ABC | Volume 113, Nº1, July 2019

Original Article Wollmann et al Characterization of decellularized pericardium Arq Bras Cardiol. 2019; 113(1):11-17 Figure 1 – Histological characterization of fresh and decellularized human pericardium. H&E and DAPI staining showed no evidence of cell in the tissue sections (Dand E). H&E and RPM staining showed that decellularization did not affect the structure of collagen bundles. Howevers there was a reduction of collagen and increased thickness of the decellularized tissue compared with the fresh one (D and F). aureus , Klebsiella oxytoca , Anaerococcus prevotii and Leuconostoc pseudomesenteroides were the bacteria identified. Other microbiological tests after decontamination and decellularization were negative. Analysis of extracellular matrix integrity and cell removal H&E and DAPI staining revealed a reduction in visible nuclei present in pericardium tissue after decellularization (Figure 1A, B, D and E). The main extracellular matrix components were examined in RMP staining (collagen - yellow, elastin - black, and glycosaminoglycans – blue). The structure of the decellularized pericardium extracellular fibers retained collagen and elastin bundles similar to fresh pericardium. However, there was increased thickness of the decellularized tissue compared with the fresh one (Figure 1 C and F). DNA quantification Fresh pericardium tissue with complete cellular contents had a DNA amount as high as 1591±726 ng per mg dry weight. The DNA contents of the decellularized pericardium were significantly reduced to less than 511.23±120.4 ng per mg dry weight. (p < 0.001) (table 1). Effect of decellularization on sulfated glycosaminoglycan and collagen content The concentration of collagen per mg of dry weight of human pericardium tissue before and after decellularization was 126.9 ± 45.8 and 138.3 ± 58.6 µg/mg of dry tissue, respectively (p = 0.716). Quantification of collagen content in fresh pericardiumdemonstrated a small increase in the collagen content of the tissue after decellularization. sGAG content was determined to be 0.64±0.50 and 0.02 ± 0.06 sGAG per mg of dry tissue, respectively. (p = 0.014) Cytotoxicity assay Decellularized human pericardium (n = 3) was incubated for 24 h (Figure 2A). The samples from the decellularized pericardium had no effect on the cells, as indicated by their normal cell morphology after incubation for 24 h. The negative control also had no effect on the cells (Figure 2B). In contrast, latex (positive control) caused cell death (Figure 2C). Biomechanical Assay Average human pericardium thickness showed no significant difference (Welch’s t test: p = 0.9518), which was 0.45 ± 0.06 mm for fresh tissue and 0.44 ± 0.05 mm for the decellularized tissue. Tensile strength, elongation and elastic modulus are presented as column scatter plot graphs (Figure 3A, B and C) showing the mean and standard deviation. The mean difference among the groups was not significant for all properties (p > 0.05). The average stress‑strain curve of fresh and decellularized tissue are presented in Figure 3D, which shows the typical behavior for biological tissues as previously described. 20 It is also possible to observe that the behavior of curves was similar in the initial phase until transition phase, keeping approximate parallels until maximum stress. Discussion The aim of decellularization is to minimize the immune response by completely removing cellular components as well as preserving the physical characteristics of the extracellular matrix. 2,21 The ideal decellularized tissue must achieve complete decellularization and preservation of the extracellular matrix for biomechanical stability and function, but it is recognized that any decellularization protocol may result in extracellular matrix disruption andpotential loss of surface structure and composition. 2 14