Biomedical Engineering Reference
In-Depth Information
throughout Europe that BSE cannot be detected by any
in vitro tests and therefore it is impossible to be confi-
dent that bovine material is BSE free unless it comes
from herds that have never been exposed to BSE.
well established in characterizing cell proliferation in 2-D
monolayer cultures of low cell densities.
The most common methods to visualize cells on bio-
materials have been limited to light microscopy or use
of dyes that compromises the viability of the cells.
Conventional techniques, such as histomorphometry,
electron microscopy, and Fourier transform infrared (FT-
IR) imaging, are capable of giving information on tissue
development, but they require that scaffolds be de-
structively sectioned. Other problems include the dis-
solution of polymeric scaffolds by the organic solvents
used in the preparation of thin sections or the disruption
of early mineral deposits by aqueous solvents. Some in-
vestigators reported on the successful application of
confocal microscopy to study cells growing in the pores
of scaffolds, but the depth penetration of this technique
is limited and it cannot be applied to scaffolds that are
optically opaque. What is required is a nondestructive
technique that can provide spatially resolved, chemically
specific, tissue-level information about the tissue formed
within the pores of scaffolds.
DNA binding dyes coupled with confocal microscopy
have been used to demonstrate the coverage of cells on
materials. In a similar method, 3-D cell distribution on
a material can be demonstrated with the fluorescence of
enhanced green fluorescent protein (EGFP) and confocal
microscopy without the need for special preparation of
the cells. This approach facilitates visual assessment of
cells as they exist in tissues and is not dependent on cell
markers. Since EGFP is stably produced by the cells, no
staining or cellular manipulation is required. Reporter
genes have been utilized for a variety of applications rang-
ing from gene expression and regulation to determination
of efficiency of gene vector delivery. The technique
allows the tracking of stem cells as they differentiate or
become specialized. A reporter gene is inserted into
a stem cell. This gene is only activated or ''reports'' when
cells are undifferentiated and is turned off once they
become specialized. Once activated, the gene directs the
stem cells to produce a protein that fluoresces in a bril-
liant green color. Two commonly used reporter genes are
EGFP and luciferase. Figure 7.2-20 illustrates the ge-
netical modification of cell to express both EGFP and
luciferase [16] . Both the fluorescent and luminescent
signals from the cells follow a linear relationship as
a function of cell number. These relationships provide
two opportunities for quantifying cellularity from simple
extracts. Both genes, when expressed in mammalian
cells, will produce a molecule that can be detected by
different techniques. A chromophore-containing protein,
EGFP, emits fluorescent light when excited with light at
a wavelength of approximately 488 nm. Qualitatively,
EGFP under excitation provides an opportunity to visu-
alize only cells expressing this protein. In contrast, the
luciferase enzyme hydrolyzes its substrate, D -luciferin, in
7.2.6.2.3 Seeding efficiency
Although there are some techniques that successfully
introduce cells into biomaterials, seeding efficiencies are
not yet at optimal levels, especially at low cell concen-
trations. Lower seeding densities affect the amount of
time and resources required to obtain scaffolds ready for
implantation. The systems developed for the seeding of
cells onto scaffolds include from simple techniques such
as static seeding, where cells and scaffolds are brought into
direct contact and allowed to sit, relatively undisturbed,
with the intention of cellular attachment and migration
into the scaffolds, to more elaborate techniques such as
pulsatile perfusion wherein medium flows under oscilla-
tory pressures to try to mimic the natural environments.
Dynamic seeding which induces medium flow within the
scaffold pores and shear stress on cells seems to be the
most applicable method when relatively thin ( < 1 mm)
scaffolds with a large 2-D surface need to be seeded.
In addition to seeding a greater number of cells into
scaffolds, it is important to achieve homogeneity in cel-
lular distribution. Achieving a confluent coating on scaf-
fold exteriors may not be ideal because of potential
problems with vascular ingrowth and nutrient/waste
transport. Such an exterior cellular coating is also unde-
sirable because of cell migration: If there is no subsequent
cell migration into the scaffold interior, a bioabsorbable
scaffold would ultimately collapse and the goal of quicker
tissue regeneration will not be achieved.
7.2.6.2.4 Assessment of cells in scaffolds
Strategies for investigating cell growth in scaffolds in-
clude cell viability, proliferation, and metabolic active
assays. In most cases, it is unknown how many cells
remain viable over time in scaffolds in vitro and in vivo
without assays in which the viability of the samples must
be compromised to perform the specific assessment.
Various assays are available for assessing cultured cell
proliferation. These include (1) mitochondrial enzyme
reduction of tetrazolium salts into their respective for-
mazon by-products [MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazo-zolium bromide) and MTS];
(2) cellular redox indicators (Alamar blue); (3) ATP quan-
tification through bioluminescence; (4) S-phase incorpor-
ation of radioactively labeled DNA precursors such
as [ 3 H]thymidine and bromodeoxyuridine (BrDU); and
(5) co-staining with a fluorescent DNA-specific dye
for live cells (Hoechst 33258 and PicoGreen), and
physical counting (hemocytometer). These assays are
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