Decellularization Following Fixation of Explanted Aortic Valves as a Strategy for Preserving Native Mechanical Properties and Function

FIGURE 1. Overall scheme and study design. (A) Porcine aortic valves are explanted and either chemically crosslinked with fixative formaldehyde (fixed), or decellularized with surfactants Triton X-100/sodium deoxycholate (decel) or treated with a combination of fixative and surfactants (fixed-decel) to optimize the biomechanical properties. Digital photos of the cut leaflets show that decel and fixed-decel groups were discolored due to the removal of cell and related membrane proteins by the surfactants. Fixed leaflets appear to be distorted in shape relative to the native valve and the original shape was recovered when fixed leaflets were decellularized (fixed-decel). (B) Digital pictures and a cross-section schematic of an aortic valve showing composition and orientation of collagen and elastin fibers in different layers during systole and diastole. (C) Fourier Transform Infrared Spectroscopy (FTIR) spectra indicate the peak intensity and location of characteristic chemical groups.

FIGURE 2. Surface characterization using scanning electron microscopy (SEM). (A–D) SEM micrographs showing the morphology of leaflets from the surface. (E–H) Micrographs showing cross-sectional morphology of leaflets that have been fixed, decellularized, and a combination thereof including the native control. The scale bar is 10 µm for all.

FIGURE 3. Evaluation of mechanical properties. (A) Set up of uniaxial tensile test performed in the radial direction. (B) Comparison of leaflet thickness and maximum stress at failure (C) Comparison of elastic modulus (i.e., stiffness) and maximum strain at failure. (D) two-dimensional comparison (Elastic modulus vs. ultimate stress at break) of mechanical properties among fixed, decellularized, and a combination thereof, including the native control groups. Data presented as mean ± Std. Dev., n = 3, p-values are calculated using one-way ANOVA with Tukey correction, *p < 0.05 in Origin Pro 2019 (64-bit); ns denotes no statistical difference).

FIGURE 4. Hydrodynamic assessment. (A) Custom-built benchtop setup with a pulsatile mock circulatory loop representing left heart circulation. (B,C) Representative pressure and flow waveforms for native aortic valves at 60 bpm and cardiac output of 3.6 L/min. (D) Representative transvalvular pressure gradients for native valves over a few cardiac cycles. (E) Digital images were captured using an endoscope camera for different groups of aortic valves at peak systole and diastole. (F) Comparison of mean transvalvular pressure gradients (ΔP) among fixed, decellularized, and a combination thereof, including the native and commercially available mechanical (St. Jude) control groups. (G) Comparison of regurgitant flow and opening orifice area at peak systole. Data presented as mean ± Std Dev, p-values are calculated using one-way ANOVA with Tukey correction, *p < 0.05 in Origin Pro 2019 (64-bit), ns denotes no statistical difference. LA = left atrium, LV = left ventricle, MV = mitral valve. Decel = decellularized.