Cellular and Molecular Biology


Ultrastructural characterization of calcification onset and progression in subdermally implanted aortic valves. Histochemical and spectrometric data

F. Ortolani1,2, A. Bonetti1, F. Tubaro3, L. Petrelli1, M. Contin1, S.L. Nori4, M. Spina5 and M. Marchini1,2

1Department of Medical Morphological Research, University of Udine, Udine, Italy, 2Interdepartmental Center for Regenerative Medicine (C.I.M.E.), University of Udine, Udine, Italy, 3Department of Chemical Sciences and Technology, University of Udine, Udine, Italy, 4Institute of Human Anatomy, Catholic University, Rome, Italy and 5Department of Experimental Biomedical Sciences, University of Padua, Padua, Italy

Offprint requests to: Dr. F. Ortolani, Department of Medical Morphological Research, University of Udine, Piazzale Kolbe 3, I-33100, Udine, Italy. e-mail: fulvia.ortolani@uniud.it

Summary. 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-week-implantated aortic valve leaflets, modified pre-embedding 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 calcium-binding 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. Histol Histopathol 22, 261-272 (2007)

Key words: Aortic valve calcification, Ultrastructural cell degeneration, Subdermal model, Cuprolinic blue, von Kossa

DOI: 10.14670/HH-22.261