Developing a Rat Model of Cardiovascular Calcification to Evaluate Tissue-Engineered Heart Valve Prostheses

Abstract

A small animal model to evaluate the in vivo degeneration and calcification of biological versus tissue-engineered cardiovascular prostheses in short time periods is warranted. Our study aims at developing a standardized rat model of accelerated cardiovascular calcification and lipid metabolism disorder. Male Wistar rats (n = 60; 200 – 250g) were fed ad libitum with 5 different regimens of procalcific diet (group 1: +300,000 units/kg vitamin D (VD) +2% cholesterol (CHOL) +1.5% calciumphosphate (PO4); group 2: +150,000 units/kg VD +1% CHOL +0.75% PO4; group 3: +300,000 units/kg VD +2% CHOL; group 4: +300,000 units/kg VD +1.5% PO4; group 5: +2% CHOL + 1.5% PO4; group 6: normal food). After 4, 8 and 12 weeks, animals were euthanized, organs explanted (left ventricular myocardium, aortic valve, ascending aorta, abdominal aorta, kidney and liver) and histology as well as immunohistochemistry conducted. During the study, body weight and chow intake were monitored. Heart function was examined by echocardiography, and blood serum level analyses were conducted at explantation. Unimpaired survival was 100% in all groups. Histology revealed calcification of the aortic valves after 4 weeks, while relevant calcium deposition in the aortic wall was observed only after week 8 (vonKossa staining). Aortic valves of rats with high doses of VD (groups 1, 3 and 4) were significantly more calcified than those of animals with a reduced dose of VD (group 2; p < 0.01) or no VD supplementation (group 5; p < 0.001). In all rats, early calcium deposition was located at the commissures, whereas the aortic sinus walls and especially the valve leaflets were diseased at later time points. Massive calcification was accompanied by chondroid cells and lipid-containing cells (oil red staining). However, animals on a diet with reduced VD or no VD presented a significantly higher amount of chow intake (each with p < 0.01 versus groups 1, 3, 4 and 6), paralleled by significantly larger increase in body as well as heart weight (each with p < 0.001 versus groups 1, 3, 4 and 6). All supplementation regimens resulted in early aortic valve and later aortic wall calcification. High doses of VD intake accelerated the calcium deposition, however, the somatic growth of these rats was impaired. A procalcific diet with moderate doses of VD + CHOL + PO4 seems to be most suitable for a comparative evaluation of calcifying degeneration in native and prosthetic cardiovascular tissues.