5.539.

From Tissue to Organ Engineering

A Atala, Wake Forest University School of Medicine, Winston-Salem, NC, USA
ã 2011 Elsevier Ltd. All rights reserved.

5.539.1. Introduction 548

5.539.2. Biomaterials 548
5.539.2.1. Naturally Derived Materials 549
5.539.2.2. Acellular Tissue Matrices 549
5.539.2.3. Synthetic Polymers 549
5.539.3. Cells for Use in Tissue Engineering 549
5.539.3.1. Embryonic Stem Cells 549
5.539.3.2. Laboratory-Generated Stem Cells 550
5.539.3.3. Amniotic Fluid and Placental Stem Cells 551
5.539.3.4. Adult Stem Cells and Native Progenitor Cells 551
5.539.4. Cell Therapy with Injectable Substances 551
5.539.4.1. Bulking Agents 551
5.539.4.2. Injectable Muscle Cells 552
5.539.4.3. Endocrine Replacement 552
5.539.5. Tissue Engineering of Specific Structures 553
5.539.5.1. Urethra 553
5.539.5.2. Bladder 554
5.539.5.3. Kidney 556
5.539.5.4. Male Reproductive Organs 557
5.539.5.5. Female Reproductive Organs 558
5.539.5.6. Blood Vessels 558
5.539.5.7. Heart 558
5.539.5.8. Liver 559
5.539.5.9. Articular Cartilage and Trachea 559
5.539.6. Summary and Conclusion 560
References 560

Glossary Embryonic stem (ES) cell A broadly multipotent cell type

Acellular matrix Collagen-rich scaffolds prepared
by removing all cellular components from a donor
tissue.
Adult stem cell An undifferentiated cell found in postnatal
tissues that can self-renew as well as produce progeny that
can differentiate into the various cell types that comprise
the tissue.
Amniotic fluid stem (AFS) cell A multipotent cell type
found in the amniotic fluid surrounding an embryo; AFS
cells may be mesenchymal in origin and differentiation to
neural, hepatic, osteogenic, and chondrogenic lineages,
among others, has been demonstrated.
found in the inner cell mass of a blastocyst; ES cells can
differentiate into nearly every cell type in the body.
Induced pluripotent stem (iPS) cell A somatic cell that has
been dedifferentiated to a stem cell-like phenotype via the
introduction of functional gene products involved in
maintaining pluripotency.
Reproductive cloning The use of cloning procedures to
produce a live offspring that is a genetic copy of an adult
animal.
Therapeutic cloning The use of cloning procedures to
produce a blastocyst in vitro in order to obtain embryonic
stem cells for cell therapy or tissue engineering applications.

Abbreviations FDA Food and Drug Administration

AFPSC Amniotic fluid and placental stem cell FLM Fluorescent latex microspheres
AFS Amniotic fluid stem GIRK G-protein-gated inwardly rectifying potassium
CDC Centers for disease control iPS Induced pluripotent state
DNA Deoxyribonucleic acid IFN Interferon
ECM Extracellular matrix MEF Murine embryonic fibroblast
EliSPOT Enzyme-linked immunosorbent spot assay MMP Matrix metalloproteinase
ES Embryonic stem mtDNA Mitochondrial DNA

