Fisher John P. e.a. (ed.) Tissue Engineering
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24-6 Tissue Engineering
offers advantages for studying the effects of specific bioactive ligands or peptides presented from the
scaffold [83,84]. Studies utilizing surface modified PEGs have demonstrated that a number of cellular
functions, including adhesion [83], migration [85], and matrix production [46] can be regulated by
ligand presentation. In general, hydrogels are an appealing scaffold material because they are structurally
similar to the highly hydrated ECM of many tissues [86]. However, the use of hydrogels is often constrained
by their limited range of mechanical properties.
The elasticity provided by elastin in SM tissues has motivated the development of elastomeric scaffolds
that can similarly provide this property to engineered SM. Elastomeric polymers can recoverfrom extensive
deformation [87–89] and are designed to resemble the incompressible nature of the ECM [90]. This
property of biomaterials may be ideal to engineer functional SM tissues that require transduction of
mechanical signals from the extracellular environment in order to elicit and activate key cellular functions
[40,53,54]. This type of biomaterial resolves the limitations of lack of pliancy that limits many synthetic
polymer scaffolds (i.e., poly[lactic acid] [PLA]).
24.4.2.2 Naturally Derived Biomaterials
Type I collagen (Figure 24.3b) has been frequently used to create polymer scaffolds for engineering SM
tissues [56,73,91,92]. Naturally derived collagen is an attractive biologic material because collagen is
the primary constituent of the ECM [58], and contains adhesion ligands that facilitate cell attachment.
Although type I collagen does not require additional surface modification to promote tissue formation,
glycosaminoglycans (GAGs) [93] and growth factors [45] can be incorporated to improve mechanical
properties and to induce specific cellular functions. Type I collagen matrices used to engineer SM tissues
have demonstrated partial elasticity and are capable of withstanding cyclic stain [56]. The high tensile
strength of type I collagen can be attributed to its molecular structure, while the elasticity is conferred
by the intermolecular cross-linking. The degradation of type I collagen scaffolds is dependent on the
extent of cross-linking, pore structure and the apparent density, which are variables that can be readily
altered to meet a desired target. Although type I collagen is typically extracted from xenogeneic sources,
it is considered biocompatible and exhibits low immunogenic responses, likely due to the similarity of
this molecule between species [94]. However, naturally derived materials may suffer from batch to batch
variations.
Another collagen based biomaterial, small intestinal submucosa (SIS), has also been widely used in
tissue engineering research [95–97]. This xenogeneic matrix is harvested from the submucosal layer of
the intestine. SIS may provide functional growth factors [98]. that contribute to SM tissue formation.
In addition, SIS matrices maintain elasticity and high strength [99]. SIS has typically been obtained from
porcine sources, but isolation from rats [100] and canines [101] has also been attempted. SIS has been
used to promote regeneration of several SM tissues, in the blood vessels [102,103] and in the bladder
[99,101,104].
24.5 Engineered Smooth Muscle Tissues
A number of studies to date have utilized a combination of scaffolding technologies and cells to reconstruct
the smooth muscle component of cardiovascular, gastrointestinal, and urinary tissues. The two primary
tissue-engineering approaches used to regenerate tissues are cell transplantation and cell recruitment
from surrounding tissue. Cell transplantation requires an initial step of procuring cells, often via biopsy
from the host, followed by dissociation and expansion in vitro. The cells are then seeded onto a scaffold
and implanted as a cell–matrix construct. Alternatively, an implanted acellular matrix may be implanted
to promote the recruitment of neighboring SMCs and possibly other cell types of interest (e.g., ECs,
urothelial cells). Work to date in engineering SM tissues is briefly summarized in this section.
