Browsing by Subject "Aortic valves"

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    Malachite green and phthalocyanine-silver reactions reveal acidic phospholipid involvement in calcification of porcine aortic valves in rat subdermal model
    (Murcia : F. Hernández, 2003) Ortolani, F.; Petrelli, L.; Nori, S.L.; Gerosa, G.; Spina, M.; Marchini, M.
    Subdermal implant models are helpful in the study of calcification "in vivo” and for testing anticalcific treatments. After implantation of porcine aortic valve leaflets in rat subcutis, we previously found that glutaraldehyde-Cuprolinic blue reactions (GA-CB) at low pH induce favourable tissue unmasking from mineral deposits, and visualize peculiar, electrondense layers that outline the calcifying cells and matrix vesiclelike structures. The layer-forming material seemed to consist of acidic phospholipids because of its anionic nature and differential susceptibility to chemical/enzymatic extractivity. In the present investigation, pre-embedding glutaraldehyde-Malachite green (GA-MG) reactions and subsequent osmium postfixation were compared with pre-embedding GA-CB reactions, combined with post-embedding von Kossa silver staining (GA-CB-S), to assess whether the layerforming material is actually composed of acidic phospholipids and exhibits calcium-binding properties. After lowering standard pH, GA-MG reactions also caused sample demineralization and the appearance of pericellular osmium-MG-reactive layers comparable to CB-reactive ones. Moreover, GA-CB-S reactions showed that major silver precipitation was superimposed to the CB-reactive layers, whereas minor metal extraprecipitation occurred at three distinct, additional sites. These results demonstrate that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface, replacing cell membrane and acting as major apatite nucleator. However, the overall observations are consistent with the hypothesis that certain phases are common to the various types of normal and/or abnormal calcification.
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    Ultrastructural characterization of calcification onset and progression in subdermally implanted aortic valves. Histochemical and spectrometric data
    (Murcia : F. Hernández, 2007) Ortolani, F.; Bonetti, A.; Tubaro, F.; Petrelli, L.; Contin, M.; Nori, S.L.; Spina, M.; Marchini, M.
    Detailed characterization of the subdermal model is a significant tool for better understanding of calcification mechanisms occurring in heart valves. In previous ultrastructural investigation on six-weekimplantated aortic valve leaflets, modified preembedding glutaraldehyde-cuprolinic-blue reactions (GA-CB) enabled sample decalcification with concurrent retention/staining of lipid-containing polyanionic material, which lined cells and cell-derived matrix-vesicle-like bodies (phthalocyanin-positive layers: PPLs) co-localizing with the earliest apatite nucleation sites. Additional post-embedding silver staining (GA-CB-S) revealed PPLs to contain calciumbinding sites. This investigation concerns valve leaflets subjected to shorter implantation times to shed light on the modifications associated with PPLs generation and calcification onset/progression. Spectrometric estimations revealed time-dependent calcium increase, for unreacted samples, and copper modifications indicating an increase in acidic, non-glycanic material, for GA-CB-reacted samples. Two-day-implant thin sections showed emission and subsequent reabsorption of lamellipodium-like protrusions by cells, originating ECM-containing vacuoles, and/or degeneration stages characterized by the appearance of GA-CB-S-reactive, organule-derived dense bodies and progressive dissolution of all cell membranes. In one-week-implants, the first PPL-lined cells were found to co-exist with cells where GA-CB-S-reactive material accumulated, or exudated towards their edges, or outcropped at the ECM milieu, so acquiring PPL features. PPL-derived material was observed increasingly to affect the ECM on thin sections of one-week- to six-week-implants. These results show an endogenous source for PPLs and reveal that a peculiar cascade of cell degenerative steps is associated with valve mineralization in the subdermal model, providing new useful parameters for more reliable comparison of this experimental calcification process versus the physiological and pathological processes.