F/A where: σ is the stress in megapascal [MPa]; F is the force in newton [N] and A is the cross-sectional area in square millimeter [mm²]. Equation 2. Calculation of elongation percentage. %El=(ΔL/Li) x 100 = ε x 100 where: %El is the elongation percentage; ΔL is the variation of sample length in millimeters [mm]; Li is the initial sample length in millimeters [mm] and ε is the strain in millimeters per millimeter [mm/mm]. Statistical analyses Continuous variables with normal distribution were expressed as mean ± SD and median and interquartile range for non-normal distribution, while categorical variables were expressed as frequencies or percentages. Mann-Whitney was used for non-normal distribution of continuous variables, t-test for normal distribution of continuous variables. All tests were two-tailed, and p value < 0.05 was considered significant. Statistical analyses were carried out using SPSS 23.0. Results Donors’ characteristics The mean age was 39 ± 14 years ranging between 17 and 59 years old. Regarding race, among the samples used, there was a preponderance of donors declared white, 86% (n = 12) of the total and 7% (n = 1) declared themselves as Afro-descendants. An additional 7% (n = 1) was not recorded in the forms. Microbiological assessment Transport solution was positive in 5 samples from 14. Corynebacterium spp . , Streptococcus salivarius , Staphylococcus 13