Supercritical carbon dioxide-based sterilization of decellularized heart valves

Abstract

Objective: The goal of this research project encompasses finding the most efficient and effective method of decellularized tissue sterilization.

Background: Aortic tissue grafts have been utilized to repair damaged or diseased valves. Although, the tissues for grafting are collected aseptically, it does not eradicate the risk of contamination nor disease transfer. Thus, sterilization of grafts is mandatory. Several techniques have been applied to sterilize grafts; however, each technique shows drawbacks. In this study, we compared several sterilization techniques: supercritical carbon dioxide, electrolyzed water, gamma radiation, ethanol-peracetic acid, and hydrogen peroxide for impact on the sterility and mechanical integrity of porcine decellularized aortic valves.

Methods: Valve sterility was characterized by histology, microbe culture, and electron microscopy. Uniaxial tensile testing was conducted on the valve cusps along their circumferential orientation to study these sterilization techniques on their integrity.

Results: Ethanol-peracetic acid and supercritical carbon dioxide treated valves were found to be sterile. The tensile strength of supercritical carbon dioxide treated valves (4.28 ± 0.22 MPa) was higher to those valves treated with electrolyzed water, gamma radiation, ethanol-peracetic acid and hydrogen peroxide (1.02 ± 0.15, 1.25 ± 0.25, 3.53 ± 0.41 and 0.37 ± 0.04 MPa, respectively).

Conclusions: Superior sterility and integrity were found in the decellularized porcine aortic valves with supercritical carbon dioxide sterilization. This sterilization technique may hold promise for other decellularized soft tissues.

Summary: Sterilization of grafts is essential. Supercritical carbon dioxide, electrolyzed water, gamma radiation, ethanol-peracetic acid, and hydrogen peroxide techniques were compared for impact on sterility and mechanical integrity of porcine decellularized aortic valves. Ethanol-peracetic acid and supercritical carbon dioxide treated valves were found to be sterile using histology, microbe culture and electron microscopy assays. The cusp tensile properties of supercritical carbon dioxide treated valves were higher compared to valves treated with other techniques. Superior sterility and integrity was found in the decellularized valves treated with supercritical carbon dioxide sterilization. This sterilization technique may hold promise for other decellularized soft tissues.

Keywords: Decontamination; decellularized; heart valve; tensile properties; tissue engineering.

Graphical abstract

Figure 1

Schematic Diagram of a Sectioned Aortic Valve Containing Cusps and Sinuses Used with permission of Mayo Foundation for Medical Education and Research, all rights reserved.

Figure 2

H&E-Stained Image and SEM Image of Sinus of a Native Aortic Valve (A) In hematoxylin and eosin (H&E) staining, nuclei are seen as blue/purple. Collagen fibrils are seen as pale pink. Scale: 5 μm. (B) Collagen fibrils were aligned and dense as shown in the scanning electron microscopy (SEM) image. Scale = 8 μm.

Figure 3

Decellularized Aortic Valve Samples Stained With H&E, B&B, and PAS for Each Sterilization Technique In hematoxylin and eosin (H&E) staining, no blue/purple-colored nuclei are seen. Collagen fibrils are seen as pale pink. In Brown and Brenn (B&B) staining, gram-positive bacteria are seen as blue. In Periodic acid-Schiff (PAS) staining, the fungus wall is seen as magenta. Arrows show the gram-positive+ bacteria. Scale = 10 μm. Decell = decellularized; Dohman = Dohman technique (Dohman et al. [27]); EOW = electrolyzed water; LHP = 6% liquid hydrogen peroxide; scCO₂ = supercritical carbon dioxide.

Figure 4

SEM Images of Sinuses After Sterilization by Various Techniques (A) Nonsterile decellularized sinuses showed the presence of microbes (arrows). (B) Sterilization with the EOW technique could not remove the microbes (arrows). (C) Microbes (arrows) were evident after sterilization with gamma radiation. (D) No microbes were found after sterilization with the EPTA technique. (E) Presence of microbes (arrows) proved that the LHP technique could not sterilize sinus tissue completely. (F) Sinus tissues were completely sterile because no microbes were present after sterilization with the scCO₂ technique. Scale = 12 μm. Abbreviations as in Figures 2 and 3.

Figure 5

Mechanical Integrity of Sterile Valve Cusps (A) Stiffness of the sterile valve leaflets was compared with that of non-sterile decellularized valve leaflet. Stiffness of native valve leaflet (∗p < 0.0001) and LHP treated leaflet (∗p = 0.002) were significantly different from decellularized leaflet by 1-way ANOVA. (B) Ultimate tensile strength (UTS) of the sterile valve leaflets was compared with that of non-sterile decellularized valve leaflet. UTS of native valve leaflet (∗p < 0.0001), ETPA treated (∗p < 0.0001), LHP treated (∗p = 0.0001), and scCO₂ treated leaflets (∗p < 0.0001) were significantly different from decellularized leaflet by 1-way ANOVA. ETPA = 96% ethanol with 2% peracetic acid; other abbreviations as in Figure 3.

Figure 6

DSC Curves of Treated and Nontreated Valve Samples Differential scanning calorimetry (DSC) curves of a leaflet of a native aortic valve (red) and glutaraldehyde-treated (Glut) (blue) valve samples. DSC curves of a leaflet of 90% ethanol–treated (EtOH) (black), PAA-treated (green), and scCO₂-treated (purple) valve samples. The treatments were performed on decellularized valves. Abbreviations as in Figure 3.