Carbodiimide Treatment Dramatically Potentiates the Anticalcific Effect of Alpha-Amino Oleic Acid on Glutaraldehyde-Fixed Aortic Wall Tissue Peter Zilla,

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Carbodiimide Treatment Dramatically Potentiates the Anticalcific Effect of Alpha-Amino Oleic Acid on Glutaraldehyde-Fixed Aortic Wall Tissue Peter Zilla, MD, PhD, Deon Bezuidenhout, PhD, Paul Human, PhD The Annals of Thoracic Surgery Volume 79, Issue 3, Pages 905-910 (March 2005) DOI: 10.1016/j.athoracsur.2003.12.026

Fig 1 Von Kossa/van Gieson stains of aortic wall tissue after 60 day subdermal implantation in the rat (A = glutaraldehyde [GA] fixed/GA stored; B = GA fixed and α-amino oleic acid [AOA] treated/GA stored; C = GA and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) fixed/GA stored; D = GA fixed and AOA treated/EDC stored; E = GA fixed, AOA treated, and EDC fixed/EDC stored). A dramatic shift in calcification pattern is seen through the concomitant presence of AOA and EDC (D, E). While the typical sandwich appearance of GA-fixed aortic wall tissue (A) is only mildly mitigated by AOA treatment (B) or additional EDC fixation (C), the presence of AOA before EDC fixation (D) leads to the formation of well-defined, oval calcium deposits within the media. Further EDC exposure through storage in EDC-based solution (E) practically abolishes the surface bands of calcification. (Original magnification A–D, ×20; E, ×40.) The Annals of Thoracic Surgery 2005 79, 905-910DOI: (10.1016/j.athoracsur.2003.12.026)

Fig 2 Histochemical double staining of calcium deposits and tissue components. A and B: von Kossa and Victoria Blue (Elastin, purple; calcium, dark brown). C and D: von Kossa and Azan (cells, red; collagen, blue; calcium, dark brown). Control tissue fixed and stored in glutaraldehyde shows distinct colocalization of cells and calcium phosphate (D) while elastic lamellae lie apparently unaffected between often heavily calcified tissue (B). In contrast, glutaraldehyde- fixed and α-amino oleic acid-treated tissue that was additionally cross-linked and stored in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide presents an almost inverted image in that, while calcium phosphate is seen to colocalize with elastin (A), both the cellular and the collagenous structures appear calcium free under light microscopy (C). (Original magnification, ×600.) The Annals of Thoracic Surgery 2005 79, 905-910DOI: (10.1016/j.athoracsur.2003.12.026)

Fig 3 Variability plot for tissue calcium levels as determined by atomic absorption spectroscopy. Box whiskers show range of data and box bounds show 25% and 75% quartiles. Enclosed error bars show mean and SEM. Tukey-Kramer honestly significant difference test: * = versus 0.2% GA control; ** = versus Freestyle. (AOA = α-amino-oleic-acid anticalcification process; EDC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA = glutaraldehyde.) The Annals of Thoracic Surgery 2005 79, 905-910DOI: (10.1016/j.athoracsur.2003.12.026)

Fig 4 Reduction of aortic wall calcium levels as a percentage of 0.2% glutaraldehyde (GA-fixed control tissue as determined by atomic absorption spectroscopy. (Freestyle is a registered trademark of Medtronic, Inc, Santa Ana, CA. AOA is a registered trademark of Biomedical Design, Atlanta, GA. Jeffamine is a registered trademark of Huntsman Performance Chemicals, Houston, TX.) The black bars represent the % reduction of calcium relative to the control tissue (represented as 100%). (AOA = α-amino-oleic-acid anticalcification process; EDC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA = glutaraldehyde.) The Annals of Thoracic Surgery 2005 79, 905-910DOI: (10.1016/j.athoracsur.2003.12.026)