Anti-calcification Effects in Decellularized and Variously Fixed Bovine Pericardium

Abstract

Background: Our goal was to evaluate anti-calcification effects of decellularization and diverse fixing methods including preincubation of the bovine pericardium with ethanol. We also assessed changes in mechanical properties. Material and Method: Harvested bovine pericardium was decellularized with 0.25% sodim dodecysulfate and then treated with 5 methods of fixation: ① 0.5% glutaraldehyde (GA) for 14 days, ② 0.5% GA for 5 days, 2% GA for 2 days and 0.25% GA for 7 days, ③ 0.5% GA for 5 days, 2% GA for 2 days, 0.25% GA for 7 days, and then 70% ethanol for 2 days, ④ 0.5% GA for 5 days, a mixture of 2% GA and 70% ethanol for 2 days, and 0.25% GA for 7 days, ⑤ 0.5% GA for 5 days, a mixture of 2% GA, 65% ethanol, and 5% octanediol for 2 days and then 0.25% GA for 7 days. All treated bovine pericardia were tested for histological variables, lipid content, and mechanical properties including tensile strength and thermal stability. A total 10 kinds of differently treated bovine pericardia were implanted into rat subdermis and harvested 8 weeks later. Harvested pericardia were evaluated for calcium content. Result: No protein denaturation was observed microscopically after decellularization. There was a 32% mean decrease in tensile strength index after decellularization in the bovine pericardium group fixed. Octanediol preincubation attenuated the decrease in tensile strength and maintained thermal stability. TG and cholesterol were not affected by decellularization but were decreased by organic solvent. Calcium content was decreased after decellularization, and organic solvent preincubation decreased calcification in the non-decellularized bovine pericardium group. Conclusion: Decellularization and organic solvent preincubation have anti-calcification effects but decellularization may cause mechanical instability. A method of decellularization and fixation that does not cause damage to matrices will be needed for evaluation of the next step in using tissue-engineering for replacement of cardiac valves.