cells were exposed to orthosilicate that had been prepared from prehydrolyzed
TEOS (Wiens et al. 2010c). The cell density was assessed by the colorimetric
XTT cell proliferation assay. Addition of orthosilicate within the concentration
range of 10–1,000 mM resulted in increased absorbance values (incubation period,
72 h), indicating a cell growth-stimulating effect of orthosilicate (Wiens et al.
2010c). A stimulatory (but less significant) effect on cell growth has also been
reported for silicon-substituted HA (Lo ´pez-Alvarez et al. 2009; Zou et al. 2009). In
parallel to the cell density, cell viability was quantified by the trypan blue exclusion
test. This assay did not reveal any silicate-induced cell toxicity during the 72h incubation period in the presence of a concentration of up to 1,000 mM
orthosilicate (Wiens et al. 2010c).
10.9 Effect of Biosilica on HA Formation
In order to determine the effect of biosilica on mineralization activity of SaOS2 cells, cover slips or bottoms of multi-well plates were coated with a biosilica layer
biocatalytically formed by immobilized recombinant silicatein. A His-tagged
fusion protein was used (Wiens et al. 2010c). Using the SaOS-2 cell system, we
could demonstrate that growth of the cells on the biosilica matrix strongly promotes
HA formation (Schr€ oder et al. 2005a; Wiens et al. 2010c). In the experiment shown
in Fig. 10.4, SaOS-2 cells were grown on silicatein/biosilica-coated slips placed
into 24-well plates for 7 days (Wiens et al. 2010c). The biosilica coating had been
prepared by incubation of the immobilized silicatein in the presence of various
concentrations of orthosilicate. Subsequently, the slips were stained with Alizarin
Red S (staining for HA), or Alcian Blue (staining for cartilaginous proteoglycans
and sulfated glycosaminoglycans), or with both dyes. The cells grown on the
biosilica-coated cover slips revealed a considerable staining with Alizarin Red S
(Fig. 10.4). The intensity of the red color correlated to the orthosilicate concentration that had been applied for biocatalytic silica formation using the immobilized
enzyme. In contrast to HA synthesis, formation of cartilaginous proteoglycans and
sulfated glycosaminoglycans (staining with Alcian Blue) is downregulated (Wiens
et al. 2010c). Cartilage damage has been shown to be inversely correlated with the
bone mineral density index (Calvo et al. 2007). Samples that had been doublestained with both dyes showed a dark red/blue color (Wiens et al. 2010c); Fig. 10.4.
Similar results were obtained when cells (SaOS-2) were grown on silicatein/
biosilica-modified bone slices or Ca-P-coated cover slips (Wiens et al. 2010b).
Immobilization of silicatein to bone HA- or Ca-P-coated cover slips was achieved
by using a Glu-tagged silicatein-a (Natalio et al. 2010). The Glu-tag consisting of
eight N-terminal glutamic acid residues allows for ionic interaction with Ca
2+ ions
at the HA surface (formation of a coordination complex between the carboxyl
groups and the Ca
2+ ions). Incubation of the immobilized enzyme with 200 mM
orthosilicate resulted in the formation of a 50–150 nm thick nano-biosilica layer on
the HA surface. Biosilica coating was confirmed by EDX analysis (Wiens et al. 2010b).
10 Biosilica-Based Strategies for Treatment of Osteoporosis and Other Bone Diseases
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TEOS (Wiens et al. 2010c). The cell density was assessed by the colorimetric
XTT cell proliferation assay. Addition of orthosilicate within the concentration
range of 10–1,000 mM resulted in increased absorbance values (incubation period,
72 h), indicating a cell growth-stimulating effect of orthosilicate (Wiens et al.
2010c). A stimulatory (but less significant) effect on cell growth has also been
reported for silicon-substituted HA (Lo ´pez-Alvarez et al. 2009; Zou et al. 2009). In
parallel to the cell density, cell viability was quantified by the trypan blue exclusion
test. This assay did not reveal any silicate-induced cell toxicity during the 72h incubation period in the presence of a concentration of up to 1,000 mM
orthosilicate (Wiens et al. 2010c).
10.9 Effect of Biosilica on HA Formation
In order to determine the effect of biosilica on mineralization activity of SaOS2 cells, cover slips or bottoms of multi-well plates were coated with a biosilica layer
biocatalytically formed by immobilized recombinant silicatein. A His-tagged
fusion protein was used (Wiens et al. 2010c). Using the SaOS-2 cell system, we
could demonstrate that growth of the cells on the biosilica matrix strongly promotes
HA formation (Schr€ oder et al. 2005a; Wiens et al. 2010c). In the experiment shown
in Fig. 10.4, SaOS-2 cells were grown on silicatein/biosilica-coated slips placed
into 24-well plates for 7 days (Wiens et al. 2010c). The biosilica coating had been
prepared by incubation of the immobilized silicatein in the presence of various
concentrations of orthosilicate. Subsequently, the slips were stained with Alizarin
Red S (staining for HA), or Alcian Blue (staining for cartilaginous proteoglycans
and sulfated glycosaminoglycans), or with both dyes. The cells grown on the
biosilica-coated cover slips revealed a considerable staining with Alizarin Red S
(Fig. 10.4). The intensity of the red color correlated to the orthosilicate concentration that had been applied for biocatalytic silica formation using the immobilized
enzyme. In contrast to HA synthesis, formation of cartilaginous proteoglycans and
sulfated glycosaminoglycans (staining with Alcian Blue) is downregulated (Wiens
et al. 2010c). Cartilage damage has been shown to be inversely correlated with the
bone mineral density index (Calvo et al. 2007). Samples that had been doublestained with both dyes showed a dark red/blue color (Wiens et al. 2010c); Fig. 10.4.
Similar results were obtained when cells (SaOS-2) were grown on silicatein/
biosilica-modified bone slices or Ca-P-coated cover slips (Wiens et al. 2010b).
Immobilization of silicatein to bone HA- or Ca-P-coated cover slips was achieved
by using a Glu-tagged silicatein-a (Natalio et al. 2010). The Glu-tag consisting of
eight N-terminal glutamic acid residues allows for ionic interaction with Ca
2+ ions
at the HA surface (formation of a coordination complex between the carboxyl
groups and the Ca
2+ ions). Incubation of the immobilized enzyme with 200 mM
orthosilicate resulted in the formation of a 50–150 nm thick nano-biosilica layer on
the HA surface. Biosilica coating was confirmed by EDX analysis (Wiens et al. 2010b).
10 Biosilica-Based Strategies for Treatment of Osteoporosis and Other Bone Diseases
293