Biomedical Engineering Reference
In-Depth Information
solution containing mammalian cells (direct contact) for 24 hours at 37.4 °C
(human body temperature).
The choice of cell types depends on desired application of a given bio-
material. For example, if the material under investigation is to be used as
bone analogue material, then human osteoblast (HOB) cell lines are to be
used. Nevertheless, as per ISO guidelines, fi broblast cells, which are normally
contained in living connective tissue, are to be used for primary assessment
of cell adhesion. This is because of the fact that these cells can easily prolif-
erate on material surfaces and also they come in contact at wound/injured
area (e.g., implant zone) at the initial stage. Another parameter for cell cul-
ture testing is the time of culture. Usually, the tests are widely reported to
be carried out for 24 hours; however, slowly growing cells, like HOB, need
to be cultured for three to seven days. In order to quantify the number of
attached cells or to study cell-material or cell-cell interactions using light/
electron microscopy, the cells were fi xed in glutaraldehyde/formaldehyde.
Before fi xing, these are washed twice in PBS (Phosphate buffer saline) to
completely remove the culture medium. The formaldehyde solution is (4%)
diluted in PBS and it is kept for 20 minutes. Finally, the samples are stored
in PBS at 4 °C. Afterward, the samples will be dried in a critical point dryer
using liquid CO
2
.
MTT assay is a standard colorimetric assay (an assay which measures
changes in color) to quantify cellular proliferation (cell growth). It is used
to determine cytotoxicity of potential medicinal agents and other toxic ma-
terials. Yellow MTT (3 - (4,5 - Dimethylthiazol - 2 - yl) - 2,5 diphenyltetrazolium
bromide, a tetrazole) is reduced to purple formazan in the mitochondria
of living cells. A solubilization solution (usually either dimethyl sulfoxide
or a solution of the detergent sodium dodecyl sulfate in dilute hydrochlo-
ric acid) is added to dissolve the insoluble purple formazan product into a
colored solution. The absorbance of this colored solution can be quantifi ed
by measuring at a certain wavelength (usually between 500 and 600 nm) by
a spectrophotometer. This reduction takes place only when mitochondrial
reductase enzymes are active, and therefore, conversion is directly related
to the number of viable (living) cells.
•
Genotoxicity:
In this
in vitro
experiment, it is primarily observed whether
any genetic mutation occurs in the cells in direct contact with the biomate-
rial surface.
•
Hemocompatibility:
Hemocompatibility evaluates the material's com-
patibility with red blood cells. In particular, thrombogenic property or
changes in RBC content in a blood stream fl owing over the biomater-
ials are assessed and a better thrombus material should ideally show
limited thrombus formation. Such evaluation is a must for cardiova-
scular implant materials. The examples of hemocompatibile materials
are PolyTetra Flouro Ethylene (PTFE), Diamond Like Carbon (DLC),
and so on.
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