Biomedical Engineering Reference
In-Depth Information
• With biodegradable scaffolds completely natural tissue and long-term biocompatibility is eventu-
ally achieved. However, the degradation rate needs to match the development of the regenerated
tissue.
Scaffold materials include decellularized extracellular matrix (Gilbert et  al. 2006), synthetic poly-
mers, for example, poly(glycolic) acid (PGA), poly(l-lactic acid) (PLLA), and natural polymers such as
collagens, gelatins, fibrin, carbohydrates, peptides, and nucleic acids (Pachence et al. 2007). A number of
methods are available for polymer scaffold fabrication, for example, fiber bonding and electrospinning
render fiber network structures similar to native tissue matrices. Solvent casting, particulate leaching,
and melt molding result in porous structures and the development of rapid prototyping techniques has
provided a tool for design of advanced three-dimensional structures. Overviews of polymer scaffold
fabrication methods can be found in textbooks, for example, Principles of Tissue Engineering (Murphy
and Mikos 2007).
Fibrous scaffold materials can also be obtained from bacteria spinning cellulose (Czaja et al. 2007)
or from worms spinning silk fibroin (Mandal and Kundu 2010). Another alternative for scaffold syn-
thesis is to use genetically encoded polypeptides, allowing for precise control of protein structure and
hence scaffold function (Chow et al. 2008, Sengupta and Heilshorn 2010). Furthermore, scaffold mate-
rial properties can be tuned by forming composites, for example, between natural and synthetic fiber
polymers (Heydarkhan-Hagvall et al. 2008).
In all, an impressive number of advanced technologies are available to produce artificial tissues, and
with innovations made within material science, nano- and biotechnologies, as well as stem cell biol-
ogy, highly sophisticated materials can be expected in the future. This sets high demands on methods
to characterize the morphology of intact constructs in three dimensions under native conditions. In
addition, it is desirable to be able to monitor their function in and interaction with host tissue in vivo .
18.1.3 Microscopy techniques in tissue engineering
A number of microscopy techniques are available for characterization of structure and morphology of
the cell-scaffold constructs synthesized in tissue engineering. Electron microscopy remains an invalu-
able tool for this purpose with the ability to provide high-resolution images of the scaffold architecture,
allowing 10 nm-sized structures to be resolved. However, electron microscopy requires extensive sample
preparation involving fixation, dehydration, freeze-drying, and material coating (Abeysekera et al. 1993,
Czaja et al. 2004), all potentially affecting the properties of soft materials and excluding the possibility
of observing the cell-scaffold matrix under native conditions. In addition, it only provides access to the
most superficial layer, requiring physical sectioning in order to examine the interior sample structure,
which in turn could introduce compression and edge artifacts to the architecture. Atomic force micros-
copy (AFM) eliminates the need for harsh sample preparation and also has the potential to render images
with very fine details resolved. However, an AFM tip has a tendency to stick to soft material, which is a
serious practical limitation for characterization of tissue (Shao et al. 1996). In addition, the information
obtained is merely of topographical character, giving no access to the morphology below the sample
surface. Access to the internal sample structure can be obtained by confocal fluorescence microscopy,
which gives an optical sectioning of the sample. However, fluorescence microscopy usually requires the
sample to be labeled with a marker molecule that specifically attaches to the structure or cellular com-
ponent to be imaged. This indirect visualization method via a tracer is dependent on the uptake and
expression efficiencies of the fluorophore in the material as well as an often unknown fluorescence yield,
which both introduce measurement uncertainties. An additional limitation of fluorescence microscopy
is photobleaching, which makes exposure times longer than the order of minutes unrealistic and long-
term studies of cell vs. scaffold interaction processes infeasible. In addition, fluorescence labeling may
also affect the viability of living matter; for example, it has been shown that it influences and inhibits the
formation of nanofibrils during cellulose synthesis by bacteria (Colvin and Witter 1983).
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