547
548 Organ Engineering
PGA Polyglycolic acid
PLA Polylactic acid
PLGA Poly(lactic-co-glycolic) acid
RGD Arginine-glycine-aspartate
5.539.1. Introduction
Patients suffering from diseased and injured organs may be
treated with transplanted organs. However, there is a severe
shortage of donor organs that is worsening yearly. As modern
medicine increases the human lifespan, the aging population
grows, and the need for organs grows with it, as aging organs
are generally more prone to failure. Scientists in the field of
regenerative medicine and tissue engineering apply the princi-
ples of cell transplantation, material science, and bioengineering
to construct biological substitutes that can prolong life by reduc-
ing mortality from these diseases. Therapeutic cloning, where the
nucleus from a donor cell is transferred into an enucleated oocyte
in order to extract pluripotent embryonic stem cells, offers a
potentially limitless source of cells for tissue engineering applica-
tions. The stem cell field is also advancing rapidly, opening new
options for therapy. This chapter reviews recent advances that
have occurred in regenerative medicine and describes applica-
tions of these new technologies that offer promise of a longer life.
The field of regenerative medicine encompasses various
areas of technology, such as tissue engineering, stem cells, and
cloning. Tissue engineering, one of the major components of
regenerative medicine, follows the principles of cell transplan-
tation, materials science, and engineering toward the develop-
ment of biological substitutes that can restore and maintain
normal function. Tissue engineering strategies generally fall
into two categories: the use of acellular matrices, which depend
on the body’s natural ability to regenerate for proper orienta-
tion and direction of new tissue growth, and the use of matrices
with cells. Acellular tissue matrices are usually prepared by
manufacturing artificial scaffolds, or by removing cellular com-
ponents from tissues via mechanical and chemical manipula-
tion to produce collagen-rich matrices.1–4 These matrices tend
to slowly degrade on implantation and are generally replaced
by the extracellular matrix (ECM) proteins that are secreted by
the ingrowing cells. Cells can also be used for therapy via
injection, either with carriers such as hydrogels, or alone.
When cells are used for tissue engineering, a small piece
of donor tissue is dissociated into individual cells. These cells
are either implanted directly into the host, or are expanded in
culture, attached to a support matrix, and then reimplanted
into the host after expansion. The source of donor tissue can be
heterologous (such as bovine), allogeneic (same species, dif-
ferent individual), or autologous. Ideally, both structural and
functional tissue replacement will occur with minimal compli-
cations. The preferred cells to use are autologous cells, where
a biopsy of tissue is obtained from the host, the cells are
dissociated and expanded in culture, and the expanded cells
are implanted into the same host.4–12 The use of autologous
cells, although it may cause an inflammatory response, avoids
rejection, and thus, the deleterious side effects of immunosup-
pressive medications can be avoided.
RNA Ribonucleic acid
RT-PCR Reverse transcription polymerase chain
reaction
SCNT Somatic cell nuclear transfer
Most current strategies for tissue engineering depend upon
a sample of autologous cells from the diseased organ of the host.
Aging itself is not a barrier to successful applications of engi-
neered tissues, as cells can be expanded from young and old
patients alike. However, for many patients with extensive end-
stage organ failure, a tissue biopsy may not yield enough normal
cells for expansion and transplantation. In other instances, pri-
mary autologous human cells cannot be expanded from a par-
ticular organ, such as the pancreas. In these situations, stem cells
are envisioned as being an alternative source of cells from which
the desired tissue can be derived. Stem cells can be derived from
discarded human embryos (human embryonic stem cells), from
fetal tissue, or from adult sources (bone marrow, fat, skin).
Therapeutic cloning has also played a role in the develop-
ment of the field of regenerative medicine. This type of cloning,
which has also been called nuclear transplantation and nuclear
transfer, involves the introduction of a nucleus from a donor
cell into an enucleated oocyte to generate an embryo with a
genetic makeup identical to that of the donor. Stem cells can be
derived from this source, which may have the future potential
to be used therapeutically.
5.539.2. Biomaterials
For cell-based tissue engineering, the expanded cells are seeded
onto a scaffold synthesized with the appropriate biomaterial.
In tissue engineering, biomaterials replicate the biologic and
mechanical function of the native ECM found in tissues in the
body by serving as an artificial ECM. Biomaterials provide a
three-dimensional space for the cells to form into new tissues
with appropriate structure and function, and also can allow
for the delivery of cells and appropriate bioactive factors (e.g.,
cell-adhesion peptides, growth factors), to desired sites in the
body.13 As the majority of mammalian cell types are anchorage-
dependent and will die if no cell-adhesion substrate is available,
biomaterials provide a cell-adhesion substrate that can deliver
cells to specific sites in the body with high loading efficiency.
Biomaterials can also provide mechanical support against in vivo
forces such that the predefined three-dimensional structure is
maintained during tissue development. Furthermore, bioactive
signals, such as cell-adhesion peptides and growth factors, can be
loaded along with cells to help regulate cellular function.
The ideal biomaterial should be biodegradable and bio-
resorbable to support the replacement of normal tissue
without inflammation. Incompatible materials are destined
for an inflammatory or foreign-body response that eventually
leads to rejection and/or necrosis. Degradation products, if
produced, should be removed from the body via metabolic
pathways at an adequate rate that keeps the concentration of
these degradation products in the tissues at a tolerable level.14
The biomaterial should also provide an environment in which

