MEDICAL DEVICES BASED ON BIOINSPIRED CERAMICS - Advanced Biomaterials: Fundamentals, Processing, and Applications - page 391

The test on extracts was conducted by measuring the cellular activity in
response to different concentrations of solvents extracted from the materials
using the MTT cell proliferation assay. This test is a colorimetric assay system
that measures the reduction of the yellow tetrazolium salt MTT (3- [4,5 - dimethyl-
tihazol - 2 - yl] - 2,5 - diphenyl tetrazolium bromide) into an insoluble purple forma-
zan crystals by the mitochondrial enzyme succinate dehydrogenase, only present
in viable cells. This cellular reduction involves the pyridine nucleotide cofactors
NADH and NADPH. The formazan crystals formed were solubilized and the
resulting coloured solution was quantifi ed using a scanning multiwell spectropho-
tometer, with the absorbance values being proportional to the number of viable
cells [Selvakumaran, 2005].
Extracts were prepared by rolling bioSiC pieces (coated and uncoated) in
EMEM culture medium supplemented with 10% foetal calf serum for 90 hours
at 37 °C. Different concentrations of these extracts were incubated for 24 hours
with MG-63 human osteoblast. Extracts obtained from Polyvinyl chloride discs
and Thermanox plastic cover slips were used as positive and negative controls
respectively. The extracts were diluted with EMEM to give 10, 20, 30, 50 and 100%
of the original concentration.
MG-63 osteoblast-like cells were seeded at a concentration of 6

1 0 5 cells per
mL, grown to confl uent layers in 96-well tissue culture plates in a fi nal volume of
0.1 mL of culture medium per well. The different concentrations of the extracts
were incubated with cells for 24 hours. Four wells per substrate and extract con-
centration were used. After incubation, cellular activity was quantifi ed by using
the MTT assay as previously described.
Normalized solvent extraction tests summarize the MG-63 osteoblast-like
cell activity after the incubation with different concentrations of the extracts
obtained from uncoated biomorphic SiC ceramics and coated bioSiC ceramics
with bioactive glass (Figure 11.25). Thermanox plastic cover slips were used as
a negative control and PVC were used as a positive toxic control. A bulk bioac-
tive glass and Ti6Al4V were used as references for the test. As can be seen,
PVC extract (positive for cytotoxicity) reduced cell viability in a progressive
manner for all tests, thus validating the solvent extraction method and dilution
procedures.
In the solvent extraction test for uncoated samples (Figure 11.25a), the refer-
ence materials present an absorbance similar to Thermanox slips, which means
the absence of cytotoxicity. All the extracts did not signifi cantly affect the cellular
activity at any of the concentrations tested, not even at 100% concentration. Only
a slight detrimental effect was found for sapeli and eucalyptus wood ceramics for
extract concentrations of 30 and 50% that disappears at 100% of extract concen-
tration. Nevertheless, the differences fall within the statistical range. This result
suggests that bioSiC is free of harmful extractable substances or has, at least, an
insuffi cient quantity of it to cause serious effects in cell monolayers under in vitro
culture conditions.
The same behaviour was found for the coated biomorphic SiC. Cells incu-
bated in the bioactive glass-coated SiC ceramics extracts (Figure 11.25b) showed

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