New insights on aortic valve interstitial cell calcification as revealed by von Kossa silver reactions adapted to electron microscopy

The silver reaction introduced in 1901 by von Kossa (vKr) persists as a method of choice for the visualisation of calcium-binding sites in light microscopy, being based on phosphate- or carbonate-bound calcium ion replacing by silver ions and subsequent reduction to metallic silver.1 Later, procedural adjustments were made to adapt vKr to electron microscopy.2 In previous ultrastructural studies on aortic valve interstitial cell (AVIC) calcification,3,4 a combined procedure consisting in vKr on semithin sections after preembedding phthalocyanine reaction on samples and before semithin re-embedding and conventional contrast of derived thin sections revealed major hydroxyapatite nucleators to consist in acidic phospholipid containing layers (PPLs) edging the degenerating AVICs. Here, analogous combined procedure was employed to assess whether ribosomal RNA (rRNA) and nuclear chromatin contribute to AVIC mineralization in in vitro pro-calcific cell cultures and in vivo aortic valve leaflets undergone experimental or pathological calcification. At early stages, mineralizing AVICs showed superimposition of metallic silver particles on both free and membrane-bound ribosomes. Subsequent melting of ribosomes with PPLs was observed, with the resulting pro-calcific substratum being additionally susceptible to decoration by anti-rRNA immunogold particles. Silver particle deposition onto nuclear chromatin was also found, in absence of apoptotic or oncotic cell death signs. In conclusion, the use of vKr so adapted to electron microscopy enabled the identification of rRNA and nuclear chromatin as additional sites of calcium salt nucleation during AVIC mineralization, providing more information on this type of pro-calcific cell death.