appropriate regulation of cell behavior (adhesion, prolifera-
tion, migration, and differentiation) can occur such that func-
tional tissue can form. Cell behavior in the newly formed tissue
has been shown to be regulated by multiple interactions of
the cells with their microenvironment, including interactions
with cell-adhesion ligands15 and with soluble growth factors.
Since biomaterials provide temporary mechanical support
while the cells undergo spatial reorganization into tissue, the
properly chosen biomaterial should allow the engineered tis-
sue to maintain sufficient mechanical integrity to support itself
in early development, while in late development, it should
have begun degradation such that it does not hinder further
tissue growth.13
In general, three classes of biomaterials are used for engi-
neering tissues: naturally derived materials (e.g., collagen and
alginate), acellular tissue matrices (e.g., bladder submucosa
and small intestinal submucosa), and synthetic polymers
such as polyglycolic acid (PGA), polylactic acid (PLA), and
poly(lactic-co-glycolic acid) PLGA). These classes of biomater-
ials have been tested with respect to their biocompatibility.16,17
Naturally derived materials and acellular tissue matrices have
the potential advantage of biological recognition. However,
synthetic polymers can be produced reproducibly on a large
scale with controlled properties such as strength, degradation
rate, and microstructure.
5.539.2.1. Naturally Derived Materials
Collagen is the most abundant and ubiquitous structural protein
in the body, and may be readily purified from both animal and
human tissues with an enzyme treatment and salt/acid extrac-
tion.18 Collagen implants, under normal conditions, degrade
through a process involving phagocytosis of collagen fibrils by
fibroblasts.19 This is followed by sequential attack by lysosomal
enzymes including cathepsins B1 and D. Under inflammatory
conditions, the implants can be rapidly degraded largely by
matrix metalloproteinases (MMPs) and collagenases.19 How-
ever, the in vivo resorption rate of a collagen implant can be
regulated by controlling the density of the implant and the extent
of intermolecular cross-linking. The lower the density, the greater
the space between collagen fibers and the larger the pores for cell
infiltration, leading to a higher rate of implant degradation.
Collagen contains cell-adhesion domain sequences (e.g., RGD)
that may assist in retaining the phenotype and activity of many
types of cells, including fibroblasts20 and chondrocytes.21
Alginate, a polysaccharide isolated from seaweed, has been
used as an injectable cell delivery vehicle22 and a cell immobi-
lization matrix23 owing to its gentle gelling properties in the
presence of divalent ions, such as calcium. Alginate is relatively
biocompatible and approved by the Food and Drug Adminis-
tration (FDA) for human use as wound dressing material.
Alginate is a family of copolymers of D-mannuronate and
L-guluronate. The physical and mechanical properties of algi-
nate gel are strongly correlated with the proportion and length
of polygluronic block in the alginate chains.22
5.539.2.2. Acellular Tissue Matrices
Acellular tissue matrices are collagen-rich matrices prepared
