We previously have been able to show that fixation at increasing concentrations of glutaraldehyde (GA) leads to mitigated rather than facilitated tissue calcification. The purpose of the present study was to introduce additional crosslinks and provide evidence that crosslink density may be an underlying inhibitory principle. Entire aortic roots were chosen to verify the concept on the challenging aortic wall tissue. Porcine aortic roots were crosslinked with 0.2% GA, 3%GA, and 3% GA containing an interim step that introduced 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(10)). Calcium levels, calcification patterns, and inflammation were assessed after 6 and 24 weeks of implantation in a sheep circulatory model. Crosslink efficiency in aortic wall tissue was moderately affected by increasing the fixative concentration from 0.2% GA to 3% GA (SrT degrees from 85.7 degrees +/- 0.3 degrees to 87.5 degrees +/- 0.3 degrees C, 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 micromol/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 degrees from 87.5 degrees +/- 0.3 degrees to 93.4 degrees +/- 0.3 degrees C, p << 0.0001; RPD from 29.1 +/- 0.7 to 68.4 +/- 1.8%, p << 0.0001; and E(10) from 2.94 +/- 0.34 to 6.80 +/- 0.61 MPa, p < 0.0003). Aortic wall calcification was reduced significantly by increasing the GA concentration 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 [84.0%, p = 0.006 (6 weeks) and 29.8%, p = 0.037 (24 weeks)]. The combined effect of increased GA concentration plus an interim diamine step on aortic wall tissue resulted in a 90% and 53.7% reduction of calcification after 6 weeks and 24 weeks, respectively. The correlation coefficients between calcification 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 diamine bridges. Our study demonstrated a distinct correlation between the mitigation of aortic wall calcification and three parameters used to assess crosslink density.