SYNERGISTIC INHIBITION OF THE CALCIFICATION OF GLUTARALDEHYDE PRETREATED BOVINE PERICARDIUM IN A RAT SUBDERMAL MODEL BY FECL3 AND ETHANEHYDROXYDIPHOSPHONATE - PREINCUBATION AND POLYMERIC CONTROLLED-RELEASE STUDIES

Calcification is a frequent cause of the clinical failure of bioprosth etic heart valves fabricated from glutaraldehyde-pretreated porcine ao rtic valves or glutaraldehyde-pretreated bovine pericardium (GPBP). We investigated the hypothesis that ferric chloride (FeCl3) and sodium-e thanehydroxydiphosphonate (EHDP) may act synergistically to prevent bi oprosthetic tissue calcification. Pre-incubations and controlled relea se systems were studied individually. FeCl3-EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated f or in vitro release kinetics at pH 7.4 and 37-degrees-C. The effects o f Fe-EHDP synergism on GPBP calcification were investigated with 21 d subdermal implants in 3 wk-old male rats. Results demonstrated that le vels of Fe3+ and EHDP uptake, measured in GPBP tissues pre-incubated f irst in an FeCl3 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 FeCl3 or Na2 EHDP 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) m) was combined with Na2EHDP (second pre-incubation, 0.1 m) (1.78 +/- 0.2 mug of Ca2+/mg o f dried tissue) compared with the other pre-incubation groups: EHDP (f irst preincubation) combined with FeCl3 (Second pre-incubation) (21.7 +/- 6.4), FeCl3 solution alone at 10(-5) m (27.9 +/- 10.7), Na2EHDP so lution 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-implante d with individual controlled release systems containing one of the fol lowing 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 matric es were the most effective for inhibiting GPBP mineralization (13.7 +/ - 3.0 mug Ca2+/Mg of dried tissue) compared with non-drug silicone co- implant controls (74.7 +/- 5.58 mug Ca2+/Mg of dried tissue) and other polymeric treatment groups (32.3 +/- 2.3-80.0 +/- 19.7). No adverse e ffects on bone or overall growth of any treatment protocols were noted . Thus, combinations of FeCl3 and EHDP, using either pre-incubations o r polymeric controlled release, were synergistic for inhibiting GPBP c alcification.