Inhibition of aortic wall calcification in bioprosthetic heart valves by ethanol pretreatment: Biochemical and biophysical mechanisms Chi-Hyun Lee, 1 Narendra Vyavahare, 2 Robert Zand, 3 Howard Kruth, 4 Frederick J. Schoen, 5 Richard Bianco, 6 Robert J. Levy 2 1 Department of Pediatrics and Communicable Diseases, The University of Michigan, Ann Arbor, Michigan 48109 2 The Joseph Stokes, Jr., Research Institute, Children’s Hospital of Philadelphia, Abramson Pediatric Research Center, 1107B, 34th Street and Civic Center Blvd., Philadelphia, Pennsylvania 19104 3 Department of Biochemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109 4 NHLBI, Bethesda, Maryland 5 Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 6 Department of Surgery, University of Minnesota Medical School, Minnesota Received 16 September 1997; 20 February 1998 Abstract: The effectiveness of ethanol pretreatment on pre- venting calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific colla- gen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calci- fication of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80 ± 8.45 g/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90 ± 7.24 g/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circula- tory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02 ± 4.42 g/mg compared to the control calcium level of 56.35 ± 6.14 g/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) stud- ies of ethanol-pretreated BPHV aortic wall (vs. control) dem- onstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T 1 ) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The op- timum condition of 80% ethanol pretreatment almost com- pletely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification oc- curred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effec- tive for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bio- prosthetic aortic wall calcification only in synergistic com- bination with another agent. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 42, 30–37, 1998. INTRODUCTION Calcification is the most frequent cause of the clini- cal failure of bioprosthetic heart valve (BPHV) replace- ments fabricated from porcine aortic valves crosslinked with glutaraldehyde. 1 Calcification refers to the deposition of calcium phosphates and associ- ated minerals in the BPHV tissues. This calcific degen- eration is usually followed by stenosis or cuspal tear- ing with regurgitation, and can result in prosthesis failure. Although calcification is always pathological in the cardiovascular system, calcium phosphate min- eral deposition and resorption occur in the course of normal bone and tooth development and metabolism. Since cell-oriented calcification and collagen calcifica- tion are prominent pathophysiologic features of bio- prosthetic calcification, inhibition of both disease pro- cesses is necessary for therapeutic efficacy. Correspondence to: R. J. Levy Contract grant sponsor: National Institutes of Health; Contract grant number: HL 38118 Contract grant sponsor: The Children’s Hospital of Phila- delphia (to R. J. Levy) © 1998 John Wiley & Sons, Inc. CCC 0021-9304/98/010030-08