A great deal of research has been performed with the goal of developing blood vessel substitutes, due
to the large impact this advance would have on the millions of patients that annually suffer from diseases
of blood vessels [105]. Strategies to engineer blood vessel must provide adequate mechanical properties,
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Engineering Smooth Muscle 24-7
(a) (b)
FIGURE 24.4 Engineered blood vessel substitutes. (a) Self-assembly approach to blood vessel formation relies on
the ability of sheets of cells to form their own ECM, and multiple cells sheets can subsequently be combined to form
tissues (b) Cyclic mechanical strain can play a prominent role in the development of engineered vascular tissues, as it
can lead to alignment of cells, as shown in this photomicrograph, and also leads to an increase in tissue mechanical
properties. (Photomicrograph was taken from Kim, B.S. et al., Nat. Biotechnol., 1999, 17: 979–983; L’Heureux, N. et al.,
FASEB J., 1998, 12: 47–56, and used with permission from FASEB and the Nature Publishing Group, respectively.)
to avoid catastrophic failure in this mechanically demanding site, and appropriate cellular components to
form the complex vascular wall. An early approach to engineer the blood vessels involved the culture of
different vascular cell populations in collagen gels to form three distinct layers, resembling the three layers
of native blood vessel [68]. However, this model did not lead to tissues with adequate mechanical strength.
A later approach exploited the ability of fibroblasts and SM cells to synthesize and secrete their own ECM
and form self assembled sheets. These sheets weresubsequently wrapped around a mandrel to form distinct
layers of the native vessels [65] (Figure 24.4a). This method led to tissues with much greater mechanical
strength, comparable to that of human vessels [69]. The increased mechanical strength of these tissues
my be partially attributed to paracrine effects between ECs and SMCs [35,39,51] that contribute to the
stability of nascent blood vessels by increasing matrix production. Also, implantation of a decellularized
SIS with additional type I bovine collagen into a rabbit artery led to the formation of a blood vessel
characterized by reasonable burst strength, cell and matrix organization [102].
Several groups have utilized externally applied mechanical stimulation to improve the mechanical
integrity of engineered SM tissues (Figure 24.4b). Blood vessel substitutes formed from allogeneic vascular
SMCs and ECs cultured on biodegradable PGA scaffolds were maintained under pulsatile stress, and this
resultedin an increased matrix production [57]. These engineered constructs were subsequently implanted
into swine for seven weeks and the explanted vessels exhibited adequate burst pressures and histology.
Several studies document that one can improve the properties of constructs engineered using collagen
through the use of mechanical stimulation [55,106]. The significance of mechanical stimulation was also
demonstrated by studies where synthetic SMCs cultured with ECs on collagen gels were found to undergo
a phenotypic reversion under contractile forces [5,107].
Currently, a common approach to replace or repair bladder tissue utilizes gastrointestinal segments,
but this method can result in mucus production, stone formation, and other abnormalities that may be
attributed to the different physiologic role of each tissue type. These complications have motivated invest-
igation into new methods for bladder replacement utilizing tissue engineering techniques. One common
acellular approach to engineer bladder tissue utilizes SIS membranes, as SIS membranes grafted during
partial cystectomy of canines have displayed development of all three layers of the bladder (urothelium,
SM, and serosa) [95]. Additionally, these regenerated tissues demonstrated contractile nerve regenera-
tion. Acellular biomaterial extracted from rat bladder also resulted in a well integrated construct when
implanted, but one that developed a compromised SM layer [108]. While these studies utilizing acellu-
lar scaffold implantation resulted in bladder regeneration, but function of the resultant tissues was not
reported. In contrast to this approach, PGA–PLGA scaffolds seeded with autologous canine cells led to
formation of a new tissue (Figure 24.5) that regained bladder function [12].
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24-8 Tissue Engineering
FIGURE 24.5 Engineered bladder formed from autologous cells cultured on polymeric scaffolds in vitro. The neo-
bladders can be implanted to replace lost bladder tissue. (Photomicrograph was taken from Oberpenning, F. et al.,
Nat. Biotechnol., 1999, 17: 149–155, and used with permission from the Nature Publishing Group.)
FIGURE 24.6 Photomicrographs of histologic section of engineered intestinal tissue. Epithelial organoid units were
isolated, seeded onto polymeric scaffolds, and implanted. Ultimately, the transplanted cells differentiate and form
new tissue. (Photomicrograph was taken from Vacanti, J.P., J. Gastrointest. Surg., 2003, 7: 831–835., and used with
permission from Elsevier Inc.)
