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Engineered Muscle Actuators 257

The strategies for generating functional muscle tissue can be broadly divided into in vitro and in vivo
strategies, the ultimate outcome of which would be a vascularized muscle construct. In any case,
once a vascular bed is established, the constructs need to be maintained in a bioreactor to provide
further electrical, mechanical, and chemical stimulation, thus guiding both the phenotype and
resulting in the development of a fully function muscle construct.

9.9.2.1 Recellularization of an Acellular Muscle Construct

This experimental approach involves harvesting muscle tissue from any natural source and
using chemical acellularization to remove myoblasts and fibroblasts leaving behind an intact
ECM. The ECM should be evaluated for structural integrity and immunogenic behavior and
its ability to support myoblast growth and differentiation. The ECM should then be used as
scaffolding material for seeding primary myoblast and the construct will be placed in a perfusion
bioreactor allowing formation of functional skeletal muscle tissue (Hall, 1997). Immunohis-
tochemical studies should be performed to determine which ECM components are present in
the acellular construct, such as collagen types I and IV, fibronectin, laminin, vitronectin,
entactin, heparin sulfate, proteoglycan, and elastin. The acellular muscle can be repopulated
by obtaining a purified sample of myogenic precursor cells, which may be injected or perfused
into the acellular muscle. Although some initial success has been reported with this
general approach, it has not yet been possible to maintain perfusion of the tissue samples in culture
for a period sufficiently long to promote and maintain full cellular infiltration into the acellular
scaffold.

9.9.2.2 Coculture Systems

Since the early 1990s, there have been reports of the use of various coculture systems to study cell–
cell interactions and the formation of tissue interfaces. For vasculogenesis, the cells in question are
presumed to be myoblast and endothelial cells. Although promising initial reports have been
published, a truly successful demonstration of a vascular bed self-organizing within a tissue
construct has yet to be demonstrated. The design of bioreactors for such a technology must
stimulate the myoblasts to form functional muscle tissue and simultaneously guide the endothelial
cells to form capillary-like structures within the newly forming muscle tissue, while providing
perfusion during development. The environment, which the bioreactor provides together with
soluble growth factor stimulation, will presumably allow formation of a functional muscle con-
struct (Vernon, 1999).

9.9.2.3 Induced Microvessel Sprouting

This approach can be attempted either in vivo or ex vivo using small vascularized tissue explants
which are cannulated and perfused while adjacent to an avascular tissue such as engineered skeletal
muscle. This is an active area of current research. For the in vivo approach, it is necessary to
mechanically support the muscle tissue while implanted to prevent hypercontraction and subse-
quent tissue damage. It is also necessary to take measures to prevent tissue rejection to implantation
into syngenic animals, or the use of immune-suppressive agents, is required. Otherwise, this method
is relatively quite simple and often yields satisfactory results. In addition to vascularization of the
implanted muscle tissue, there are collateral effects, as yet not fully understood, that also tend to
drive the muscle phenotype toward an adult phenotype, with enhanced contractility. For this reason,
it is likely that the future of tissue engineering will see increasingly common application of the
approach where the intended recipient is used as a ready-made bioreactor vessel. The engineered
tissues would be implanted within the person, presumably along with means to enhance tissue
development and to prevent tissue degeneration or resorption while implanted. The tissue need not