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.
