Prevention of calcification of glutaraldehyde-crosslinked porcine aortic cusps by ethanol preincubation: mechanistic studies of protein structure and water-biomaterial relationships.

Clinical usage of bioprosthetic heart valves (BPHVs) fabricated from glutaraldehyde-pretreated porcine aortic valves is restricted due to calcification-related failure. We previously reported a highly efficacious ethanol pretreatment of BPHVs for the prevention of cuspal calcification. The aim of the present study is to extend our understanding of the material changes brought about by ethanol and the relationship of these material effects to the ethanol pretreatment anticalcification mechanism. Glutaraldehyde-crosslinked porcine aortic valve cusps (control and ethanol-pretreated) were studied for the effects of ethanol on tissue water content and for spin-lattice relaxation times (T1) using solid state proton NMR. Cusp samples were studied for protein conformational changes due to ethanol by ATR-FTIR spectroscopy. The changes in cuspal tissue-cholesterol (in vitro) interactions also were studied. Cusp material stability was assessed in terms of residual glutaraldehyde content and collagenase degradation. Water content of the cusp samples was decreased significantly due to ethanol pretreatment. The cuspal collagen conformational changes (per infrared spectroscopy) brought about by ethanol pretreatment were persistent even after rat subdermal implantation of cusp samples for 7 days. In vitro cholesterol uptake by cusps was greatly reduced as a result of ethanol pretreatment. Ethanol pretreatment of cusps also resulted in increased resistance to collagenase digestion. Cuspal glutaraldehyde content was not changed by ethanol pretreatment. We conclude that ethanol pretreatment of bioprosthetic heart valve cusps causes multi-component effects on the tissue/material and macromolecular characteristics, which partly may explain the ethanol-pretreatment anticalcification mechanism.