Development of a New Combined Test Setup for Accelerated Dynamic pH-Controlled in vitro Calcification of Porcine Heart Valves

Abstract

Fifty years after their first implantation, bioprosthetic heart valves still suffer from tissue rupture and calcification. Since new bioprostheses exhibit a lower risk of calcification, fast and reliable in vitro methods need to be evaluated for testing the application of new anti-calcification techniques. This report describes a modification of the well-known in vitro dynamic calcification test method (Glasmacher et al, Leibniz University Hannover (LUH)), combined with the pH-controlled, constant solution supersaturation (CSS) method (University of Patras (UP)). The CSS method is based on monitoring the pH of the solution and the addition of calcium and phosphate ion solutions through the implementation of two syringe pumps. The pH and the activities of all ions in the solutions are thus kept constant, resulting in higher calcification rates compared to conventional in vitro methods in which solution supersaturation is allowed to decrease without any further control. To verify this hypothesis, five glutaraldehyde preserved porcine aortic valves were tested. Three of the valves were tested according to a free-drift methodology: the valves were immersed in a supersaturated calcification solution, with an initial total calcium times total phosphate product of (CaxP)=10.5 (mmol/L)² renewed weekly. Two valves were tested by the new pH-controlled loop system, implementing the CSS methodology. All valves were tested for a 4-week period, loaded at 300 cycles per minute, resulting in a total of 12 million cycles at the end of the testing period. The degree of calcification was determined weekly by means of μx-ray, and by conventional, clinical and micro-computer tomography (CT, μCT). The results showed that the valves mineralizing at constant solution supersaturation in vitro yielded higher rates of calcification compared to the valves tested at conditions of decreasing solution supersaturation without any control, indicating the development of a new, accelerated, controllable in vitro calcification method.