Biomedical Engineering Reference
In-Depth Information
488
nm
RetroLuciferase
RetroEGFP
525
nm
EGFP
Luciferase
D
-Luciferin
Fig. 7.2-20 Schematic depiction of cell genetically modified by retroviral vectors to express the reporter genes EGFP and luciferase.
EGFP when excited with at
w
488 nm will emit at
w
525 nm. Luciferase in the presence of the substrate, D-luciferin, will emit light.
the presence of ATP and oxygen to produce photons of
the emission spectra ranging between 400 and 620 nm.
This stable retroviral transfection of cells with green
fluorescent protein (GFP) offers a pathway to study
tissue development, with emphasis on distinguishing
between cellular components initially seeded onto a
construct and those occurring as a result of cell ingrowth
from surrounding tissue.
Various seeding techniques can be assessed using
a DNA assay to estimate the total number of cells within
a scaffold. This approach, however, yields very little in-
formation about the distribution of the cells throughout
the scaffold. For destructive cellularity assays which
quantify cellular DNA, the presence of ECM and/or
material particulates in the sample extracts can interfere
with these analyzes by making it difficult to extract the
DNA. A critical issue in tissue engineering concerns
whether the cellular components of tissue-engineered
structures are derived from cells harvested and seeded
onto an acellular scaffold or from cells originating from
surrounding tissue (
e.g.,
proximal and distal anastomosis
in the case of cardiovascular tissue engineering), or from
circulating pluripotent stem cells. To clarify this issue,
some studies have utilized fluorescent carbocyanine dyes.
It was possible to identify cells labeled by this technique
in vivo
for up to 6 weeks after transplantation, but more
stable long-term labeling and tracing without adverse
effects, such as cell toxicity, are required. Current
histomorphometric techniques evaluating rates of tissue
ingrowth tend either to measure the overall tissue con-
tent in an entire sample or to depend on the user to in-
dicate a front of tissue ingrowth. Neither method is
particularly suitable for the assessment of tissue ingrowth
rates, as these methods either lack the sensitivity re-
quired or are problematic when there is a tissue ingrowth
gradient rather than an obvious tissue ingrowth front.
Cells interacting with scaffolds may exhibit distinct
patterns of gene expression depending on the molecular
nature of the surface they are contacting. Many cell types
grown in 3-D culture exhibit phenotypes drastically
different from those of their plate-grown counterparts.
In these cases gene expression studies are beneficial.
7.2.6.2.5 Gene expression of cells
Expression of specific genes is enhanced within cell-
seeded constructs or engineered tissue. For instance, in
the bone tissue engineering, Runx 2, alkaline phosphatase
(ALP), and osteocalcin (OCN) that are important mar-
kers of different stages of bone matrix production are
expressed. Runx 2 is a transcriptional activator essential
for initial osteoblast differentiation and subsequent bone
formation. The ALP is expressed by pre-osteoblasts and
osteoblasts before the expression of OCN. Finally, OCN
is a late marker that binds HAp and is produced by os-
teoblasts just before and during matrix mineralization.
7.2.6.3 Bioreactors
When 3-D cellular constructs are grown in static culture,
cells on the outer surface of the constructs are typically
viable and proliferate readily while cells within the con-
structs may be less active or necrotic. In the absence of
a vascular blood supply
in vitro,
nutrient delivery to cells
throughout 3-D tissue engineered constructs grown in
static culture must occur by diffusion. As a result, thin
tissues (e.g., skin) and tissues that are naturally avascular
(e.g., cartilage) have been more readily grown
in vitro
than thicker, vascular tissues such as bone. The engi-
neering of tissues
ex vivo
in a bioreactor offers several
benefits, such as better understanding of tissue de-
velopment and mechanisms of disease, off-the-shelf re-
vision of transplantable tissues, and possible scale-up for
commercial production of engineered tissue. The bio-
reactors in tissue engineering are utilized for cell expan-
sion on a large scale and production of 3-D tissues
in vitro.
Bioreactors offer several advantages over culture
in plates and flasks. Bioreactors can be custom designed
