Diamine extension of glutaraldehyde crosslinks mitigates bioprosthetic aortic wall calcification in the sheep model

We previously have been able to show that fixation at increasing concentrat ions of glutaraldehyde (GA) leads to mitigated rather than facilitated tiss ue calcification. The purpose of the present study was to introduce additio nal crosslinks and provide evidence that crosslink density may be an underl ying inhibitory principle. Entire aortic roots were chosen to verify the co ncept on the challenging aortic wall tissue. Porcine aortic roots were cros slinked with 0.2% GA, 3%GA, and 3% GA containing an interim step that intro duced diamine bridges. Crosslink efficiency was determined on the basis of shrinkage temperature (SrT degrees), resistance to protease digestion (RPD) , residual amine analysis (RA), and tensile modulus (E,,,). Calcium levels, calcification patterns, and inflammation were assessed after 6 and 24 week s of implantation in a sheep circulatory model. Crosslink efficiency in aor tic wail tissue was moderately affected by increasing the fixative concentr ation from 0.2% GA to 3% GA (SrT degrees from 85.7 degrees +/- 0.3 degrees to 87.5 degrees +/- 0.3 degreesC, p < 0.002; RPD from 24.2 +/- 1.2 to 29.1 +/- 0.7%, p < 0.003; RA from 0.069 +/- 0.004 to 0.058 +/- 0.003 mu mol/mg, p < 0.03, and E-10, from 1.9 +/- 0.11 to 2.94 +/- 0.34 MPa, p < 0.01), but it was distinctly enhanced when diamine bridges were introduced (SrT degree s from 87.5 degrees +/- 0.3 degrees to 93.4 degrees +/- 0.3 degreesC, p muc h less than 0.0001; RPD from 29.1 +/- 0.7 to 68.4 +/- 1.8%, p much less tha n 0.0001; and E-10 from 2.94 +/- 0.34 to 6.80 +/- 0.61 MPa, p < 0.0003). Ao rtic wall calcification was reduced significantly by increasing the GA conc entration from 0.2 to 3% [37.8%, p = 0.076 (6 weeks) and 34.0%, p = 0.008 ( 24 weeks)] and further reduced by the introduction of additional diamine [8 4.0%, p = 0.006 (6 weeks) and 29.8%, p = 0.037 (24 weeks)]. The combined ef fect of increased GA concentration plus an interim diamine step on aortic w all tissue resulted in a 90% and 53.7% reduction of calcification after 6 w eeks and 24 weeks, respectively. The correlation coefficients between calci fication and SrT degrees, RDP, and E-10 was -0.9767, -0.9460, and -0.9740, respectively (6 weeks). The inflammatory host reaction regularly found in 0 .2%; fixed tissue was practically abolished through the introduction of dia mine bridges. Our study demonstrated a distinct correlation between the mit igation of aortic wall calcification and three parameters used to assess cr osslink density. (C) 2001 John Wiley & Sons, Inc.