Inhibition of CEM calcification by the sequential pretreatment with ethanol and EDTA

The major object of the present study is to optimize the anticalcification activity of ethanol on bioprosthetic heart valve (BHV) calcification. We hypothesize that the chelating agent, in combination with ethanol, will synergistically prevent aortic wall calcification. Collagen-elastin matrix (CEM) was developed as a calcifiable matrix for simulating the calcification process of implantable biomaterials. The efficacy of the combination effects of ethanol and EDTA on the calcification process of CEMs was investigated by implanting them after pretreatment with various conditions of ethanol and EDTA in the rat subdermal model. The relationship between calcium concentrations and pretreatment conditions (a series vs. simultaneous, i.e., first ethanol and then EDTA in water solution, the reverse, or EDTA in ethanol) was established and the optimal condition for prevention of BHV calcification was determined. The mechanistic studies on anticalcification effects exerted by particular pretreatment sequences were also conducted using FTIR and differential scanning calorimetry (DSC). The sequential pretreatment of CEM first with ethanol and then EDTA in water solution significantly decreased the calcification rate of CEM compared the control. The percentage of prevention of calcification by the serial treatment of ethanol (80% v/v) and then EDTA in water solutions decreased, as the concentration of elastin in the CEM increased. The percentage of preventing calcification was 42%, 28.6%, and 22.9% for CEM containing collagen and elastin ratios of 90:10, 50:50, 20:80, respectively. These results indicate that elastin is the major regulatory component of BHV calcification, and preventive effects on calcification increased only when CEM were pretreated with first ethanol and then EDTA in water solution. Moreover, the sequential effect is more apparent in the matrix of less elastin content, which is close to the physiological range. The sequential inhibitory effects of ethanol and EDTA could occur due to the distinct separate actions of each agent, thereby achieving a relatively greater inhibition of calcification.