steady-state level of expression of RANKL remained unchanged (Wiens et al.
2010c). In controls, the expression of OPG and RANKL changed only
unsignificantly during day 1–7 (Fig. 10.8a). Hence, the ratio of OPG/RANKL
expression markedly increased from day 1 to 7 (Fig. 10.8b). In these experiments,
the expression levels of RANKL and OPG were correlated with the expression of the
housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) which
was used as reference.
The differential expression of OPG and RANKL induced by biosilica could also
be demonstrated on the protein level (Wiens et al. 2010c). ELISA assays revealed
a significant increase in the level of OPG released from SaOS-2 cells in the presence
of biosilica from 0.8 fg/cell at 12 h to 3.1 fg/cell after 7 days (Wiens et al. 2010c).
In contrast, the concentration of RANKL did not markedly change during that time
period.
These results indicate that through stimulation of OPG synthesis, biosilica
impairs the function of RANKL, i.e. pre-osteoclasts maturation and osteoclasts
activation.
A schematic presentation of the RANK/RANKL/OPG system and of the effect
of the biosilica on this system is depicted in Fig. 10.7. Biosilica increases the
expression of OPG, whereas the expression of RANKL is not affected. By binding
to OPG (increased in the presence of biosilica), RANKL is sequestered and
unavailable to bind to its receptor, RANK. Hence, biosilica negatively affects, via
consequential secondary effects, pre-osteoclast maturation and osteoclast activation. Based on these in vitro data which must be corroborated by future studies
in vivo, it is reasonable to assume that biosilica has a potential in therapy and
prophylaxis of osteoporosis.
10.14 Effect of Biosilica on BMP-2 and TRAP Expression
In further experiments, the expression of the two further marker genes, BMP-2 (bone
morphogenetic protein 2) and TRAP (tartrate-resistant acid phosphatase), was
assessed in osteoblast-like cells growing on the biosilica substrate (Wiens et al.
2010b). BMP-2 is an inducer of bone formation. Upregulation of expression of this
cytokine is an indicator for osteoinductive activity (Eliseev et al. 2006; Katz et al.
2008). TRAP is a modulator of bone resorption. High levels of TRAP are found
in osteoclasts, but it is also expressed in SaOS-2 cells (Matsuzaki et al. 1999).
Increased expression of TRAP has been associated with the development of
osteoporosis and other bone diseases (Hollberg et al. 2005; Oddie et al. 2000).
On the other hand, TRAPÀ/À knockout mice have a reduced osteoclast activity
with the signs of osteopetrosis (Hayman et al. 1996).
SaOS-2 cells were cultivated in mineralization medium containing ascorbic acid
and ß-glycerophosphate, either on silicatein/biosilica-coated Ca-P cover slips,
untreated Ca-P cover slips, or glass cover slips (Wiens et al. 2010b). qRT-PCR
analyses demonstrated that BMP2 expression strongly increased at day 3 and day 5
in cultures grown on silicatein/biosilica-coated slips (Fig. 10.9). After 7 days, the
300
H.C. Schr€ oder et al.
2010c). In controls, the expression of OPG and RANKL changed only
unsignificantly during day 1–7 (Fig. 10.8a). Hence, the ratio of OPG/RANKL
expression markedly increased from day 1 to 7 (Fig. 10.8b). In these experiments,
the expression levels of RANKL and OPG were correlated with the expression of the
housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) which
was used as reference.
The differential expression of OPG and RANKL induced by biosilica could also
be demonstrated on the protein level (Wiens et al. 2010c). ELISA assays revealed
a significant increase in the level of OPG released from SaOS-2 cells in the presence
of biosilica from 0.8 fg/cell at 12 h to 3.1 fg/cell after 7 days (Wiens et al. 2010c).
In contrast, the concentration of RANKL did not markedly change during that time
period.
These results indicate that through stimulation of OPG synthesis, biosilica
impairs the function of RANKL, i.e. pre-osteoclasts maturation and osteoclasts
activation.
A schematic presentation of the RANK/RANKL/OPG system and of the effect
of the biosilica on this system is depicted in Fig. 10.7. Biosilica increases the
expression of OPG, whereas the expression of RANKL is not affected. By binding
to OPG (increased in the presence of biosilica), RANKL is sequestered and
unavailable to bind to its receptor, RANK. Hence, biosilica negatively affects, via
consequential secondary effects, pre-osteoclast maturation and osteoclast activation. Based on these in vitro data which must be corroborated by future studies
in vivo, it is reasonable to assume that biosilica has a potential in therapy and
prophylaxis of osteoporosis.
10.14 Effect of Biosilica on BMP-2 and TRAP Expression
In further experiments, the expression of the two further marker genes, BMP-2 (bone
morphogenetic protein 2) and TRAP (tartrate-resistant acid phosphatase), was
assessed in osteoblast-like cells growing on the biosilica substrate (Wiens et al.
2010b). BMP-2 is an inducer of bone formation. Upregulation of expression of this
cytokine is an indicator for osteoinductive activity (Eliseev et al. 2006; Katz et al.
2008). TRAP is a modulator of bone resorption. High levels of TRAP are found
in osteoclasts, but it is also expressed in SaOS-2 cells (Matsuzaki et al. 1999).
Increased expression of TRAP has been associated with the development of
osteoporosis and other bone diseases (Hollberg et al. 2005; Oddie et al. 2000).
On the other hand, TRAPÀ/À knockout mice have a reduced osteoclast activity
with the signs of osteopetrosis (Hayman et al. 1996).
SaOS-2 cells were cultivated in mineralization medium containing ascorbic acid
and ß-glycerophosphate, either on silicatein/biosilica-coated Ca-P cover slips,
untreated Ca-P cover slips, or glass cover slips (Wiens et al. 2010b). qRT-PCR
analyses demonstrated that BMP2 expression strongly increased at day 3 and day 5
in cultures grown on silicatein/biosilica-coated slips (Fig. 10.9). After 7 days, the
300
H.C. Schr€ oder et al.