Synergistic inhibition of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model by FeCl3 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 (FeCl3) and sodiumethanehydroxydiphosphonate (EHDP) may act synergistically to prevent bioprosthetic tissue calcification. Pre-incubations and controlled release systems were studied individually. FeCl3-EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated for in vitro release kinetics at pH 7.4 and 37[deg]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 Fe3+ and EHDP uptake, measured in GPBP tissues pre-incubated first in an FeCl3 solution (10-5 ) followed by an EHDP solution (0.1 ), were higher than in the reverse order of incubation. In the first series of rat implants, GPBP was pre-incubated in either FeCl3 or Na2EHDP solutions, or sequential pre-incubations of first FeCl3 and then Na2EHDP solutions, or the reverse. The inhibition of calcification was greatest when FeCl3 (first preincubation, 10-5 ) was combined with Na2EHDP (second pre-incubation, 0.1 ) (1.78 +/- 0.2 [mu]g of Ca2+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first preincubation) combined with FeCl3 (second pre-incubation) (21.7 +/- 6.4), FeCl3 solution alone at 10-5 (27.9 +/- 10.7), Na2EHDP solution alone at 0.1 (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% FeCl3, 20% CaEHDP, 20% protamine sulphate, 1% FeCl3-20% CaEHDP, and 1% FeCl3-20% protamine sulphate. The 1% FeCl3-20% CaEHDP silicone-rubber matrices were the most effective for inhibiting GPBP mineralization (13.7 +/- 3.0 [mu]g Ca2+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 +/- 5.58 [mu]g Ca2+/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 FeCl3 and EHDP, using either pre-incubations or polymeric controlled release, were synergistic for inhibiting GPBP calcification.