ABC | Volume 113, Nº1, July 2019

Original Article Wollmann et al Characterization of decellularized pericardium Arq Bras Cardiol. 2019; 113(1):11-17 a biomaterial remains elusive. 18 Several procedures of decellularization has been described, but this study evaluated low concentration of detergent. The aim of this study was to evaluate the effectiveness of cell removal, maintenance of extracellular matrix properties and mechanical integrity of decellularized human pericardium using a low concentration solution of sodium dodecyl sulfate. Methods All procedures followed the Brazilian National Regulations and Guidelines according to Regulatory Ordinance nº 2.600, published on October 21, 2009 and ANVISA Resolution - RDC n° 55, published on December 11, 2015. This study was approved by the Ethical Committee of Pontifícia Universidade Católica do Paraná (approval number 1.455.773). Donors Pericardium from 14 non heart-beating and heart-beating donors were obtained by convenience sampling under aseptic conditions. After heart explantation, the organ was placed in a tub with saline solution at 2-8 ºC and the intracavitary blood clots washed. The organ was placed in a sterilized plastic bag, immersed in ice-cold isotonic solution in sufficient quantity to maintain the organ totally immersed. This packaging was sealed, then packaged in two more plastic bags (configuring triple packaging), as each bag must be properly tied or sealed. The triple package containing the organ was placed in an airtight container and placed inside the thermal box in the middle of ice and transported to the human tissue bank (HTB). Dissection was carried out within 48 h after cardiac arrest. Tissue underwent dissection to remove connective tissue and the pericardial sac was cut into strips individually stored in saline solution at 2-8 ºC. Blood samples were tested for HIV, HTLV I/II, HBV, HCV, syphilis, cytomegalovirus (IgM and IgG), toxoplasmosis (IgM and IgG) and Chagas’ disease. Donors data were retrieved from their medical records. Positive serology was criteria for rejection except toxoplasma IgG and cytomegalovirus IgG. Microbiological control Before tissue preparation, 30 mL of transport solution (sterile NaCl 0.9% solution) were obtained aseptically in a Class II-A laminar airflow cabinet with a syringe and distributed equally over to a recipient with 90 mL of thioglycolate (Laborclin, Pinhais, Brazil), 90 mL of tryptic soy broth (Laborclin, Pinhais, Brazil) and 90 mL of Sabouraud (Laborclin, Pinhais, Brazil) for bacteriological and mycological testing. The samples were cultured for 14 days at 35°C, 22°C and 22°C, respectively. Cultures were examined daily for visual evidence of turbidity. The ones that showed microorganisms growth were identified for genus and species level. Tissue preparation and decontamination Adherent adipose tissue was dissected with the aid of scissors and forceps. The pericardium was divided into two portions to characterize fresh pericardium versus decellularized pericardium. The pericardia were decontaminated in a RPMI 1640 (Sigma-Aldrich, St. Louis, USA) medium with antibiotics (240 µg/mL cefoxitin, 50 µg/mL vancomycin, 120 µg/mL lincomycin and 100 µg/mL polymyxin B) and were kept between 24 and 48 hours at 2-8°C. Persistent positive cultures after decontamination were used as exclusion criteria. Decellularization Briefly, the pericardia were treated under shaking conditions with a solution of 0.1% sodium dodecyl sulfate (SDS) (w/v) (Sigma-Aldrich, St. Louis, USA) and 7 mM ethylenediaminetetraacetic acid (Sigma-Aldrich, St. Louis, USA) for 24 hours at room temperature. Then, they were washed with 70% ethanol (v/v) for 24 hours at room temperature followed by 10 days of washing with sodium chloride (NaCl) 0.9% solution (Baxter International, Deerfield, USA) to remove residual substances and cellular debris. Histological analysis Longitudinal 3.0 mm cuts with were fixed with neutral buffered formaline 10%, subsequently embedded in paraffin and 4-μm thick sections were taken. Pericardial morphological integrity was analyzed with hematoxylin and eosin (H&E) and Russell-Movat pentachrome (RMP) dyes using an optical microscope BX51 (Olympus Tokyo, Japan). Fresh pericardium was used as control to evaluate changes caused by decellularization. Slides were scanned using an Axio Scan.Z1 slide scanner (Carl Zeiss Microscopy GmbH, Jena, Germany) and subsequent image preparation was performed using software Zen lite (Carl Zeiss, Jena, Germany). DNA extraction and quantification DNAquantificationwas used to determine the total remaining DNA by comparing the levels in fresh and decellularized pericardium. Tissue samples were weighted, purified and rehydrated using a QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, Valencia, USA) according to the recommended protocol. The concentration of extracted DNA was determined using a Nanodrop spectrophotometer (ThermoFisher Scientific, Wilmington, USA). Extracted DNA sample (1µL) was loaded onto the Nanodrop and the absorbance was determined at 260 nm. Four readings were taken for each sample and the mean was considered as the absorbance of the sample. DNA concentrationwas calculated and expressed as micrograms per milligram of dry tissue. Biochemical Characterization Sulfated Glycosaminoglycans (sGAG) The pericardium was digested with papain solution at 60°C for 6 h. Papain (Sigma, St. Louis, MO) was dissolved at 400 mg/mL in 0.1 M phosphate buffer (pH 6.0) with 5 mM cysteine hydrochloride and 5 mM EDTA. The lysates were used for detection of the sGAG amount. The amount of sGAG was measured using a Blyscan sGAG assay kit (Biocolor, Newtownabbey, UK) according to the manufacturer’s manual. The tissue lysate was mixed with Blyscan dye to 12