Effects of glutaraldehyde concentration, pretreatment time, and type of tissue (porcine versus bovine) on postimplantation calcification

Abstract

Our objective was to evaluate the effects of glutaraldehyde (GA) concentration, time of pretreatment, and type of tissue (porcine vs bovine) on quantitative and qualitative postimplant calcification of tissues. Freshly obtained porcine and bovine pericardial tissues were each treated with increasing concentrations of GA (controls, 0.3125%, 0.625%, and 1.2%) for a fixed time (15 minutes) or increasing exposure times (5, 10, 20, and 30 minutes) at a fixed concentration of GA (0.625%). Pretreated tissues were subcutaneously implanted in 10-week-old Sprague-Dawley rats for 45 days before explantation. Quantitative calcium analysis was performed by flame atomic spectrophotometry. Histologic examination of tissue samples with hematoxylin and eosin and von Kossa staining was performed for cellular and inflammatory response, autolysis, and calcification. Two-way analysis of variance indicated significantly greater calcium levels at 1.2% compared with each lower concentration for both porcine and bovine samples (P<.01). Significantly lower calcium levels were detected with increased exposure time in porcine samples (F=6.97; P<.001); however, no significant differences in calcium levels were observed between different exposure times for bovine samples (F=1.46; P=.23). Histologic evidence of inflammatory response with infiltration with mononuclear cells, fibroblasts, and histiocytes was seen in all grafts; however, it varied from mild to severe without any pattern. There were no differences in degree or pattern of inflammatory response according to GA concentration or time of exposure. Estimation of amount of calcification by histologic examination correlated with the quantitative assay. Increasing GA concentration leads to greater calcification with a sharp rise in calcium levels abovea concentration of 0.625%, in both bovine and porcine pericardial tissues. At a concentration of 0.625%, increasing pretreatment time is inversely related to tissue calcification for porcine pericardium but not for bovine pericardium. Differences in the tissue composition in terms of cellular content and composition of the extracellular matrix could account for the observed findings.