by removing cellular components from tissues. The matrices
From Tissue to Organ Engineering 549
are often prepared by mechanical and chemical manipula-
tion of a segment of tissue.1–4 The matrices slowly degrade
upon implantation, and are replaced and remodeled by
ECM proteins synthesized and secreted by transplanted or
ingrowing cells.
5.539.2.3. Synthetic Polymers
Polyesters of naturally occurring a-hydroxy acids, including
PGA, PLA, and PLGA, are widely used in tissue engineering.
These polymers have gained FDA approval for human use in
a variety of applications, including sutures.24 The ester bonds
in these polymers are hydrolytically labile, and these polymers
degrade by nonenzymatic hydrolysis. The degradation pro-
ducts of PGA, PLA, and PLGA are nontoxic natural metabolites
and are eventually eliminated from the body in the form
of carbon dioxide and water.24 The degradation rate of these
polymers can be tailored from several weeks to several years
by altering crystallinity, initial molecular weight, and the
copolymer ratio of lactic to glycolic acid. Since these polymers
are thermoplastics, they can be easily formed into a three-
dimensional scaffold with a desired microstructure, gross
shape, and dimension by various techniques, including mold-
ing, extrusion, solvent casting,25 phase separation techniques,
and gas foaming techniques.26 Many applications in tissue
engineering often require a scaffold with high porosity and
ratio of surface area to volume. Other biodegradable synthetic
polymers, including poly(anhydrides) and poly(ortho-esters),
can also be used to fabricate scaffolds for tissue engineering
with controlled properties.27
5.539.3. Cells for Use in Tissue Engineering
5.539.3.1. Embryonic Stem Cells
Human embryonic stem cells exhibit two remarkable pro-
perties: the ability to proliferate in an undifferentiated but
pluripotent state (self-renewal), and the ability to differentiate
into many specialized cell types.28 They can be isolated by
aspirating the inner cell mass from the embryo during the
blastocyst stage (5 days postfertilization), and are usually
grown on feeder layers consisting of mouse embryonic fibro-
blasts (MEFs) or human feeder cells.29 More recent reports
have shown that these cells can be grown without the use of
a feeder layer30 and can thus avoid the exposure of these
human cells to mouse viruses and proteins. These cells have
demonstrated longevity in culture by maintaining their undif-
ferentiated state for at least 80 passages when grown using
current published protocols.31,32
Human embryonic stem cells have been shown to differen-
tiate into cells from all three embryonic germ layers in vitro.
Skin and neurons have been formed, indicating ectodermal
differentiation.33–36 Blood, cardiac cells, cartilage, endothelial
cells, and muscle have been formed, indicating mesodermal
differentiation.37–39 Pancreatic cells have been formed, indicat-
ing endodermal differentiation.40 In addition, as further evi-
dence of their pluripotency, embryonic stem cells can form
embryoid bodies, which are cell aggregations that contain all
three embryonic germ layers while in culture, and can form
teratomas in vivo.41
550 Organ Engineering

