Combined anti-calcification treatment of bovine pericardium with amino compounds and solvents

Abstract

The objective of this study was to evaluate the effect of treatment with amino compounds and solvents in vivo on calcifications of glutaraldehyde (GA)-fixed pericardium. Groups of bovine pericardium samples were fixed with 0.5% GA. We used urazole and glutamate to neutralize the free aldehyde and some solvents (ethanol with octanol or octanediol) to reduce the phospholipid content in the bovine pericardial tissue. Tensile strength and thermal stability were evaluated before implantation. Twelve weeks after rat subdermal implantation, the pericardial samples were harvested from eight juvenile rats. Urazole [calcium (Ca(2+)): 11.86 ± 2.85 μg/mg; inorganic phosphorus (IP): 32.59 ± 7.73 μg/mg] or glutamate (Ca(2+): 7.95 ± 1.21 μg/mg; IP: 21.76 ± 3.48 μg/mg) alone significantly decreased the Ca(2+) and IP concentrations (without any anti-calcification treatment, Ca(2+): 277.85 ± 17.51 μg/mg; IP: 147.07 ± 8.32 μg/mg), but when used with organic solvents, the Ca(2+) and IP concentrations were the lowest (Ca(2+): 0.05 ± 0.04 μg/mg; IP: 3.36 ± 0.61 μg/mg). After anti-calcification treatment, the calcifications in microscopic images were dramatically decreased. Anti-calcification treatment with glutamate, urazole, and solvents did not worsen the physical properties of bovine pericardium, and significantly prevented in vivo calcifications compared to GA fixation only. There should be additional studies done to understand the other mechanism underlying xenograft tissue calcification.

Valved Conduit with Glutaraldehyde-Fixed Bovine Pericardium Treated by Anticalcification Protocol

“…Previously, we proved the efficacy of our combined anti-calcification treatments for GA-fixed cardiac xenografts in small-animal experiments [6,8,9]. However, these anti-calcification treatments have not always been safe and efficacious if used on valves placed under in vitro mock circulation or large-animal in vivo circulation [2].…”

“…Our treatment targeted at the prevention of calcification includes the extraction of phospholipids [6,8,9], neutralization of residual unbound aldehyde groups of GA [6,8,9], and decellularization [6,10,11]. We also developed a specially designed mold for the manufacture of the valved conduit [12] and produced a valved conduit with the GA-fixed bovine pericardia treated with our combined anticalcification protocol, which had been proven effective in small-animal experiments [6].…”

Early Results of Novel Bovine Pericardial Patch Using Comprehensive Anticalcification Procedure in a Swine Model

“…We further demonstrated the GA cross-linked bovine pericardium with a combination of short-and long-chain alcohol had a superior anticalcification effect. 6 Our previous studies showed that GA and organic solvent treatment had better mechanical durability than GA treatment alone and did not cause a loss in the tensile strength, elasticity, and thermostability. 18,19 Amino groups can improve protein cross-linking and neutralize toxicity of free aldehyde groups.…”

“…11 Treatment with organic solvents, such as ethanol, octanol, and octanediol, are known to reduce the phospholipid content and prevent calcium phosphate nucleation. 6 We used a short-and long-chain alcohol combination organic solvent. Pathak et al 17 reported that bovine pericardium treated with a short-and long-chain alcohol combination shows a reduction in phospholipid content.…”

“…Therefore, there have been many studies to decrease calcification and improve the durability of xenograft. [2][3][4][5] We have been involved in the study of various issues of xenograft tissue engineering, [6][7][8][9] such as decellularization, space filler treatment, cross-linking, and detoxification. On the basis of these studies, we developed a comprehensive anticalcification procedure and produced novel bovine pericardial patch using this technique.…”