Although "in vivo" subdermal model is very usefully to predict tissue propensity to calcification, the inherent calcification mechanism is unclear and the reliability of this "in vivo" simulation is still debated. Samples were obtained from (i) native aortic valve leaflets excised from pig (AVLs), (ii) AVLs undergone pre-implantation treatment alone, including fixation in 0.6% glutaraldehyde under nitrogen atmosphere, (pre-TAVLs), and (iii) AVLs implanted into rat subcutis for 2 days, 2 weeks, and 6 weeks (SI-AVLs). TUNEL-reactivity was found for pre-T-AVLs and all SI-AVLs, and ultrastructural apoptosisrelated features were observed. Immunoreactivity was found for Annexin-V, with diffuse intracytoplasmic distribution, for AVLs, pre-T-AVLs and 2d-SI-AVLs, and pericellular localization, for 2w- and 6w-SI-AVLs. Ultrastructural colocalization was evident between Annexin-V (immunogold labeling) and endogenous acidic lipids clustering at cell surfaces (modified pre-embedding reactions with cuprolinic blue) and acting as apatite nucleators (additional post-embedding von Kossa silver staining). These data indicate that hypoxic/anoxic conditions characterizing the pre-T-step prime uncomplete, apoptotic cell death and a distinct cascade of cell reaction/degradation steps leading to initial apatite precipitation. New parameters are now available for more proper comparison between the experimental calcification occuring in the subdermal model and physiological or pathological calcific processes.