5.539.3.2. Laboratory-Generated Stem Cells because of immunologic incompatibility, and immunosup-

pressive drugs are usually required.55 The use of transplantable
In addition, stem cells for tissue engineering can be generated
tissue and organs derived from therapeutic cloning may poten-
through cloning procedures. There has been tremendous inter-
tially lead to the avoidance of immune responses that typically
est in the field of nuclear cloning since the birth of Dolly in
are associated with transplantation of nonautologous tissues.56
1997, but frogs were the first successfully cloned vertebrates
Although it looks promising, somatic cell nuclear trans-
derived from nuclear transfer42–45 but the nuclei were derived
fer technology has certain limitations that require further
from nonadult sources. In the past 15 years, tremendous
improvements before therapeutic cloning can be applied widely
advances in nuclear cloning technology have been reported,
in replacement therapy. Currently, the efficiency of the overall
indicating the relative immaturity of the field. Dolly was not
cloning process is low. The majority of embryos derived from
the first cloned mammal to be produced via nuclear transfer;
animal cloning do not survive after implantation.58–60 To
in fact, live lambs were produced in 1996 using nuclear transfer
improve cloning efficiency, further improvements are required
and differentiated epithelial cells derived from embryonic
in the multiple complex steps of nuclear transfer, such as enu-
discs.46 The significance of Dolly was that she was the first
cleation and reconstruction, activation of oocytes, and cell cycle
mammal to be derived from an adult somatic cell using nuclear
synchronization between donor cells and recipient oocytes.61
transfer.47 Since then, animals from several species have been
Recently, exciting reports of the successful transformation
grown using nuclear transfer technology, including cattle,48
of adult cells into pluripotent stem cells through a type of
goats,49 mice,50 and pigs.51,52
genetic “reprogramming” have been published. Reprogram-
Two types of nuclear cloning, reproductive cloning and
ming is a technique that involves dedifferentiation of adult
therapeutic cloning, have been described, and a better under-
somatic cells to produce patient-specific pluripotent stem
standing of the differences between the two types may help
cells, without the use of embryos. Cells generated by repro-
to alleviate some of the controversy that surrounds these
gramming would be genetically identical to the somatic cells
technologies.53,54 Banned in most countries for human appli-
(and thus, the patient who donated these cells) and would
cations, reproductive cloning is used to generate an embryo
not be rejected. Yamanaka was the first to discover that MEFs
that has the identical genetic material as its cell source. This
and adult mouse fibroblasts could be reprogrammed into
embryo can then be implanted into the uterus of a female
an ‘induced pluripotent state (iPS)’.62 This group used MEFs
to give rise to an infant that is a clone of the donor. On the
engineered to express a neomycin resistance gene from the
other hand, therapeutic cloning is used to generate early stage
Fbx15 locus, a gene expressed only in ES cells. They examined
embryos that are explanted in culture to produce embryonic
24 genes that were thought to be important for embryonic
stem cell lines whose genetic material is identical to that of its
stem cells and identified four key genes that, when introduced
source. These autologous stem cells have the potential to
into the reporter fibroblasts, resulted in drug-resistant cells.
become almost any type of cell in the adult body, and thus
These were Oct3/4, Sox2, c-MYC, and Klf4. This experiment
would be useful in tissue and organ replacement applica-
indicated that expression of the four genes in these transgenic
tions.55 Therefore, therapeutic cloning, which has also been
MEFs led to expression of a gene specific for ES cells. MEFs and
called somatic cell nuclear transfer, may provide an alternative
adult fibroblasts were cotransduced with retroviral vectors,
source of transplantable cells. Figure 1 shows the strategy
each carrying one of the four genes, and transduced cells were
of combining therapeutic cloning with tissue engineering to
selected via drug resistance. The resultant iPS cells possessed
develop tissues and organs. According to data from the Centers
the immortal growth characteristics of self-renewing ES cells,
for Disease Control (CDC), an estimated 3000 Americans
expressed genes specific for ES cells, and generated embryoid
die every day of diseases that could have been treated with
bodies in vitro and teratomas in vivo. When iPS cells were
stem cell-derived tissues56,57 With current allogeneic tissue
injected into mouse blastocysts, they contributed to a variety
transplantation protocols, rejection is a frequent complication
of cell types. However, although iPS cells selected in this way
were pluripotent, they were not identical to ES cells. Unlike ES
cells, chimeras made from iPS cells did not result in full-term
pregnancies. Gene expression profiles of the iPS cells showed
Therapeutic Cloning Strategies
that they possessed a distinct gene expression signature that
was different from that of ES cells. In addition, the epigenetic
Somatic
cell biopsy
Enucleated state of the iPS cells was somewhere between that found in
oocyte
somatic cells and that found in ES cells, suggesting that the
Patient Nuclear transfer reprogramming was incomplete.
These results were improved significantly by Wernig and
Transplantation Embryonic stem Jaenisch in July 2007.63 Fibroblasts were infected with retroviral
cells
vectors and selected for the activation of endogenous Oct4 or
Hematopoietic Nanog genes. Results from this study showed that DNA methyl-
cells Genetically matched tissue
Neurons ation, gene expression profiles, and the chromatin state of the
reprogrammed cells were similar to those of ES cells. Teratomas
Pancreatic
islet cells Hepatocytes induced by these cells contained differentiated cell types repre-
Cardiomyocytes senting all three embryonic germ layers. Most importantly, the
Renal cells
reprogrammed cells from this experiment were able to form
Figure 1 Strategy for therapeutic cloning and tissue engineering. viable chimeras and contribute to the germline-like ES cells,

