Model experimental system for investigation of heart valve calcification in vitro

A model system was developed for the in vitro quantitative investigation of the calcification process occurring in heart valves. The process of heart valve calcification consists of the formation of calcium phosphates at the heart valve-biological fluid interface. Calcium phosphate deposits may consist of more than one calcium phosphate mineral phase, differing with respect to their physical and chemical properties. The kinetics of the formation of hydroxyapatite, the model inorganic compound for the calcified deposits, was precisely monitored in a reactor containing supersaturated calcium phosphate solutions in which the heart valves were immersed after being treated with glutaraldehyde and mounted on special racks. The precipitation process, accompanied with proton release in the solution, was monitored by a pair of glass-saturated calomel electrodes. Upon initiation of the formation of calcium phosphate deposits, the supersaturation in the working solution was reestablished through the addition of titrant solutions made with the appropriate concentration to compensate for the ions precipitated. With this methodology, not only the rates were measured very precisely but also the nucleation capability of the various substrates could be evaluated. Moreover, it was possible to identify the formation of intermediate calcium phosphate phases formed during the calcification process. Valves previously treated with glutaraldehyde were shown to nucleate octacalcium phosphate, which at lower supersaturations converted to the thermodynamically more stable hydroxyapatite. The rates measured were found to depend on the solution supersaturation, while the apparent order of the precipitation process was found to be 1.