A number of studies suggest it may be feasible to engineer functional gastrointestinal tissues. Isolated
crypt cells implanted on PGA tubes formed epithelial-lined tubular structures lacking in a SM component
[109] (Figure 24.6). The transplantation of intestinal organoid units, in place of individual cells on PLGA
scaffolds led to the development of a neomucosa layer [14] and SM layers [109]. However, the neomucosa
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may have been lacking in its ability to control nutrient. SIS patch implantation, without cells, into canines
led to formation of both the epithelial and SM layer. However, a large percentageof animals died and a large
number of inflammatory cells were found in explanted SIS patches. One study utilized transplantation of
precursor cells derived from bone marrow in the place of differentiated SMCs [110], but the engineered
tissues did not regenerate a functional muscle layer, potentially due to a lack of appropriate extracellular
signals to induce the differentiation of the mesenchymal stem cells into SMCs.
24.6 Conclusion/Future Directions
Impressiveprogresshas been made to date in SM engineering, and these tissues may havesignificant clinical
impact and provide models to study basic biological processes. However our current understanding of the
complex interplay of factors that modulate the SM function are far from comprehensive, and advances
in this knowledge will likely translate to improved systems for engineering functional SM tissues. One
important issue yet to be addressed is whether approaches that successfully regenerate one type of SM tissue
will necessarily be successful for other organ systems. Physiologically, there are distinct differences between
the SM in cardiovascular, gastrointestinal, and urinary tissues. Similar approaches have been utilized to
date in most SM engineering approaches. However, inherent differences exist in the microenvironment
of different SM tissues that may have a significant effect on the developing SM tissue. The substratum to
which SMCs adhere also plays an important role in the presentation of paracrine signals and that may
regulate SMC phenotype [35,37,111]. An ideal cell source is also currently lacking, but stem cells may fill
this need. BMSCs contain a population of SM progenitors, but the difficulties in reproducibly isolating
and regulating the differentiation of these cell populations pose as an obstacle to their use. Embryonic stem
cells may provide a more homogeneous population of pluripotential cells, but these cells have not yet been
demonstrated to form functional SM tissue. In addition, for engineered SM tissues to be truly functional,
they must also be innervated and vascularized. Current research in the therapeutic angiogenesis field may
provide methods to induce formation of vascular networks [81]. However, development of fully functional
SM tissues will also require a means to transduce neural signals critical for blood vessel vasoactivity.
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25
Esophagus: A Tissue
Engineering Challenge
B.D. Ratner
B.L. Beckstead
University of Washington
K.S. Chian
A.C. Ritchie
Nanyang Technological University
25.1 Medical Need/Clinical Problem......................... 25-1
25.2 Anatomy and Physiology of the Esophagus ............ 25-3
25.3 Criteria for a Tissue-Engineered Esophagus ........... 25-4
25.4 Scaffold Possibilities ..................................... 25-5
Background • Materials Selection • Scaffold Design
25.5 Fabrication Processes .................................... 25-7
Electrostatic Spinning • Cryogenic Molding • Rapid Freeze
Prototyping
25.6 Cell Possibilities .......................................... 25-10
Epithelial Characteristics • Epithelial Cell Source for Tissue
Engineering • Muscle Component of the Esophagus •
Engineering the Muscularis Mucosa and Externa •
Esophageal Regeneration
25.7 Bioreactors for Esophageal Tissue Engineering........ 25-15
Mechanical Conditioning of Smooth Muscle Tissue
Constructs
25.8 Conclusions and Prognostications ..................... 25-18
Acknowledgments............................................... 25-19
References ....................................................... 25-19
25.1 Medical Need/Clinical Problem
The esophagus, a muscular/mucosal tube connecting the mouth and pharynx to the stomach, is critical for
life (and good quality of life) (Figure 25.1). This seemingly simple organ is surgically challenging to repair
or replace. There are a number of conditions where surgical repair of the esophagus is indicated. These
include accident and trauma, congenital defects such as esophageal atresia (incomplete formation of the
esophagus) and tracheoesophageal fistulas and cancer. In 2003, roughly 14,000 people in the United States
were diagnosed with esophageal cancer. The prevalence of esophageal cancer in the general population
can be 10 to 100 times higher in Iran, China, Singapore, India, and South Africa. Worldwide, cancer of
the esophagus is the seventh leading cause of cancer death.
Surgical removal of a section of the esophagus and reconnection with the stomach, the most common
strategy for more advanced cancers, leads to complication rates as high as 40%. Strictures, dilation,
leakage, and infection are often observed. Attempts to use synthetics such as polyethylene, polypropylene,
teflon, or elastomers have also met with very limited success, problems being stenosis, leakage, infection,
25-1