suggesting that these iPS cells were completely reprogrammed.
Wernig et al. observed that the number of reprogrammed colo-
nies increased when drug selection was initiated later (day 20
rather than day 3 posttransduction). This suggests that repro-
gramming is a slow and gradual process and may explain why
previous attempts resulted in incomplete reprogramming.
It has recently been shown that reprogramming of human
cells is possible.64,65 Yamanaka showed that retrovirus-mediated
transfection of OCT3/4, SOX2, KLF4, and c-MYC generates
human iPS cells that are similar to hES cells in terms of mor-
phology, proliferation, gene expression, surface markers, and
teratoma formation. Thompson’s group showed that retroviral
transduction of OCT4, SOX2, NANOG, and LIN28 could gener-
ate pluripotent stem cells without introducing any oncogenes
(c-MYC). Both studies showed that human iPS were similar but
not identical to hES cells.
Another concern is that these iPS cells contain three
to six retroviral integrations (one for each factor), which
may increase the risk of tumorigenesis. Yamanaka et al.
studied the tumor formation in chimeric mice generated
from Nanog-iPS cells and found 20% of the offspring devel-
oped tumors due to the retroviral expression of c-MYC.66 An
alternative approach would be to use a transient expression
method, such as adenovirus-mediated system, since both
Jaenisch and Yamanaka showed strong silencing of the
viral-controlled transcripts in iPS cells.66,67 This indicates that
these viral genes are only required for the induction, not the
maintenance, of pluripotency.
Another concern is the use of transgenic donor cells for
reprogrammed cells in the mouse studies. Both studies used
donor cells from transgenic mice harboring a drug resistance
gene driven by Fbx15, Oct3/4, or Nanog promoters so that, if
these ES cell-specific genes were activated, the resulting cells
could be easily selected using neomycin. However, the use of
genetically modified donors hinders clinical applicability for
humans. To assess whether iPS cells can be derived from non-
transgenic donor cells, wild-type MEF and adult skin cells were
retrovirally transduced with Oct3/4, Sox2, c-MYC, and Klf4
and ES-like colonies were isolated by morphology alone, with-
out the use of drug selection for Oct4 or Nanog.67 iPS cells from
wild-type donor cells formed teratomas and generated live
chimeras. This study suggests that transgenic donor cells are
not necessary to generate iPS cells.
Although this is an exciting phenomenon, it is unclear why
reprogramming adult fibroblasts and mesenchymal stromal
cells have similar efficiencies.62 It would seem that cells that
are already multipotent could be reprogrammed with greater
efficiency, since the more undifferentiated donor nucleus the
better SCNT performs.68 This further emphasizes our limited
understanding of the mechanism of reprogramming, yet the
potential for this area of study is exciting.
5.539.3.3. Amniotic Fluid and Placental Stem Cells
An alternate source of stem cells is the amniotic fluid and
placenta. Amniotic fluid and the placenta are known to con-
tain multiple partially differentiated cell types derived from the
developing fetus. We isolated stem cell populations from these
sources, called amniotic fluid and placental stem cells (AFPSC)
that express embryonic and adult stem cell markers.69
From Tissue to Organ Engineering 551
The undifferentiated stem cells expand extensively without fee-
ders and double every 36 h. Unlike human embryonic stem
cells, the AFPSC do not form tumors in vivo. Lines maintained
for over 250 population doublings retained long telomeres and
a normal karyotype. AFS cells are broadly multipotent. Clonal
human lines verified by retroviral marking can be induced to
differentiate into cell types representing each embryonic germ
layer, including cells of adipogenic, osteogenic, myogenic, endo-
thelial, neuronal, and hepatic lineages. In this respect, they meet
a commonly accepted criterion for pluripotent stem cells, with-
out implying that they can generate every adult tissue. Examples
of differentiated cells derived from AFS cells and displaying
specialized functions include neuronal lineage secreting the
neurotransmitter L-glutamate or expressing G-protein-gated
inwardly rectifying potassium (GIRK) channels, hepatic lineage
cells producing urea, and osteogenic lineage cells forming
tissue-engineered bone. The cells could be obtained either
from amniocentesis or chorionic villous sampling in the devel-
oping fetus, or from the placenta at the time of birth. The cells
could be preserved for self-use, and used without rejection, or
they could be banked. A bank of 100,000 specimens could
potentially supply 99% of the US population with a perfect
genetic match for transplantation. Such a bank may be easier
to create than with other cell sources, since there are approxi-
