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

Abstract

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.

Elastin as a biomaterial for tissue engineering

“…Calcifi cation may be genetically controlled by molecules that actively inhibit calcifi cation and may occur passively when these inhibitors are absent [103]. Calcifi cation can be prevented or reduced by aluminium chloride treatment [104], acyl azide crosslinking [105], ethanol/EDTA treatment [106], and presence of glycosaminoglycans [107]. Elastin can be used in biomaterials in different forms, including insoluble elastin in autografts, allografts, xenografts, decellularised extracellular matrix, and in purifi ed elastin preparations.…”

Elastomeric polypeptide-based biomaterials

“…[25][26][27] However, due to its poorly defined purification, batchto-batch variations, high propensity to calcify upon cardiovascular prosthetic implants, 28,29 as well as the fact that cross-linked elastin may serve as a nucleation site for mineralization, 25,30 natural elastin is not used that often as a tissue engineering scaffold. Some of these issues, such as calcification, can be minimized by aluminium chloride treatment, 31 ethanol/EDTA treatment, 32 the presence of glycosaminoglycans, 33 or the presence of basic fibroblast growth factor. 34 Therefore, interest in further understanding the properties of natural elastin and in emulating its highly elastomeric behavior has motivated the investigation of elastin-like polypeptide-based biomaterials.…”

Effects of periodate and chondroitin 4-sulfate on proteoglycan stabilization of ostrich pericardium. Inhibition of calcification in subcutaneous implants in rats

“…However, its use promotes calcification and is potentially cytotoxic. For this reason, alternative stabilizing reagents or procedures are being developed [4,5].…”