Dynamics of Cryoprotectant Permeation in Porcine Heart Valve Leaflets

Abstract

Valve replacement is a common cardiovascular procedure for the treatment of a variety of congenital and acquired defects. Many surgical programs rely on cryopreserved heart valves from regional tissue bank programs to meet clinical demands. Current cryopreservation strategies for heart valves are empirically derived. The aim of this study was to use proton nuclear magnetic resonance spectroscopy (NMR) to monitor changes in cryoprotectant concentration in isolated heart valve leaflets. Porcine aortic valves were locally obtained, freshly isolated, and allowed to equilibrate at various experimental temperatures (22°C, 10°C, 4°C) for 1 h prior to immersion in 1 M Me 2 SO solution. At defined intervals (0, 0.25, 0.5, 1, 2, 6, and 24 h) the valves were removed from the Me 2 SO and the leaflets were rapidly dissected and equilibrated in deuterium oxide (D 2 O). Using previously described techniques the Me 2 SO concentration in the heart valve leaflets was determined by NMR and the diffusion coefficient was calculated as a function of time and temperature. Heart valve leaflets were fully equilibrated with Me 2 SO after approximately 2 h of exposure at 22°C while equilibrium was not reached >6 h or more at 10°C and 4°C. These results indicate that that permeation of Me 2 SO in heart valves is strongly temperature dependent. Furthermore, this study provides a quantitative measure of Me 2 SO permeation and cryoprotectant at equilibration in heart valve leaflets. The clinical applications of these findings may help to optimize the balance between the protective and toxic effects of cryoprotectants and lead to improved methods of preservation of heart valves.Key words: Cryopreservation; Heart valve; Me 2 SO; Cryoprotectant permeation; Proton NMR spectroscopy INTRODUCTIONthe tissue for a minimal exposure to decrease the effect of toxicity. All cryoprotectants are toxic and increase in toxicity with concentration, time, and temperature of Heart valve replacement is a common cardiovascular procedure for the treatment of a variety of congenital diffusion. Overexposure to cryoprotectants may not only affect tissue viability in the short term, but over an exand acquired defects, but is limited by the supply of tissue for transplantation. As a result, many surgical protended period of time the cryoprotectant may have adverse toxic effects on tissue biological function. grams rely on cryopreserved heart valves from regional tissue banks to meet clinical demands (23). The use of Cryoinjury during cooling is associated with the presence of intracellular ice (12), but the probability of intraMe 2 SO as a cryoprotectant has been shown to be useful in preventing the damage associated with the freezing cellular ice formation can be reduced by increasing the concentration of intracellular solutes. Slow cooling, in process in heart valves and other tissues, and the use of cryopreserved valves for transplantation has been shown the presence of external ice, may allow this as solutes concentrate in the diminishing unfrozen fraction of wain s...

NMR Assessment of Me2SO in Decellularized Cryopreserved Aortic Valve Conduits

“…This finding suggests that the equilibration period for the cryoprotectant may have been too short to permit complete permeation prior to freezing even though we followed the freezing protocol of our local tissue bank. Previous work suggests that 360 min are required to attain greater than 90% of equilibrium diffusion in aortic valve leaflets [13] and conduits [31], respectively. These reports as well as our data presented herein demonstrate that current heart valve cryopreservation protocols may not permit adequate Me 2 SO permeation for optimal tissue preservation.…”

“…1 H-NMR spectroscopy was used to determine Me 2 SO concentration in the aqueous compartment of the tissue as described below [13]. Each tissue sample was placed in 10 mL of 100% methanol.…”

“…Me 2 SO is used as a cryoprotectant to limit the development of ice within tissue during cryopreservation, but may adversely affect tissue viability and biological function [13,14]. Residual Me 2 SO in thawed decellularized tissue may also inhibit repopulation by altering normal function of endothelial cells [15] and fibroblasts [16,17].…”

“…1 H-NMR measures the number of protons for each species and therefore allows for the direct measurement of the Me 2 SO concentration in biological tissues including cornea [24], cartilage [25], and heart valve leaflets [13].…”

MR spectroscopy measurement of the diffusion of dimethyl sulfoxide in articular cartilage and comparison to theoretical predictions

“…Different methods have been employed to characterize cartilage water permeability and the diffusion of solutes, including freezesubstitution 3 , radiotracers 4 and spectroscopic MR 5,6,7,8,9,10,11,12 . All of these studies provide an estimate of the "overall" CPA concentration in the tissue.…”