IDENTIFICATION OF SPECIFIC CALCIUM-BINDING NONCOLLAGENOUS PROTEINS ASSOCIATED WITH GLUTARALDEHYDE-PRESERVED BOVINE PERICARDIUM IN THE RAT SUBDERMAL MODEL
Ta. Gura et al., IDENTIFICATION OF SPECIFIC CALCIUM-BINDING NONCOLLAGENOUS PROTEINS ASSOCIATED WITH GLUTARALDEHYDE-PRESERVED BOVINE PERICARDIUM IN THE RAT SUBDERMAL MODEL, Journal of biomedical materials research, 35(4), 1997, pp. 483-495
Calcification of glutaraldehyde-preserved bioprosthetic heart valves (
BHVs) results in their clinical failure. The mechanism of this patholo
gic calcification is not well. defined. Since serum proteins are known
to be taken up in mineralized tissue, we hypothesized that serum prot
eins derived from several calcium-binding noncollagenous proteins (NCP
s) of bone and teeth also may be associated with pathologically minera
lized BHVs. Using a rat subdermal model of BHV calcification glutarald
ehyde-preserved bovine pericardium (GPBP) was implanted for 1, 3, 14,
and 60 days, and then subjected to an extraction procedure designed to
isolate only NCPs tightly bound to the mineral phase. Gel electrophor
esis and Coomassie Brilliant Blue staining demonstrated that these pro
teins became associated with GPBP over time, paralleling reported calc
ium uptake by the tissue. Stains-All staining demonstrated a marked ac
cumulation of highly acidic, phosphorylated NCPs associated with 60-da
y GPBP extracts. Some of these proteins were detected in rat serum but
were absent from extracts of GPBP incubated in rat serum ipl vitro. W
estern blotting with antibodies to three NCPs found in bone and teeth-
bone acidic glycoprotein 75 (BAG 75), osteopontin, and SPARC-demonstra
ted that these NCPs were tightly bound to the mineral phase of calcifi
ed GPBP. A fourth NCP, bone sialoprotein II (BSP IT) was barely detect
able. Thus each identified NCP showed a different pattern of GPBP asso
ciation relative to mineral deposition, suggesting unique roles for ea
ch in pathologic calcification. SPARC increased within 3 days of GPBP
implantation but decreased by 2 weeks. BAG 75 and osteopontin uptake w
as detected in the initial mineral deposits and increased mineralizati
on proceeded. BSP II never increased significantly over the entire per
iod. Further studies, which should include immunohistochemistry, will
be important for delineating the source, location, and function of the
se three NCPs and for identifying others that also may be involved in
this pathological process. Most important, the new insights into the m
echanism of pathologic calcification described here present exciting o
pportunities for novel approaches to BHV calcification prevention. (C)
1997 John Wiley & Sons, Inc.