Synergistic inhibition of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model by FeCl 3 and ethanehydroxydiphosphonate: pre-incubation and polymeric controlled release studies

Abstract

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves or glutaraldehyde-pretreated bovine pericardium (GPBP). We investigated the hypothesis that ferric chloride (FeCl 3) and sodiumethanehydroxydiphosphonate (EHDP) may act synergistically to prevent bioprosthetic tissue calcification. Pre-incubations and controlled release systems were studied individually. FeCl 3-EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated for in vitro release kinetics at pH 7.4 and 37°C. The effects of Fe-EHDP synergism on GPBP calcification were investigated with 21 d subdermal implants in 3 wk-old male rats. Results demonstrated that levels of Fe 3+ and EHDP uptake, measured in GPBP tissues pre-incubated first in an FeCl 3 solution (10 −5 m) followed by an EHDP solution (0.1 m), were higher than in the reverse order of incubation. In the first series of rat implants, GPBP was pre-incubated in either FeCl 3 or Na 2EHDP solutions, or sequential pre-incubations of first FeCl 3 and then Na 2EHDP solutions, or the reverse. The inhibition of calcification was greatest when FeCl 3 (first preincubation, 10 −5 m) was combined with Na 2EHDP (second pre-incubation, 0.1 m) (1.78 ± 0.2 μg of Ca 2+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first preincubation) combined with FeCl 3 (second pre-incubation) (21.7 ± 6.4), FeCl 3 solution alone at 10 −5 m (27.9 ± 10.7), Na 2EHDP solution alone at 0.1 m (52.3 ± 11.9) and the control group (72.3 ± 10.2). In a second series of implants, GPBP specimens were co-implanted with individual controlled release systems containing one of the following formulations (weight percentage in silicone rubber): 1% FeCl 3, 20% CaEHDP, 20% protamine sulphate, 1% FeCl 3-20% CaEHDP, and 1% FeCl 3-20% protamine sulphate. The 1% FeCl 3-20% CaEHDP silicone-rubber matrices were the most effective for inhibiting GPBP mineralization (13.7 ± 3.0 μg Ca 2+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 ± 5.58 μg Ca 2+/mg of dried tissue) and other polymeric treatment groups (32.3 ± 2.3–80.0 ± 19.7). No adverse effects on bone or overall growth of any treatment protocols were noted. Thus, combinations of FeCl 3 and EHDP, using either pre-incubations or polymeric controlled release, were synergistic for inhibiting GPBP calcification.