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Encyclopedia of Physical Science and Technology EN016J-783 August 1, 2001 10:58

828 Tissue Engineering

with the surface density of extracellular matrix molecules, The first application may be part of a gene therapy protocol
which is typically accompanied by an elevation in DNA aimed at providing a patient who has a single enzyme defi-
synthesis and proliferation rates. ciency (e.g., adenosine deaminase) with implantable cells
A general rule of thumb in cell culture techniques is to perform the missing function. Another important appli-
that proliferation and differentiation are mutually exclu- cation is the (over)expression of angiogenic factors that
sive. In other words, conditions promoting the expres- promote the rapid invasion by blood vessels and vascu-
sion of differentiated functions are often not optimal for larization of implantable tissue constructs. Immortalizing
replicating cells. For example, fibroblast growth factor- genes, such as the viral SV40 T antigen and telomerase
stimulated capillary endothelial cells plated on nonad- are primarily used to promote the replication of cells typ-
hesive surfaces coated with decreasing concentrations of ically very difficult to grow in vitro, such as hepatocytes,
fibronectin switch from a spreading to a tubular capillary- pancreatic beta cells, etc. The use of anti-apoptotic genes
like shape, with a concomitant reduction in cell growth. in tissue engineering is a relatively new trend stimulated
In some cases, cell differentiation can also be induced by the difficulties of maintaining cell viability in large
by altering the culture environment so as to mimic a subset tissue constructs made of cells sensitive to the depletion
of in vivo conditions. For example, keratinocytes, a type of of nutrients—for example, in the case of hepatocytes in
epithelial cell which forms the epidermal component of bioartificial livers.
the skin, can be propagated in vitro using a serum-free One of the issues raised by the use of genetic engineer-
culture medium; a single human neonatal foreskin can ing in tissue engineered products is the unknown effects
2
provide enough cells to yield over 100 m of graftable of persistent expression of the transgene in the implanted
tissue. Cells in cultured epidermal sheets are not well dif- cells. For example, overexpressing growth factors may be
ferentiated but exposure to air while in culture or after beneficial to the process of growth and integration of a
grafting onto the host induces the formation of a stratified engineered tissue implanted in a host; however, the long-
differentiated epidermis. term effects of high levels of growth factors are unknown
One of the challenges of tissue engineering is to produce and could perhaps be detrimental. This problem may be
large cell masses that are well differentiated. Although resolved soon, however, with the advent of new molecular
differentiated cells do notalways proliferate easilyin vitro, biology techniques that allow for the “excision” at will of
it may be possible to optimize culture conditions to stim- the transgenes in order to restore the native state of the
ulate cell propagation and then to change these conditions cells.
so that a stable and functional phenotype is exhibited by
the cells. For example, chondrocytes seeded on plastic in
3. Metabolic Engineering
the presence of serum proliferate but secrete a significant
amount of type I collagen and small proteoglycans, which Metabolic engineering has been defined as the introduc-
are not normally found in cartilage. Embedding these cells tionofspecificmodificationstometabolicnetworksforthe
in an agarose gel induces the re-expression of the normal purpose of improving cellular properties. In recent years,
phenotype found in vivo, which is characterized by the metabolic engineering has gained importance in biotech-
production of type II collagen and deposition of large ag- nology, being used largely to improve existing processes
gregating proteoglycans. involving the production of chemicals using microorgan-
isms. Although less widely appreciated, metabolic engi-
neering techniques can be applied to study physiological
2. Genetic Engineering
systems and isolated whole organs in vivo to elucidate
While control of the extracellular environment remains the the metabolic patterns that occur in different physiolog-
primary means of modulating cell function and prolifer- ical states, such as fed, fasted, or in disease. Metabolic
ation in tissue engineering, it is sometimes advantageous engineering techniques are also finding important uses in
to alter the genetic make-up of cells to extend their ba- tissue engineering, where they can be used to monitor the
sic capacity to perform specific functions. Describing the metabolic response of cells and tissues to perturbations in
techniques used for genetically altering cells is beyond the the environment and rationally design culture media that
scope of this chapter, and the reader is referred to the nu- enhance cell function and proliferation.
merous texbooks and reviews on the subject. Genetic mod- In metabolic engineering, the notion of cellular
ification of cells in tissue engineering has included the fol- metabolism as a network is of central importance. Also,
lowingapplications:(1)expressingfunctionsnotnormally fundamental to metabolic engineering is the idea that
present in a particular cell type or overexpressing existing metabolic processes, systemic or cellular, are coupled
functions, and (2) expressing “immortalizing” genes or and as such cannot be considered separately. The major
genes that protect cells against death caused by apoptosis. metabolic pathways (e.g., glycolysis, gluconeogenesis,