mately 4.5 million births per year in the United States.69
5.539.3.4. Adult Stem Cells and Native Progenitor Cells
One of the limitations of applying cell-based regenerative
medicine techniques to organ replacement has been the inher-
ent difficulty of growing specific cell types in large quantities.
Even when some organs, such as the liver, have a high regener-
ative capacity in vivo, cell growth and expansion in vitro may be
difficult. By studying the privileged sites for committed precur-
sor cells in specific organs, as well as exploring the conditions
that promote differentiation, one may be able to overcome
the obstacles that limit cell expansion in vitro. For example,
urothelial cells could be grown in the laboratory setting in
the past, but only with limited expansion. Several protocols
were developed over the past two decades that identified the
undifferentiated cells, and kept them undifferentiated during
their growth phase.10,70–73 Using these methods of cell culture,
it is now possible to expand a urothelial strain from a single
specimen that initially covered a surface area of 1 cm2 to
one covering a surface area of 4202 m2 (the equivalent of one
football field) within 8 weeks.10 These studies indicated that
it should be possible to collect autologous bladder cells
from human patients, expand them in culture, and return
them to the donor in sufficient quantities for reconstructive
purposes.10,71–76 Major advances have been achieved within
the past decade on the possible expansion of a variety of primary
human cells, with specific techniques that make the use of
autologous cells possible for clinical application.
5.539.4. Cell Therapy with Injectable Substances
5.539.4.1. Bulking Agents
Injectable bulking agents can be endoscopically used in the
treatment of both urinary incontinence and vesicoureteral
552 Organ Engineering
reflux. The advantages in treating urinary incontinence and
vesicoureteral reflux with this minimally invasive approach
include the simplicity of this quick outpatient procedure and
the low morbidity associated with it. Several investigators are
seeking alternative implant materials that would be safe for
human use.77
The ideal substance for the endoscopic treatment of reflux
and incontinence should be injectable, nonantigenic, nonmi-
gratory, volume stable, and safe for human use. Toward this
goal long-term studies were conducted to determine the effect
of injectable chondrocytes in vivo.78 It was initially determined
that alginate, a liquid solution of gluronic and mannuronic
acid, embedded with chondrocytes, could serve as a synthetic
substrate for the injectable delivery and maintenance of carti-
lage architecture in vivo. Alginate undergoes hydrolytic biodeg-
radation and its degradation time can be varied depending on
the concentration of each of the polysaccharides. The use of
autologous cartilage for the treatment of vesicoureteral reflux
in humans would satisfy all the requirements for an ideal
injectable substance.
Chondrocytes derived from an ear biopsy can be readily
grown and expanded in culture. Neocartilage formation can be
achieved in vitro and in vivo using chondrocytes cultured on
synthetic biodegradable polymers. In these experiments, the
cartilage matrix replaced the alginate as the polysaccharide
polymer underwent biodegradation. This system was adapted
for the treatment of vesicoureteral reflux in a porcine model.79
These studies showed that chondrocytes can be easily harvested
and combined with alginate in vitro, the suspension can be
easily injected cystoscopically, and the elastic cartilage tissue
formed is able to correct vesicoureteral reflux without any
evidence of obstruction.
Two multicenter clinical trials were conducted using this
engineered chondrocyte technology. Patients with vesicoureteral
reflux were treated at ten centers throughout the US. The
patients had a similar success rate as with other injectable sub-
stances in terms of cure (Figure 5). Chondrocyte formation was
not noted in patients who had treatment failure. It is supposed
that the patients who were cured have a biocompatible region of
engineered autologous tissue present, rather than a foreign
material.80 Patients with urinary incontinence were also treated
endoscopically with injected chondrocytes at three different
medical centers. Phase 1 trials showed an approximate success
rate of 80% at follow-up 3 and 12 months postoperatively.81
Several of the clinical trials involving bioengineered products
have been placed on hold because of the costs involved with the
specific technology. With a bioengineered product, costs are
usually high because of the biological nature of the therapies
involved. As with any therapy, the cost that the medical health
care system can allow for a specific technology is limited. There-
fore, the costs of bioengineered products have to be lowered for
them to have an impact clinically. This is currently being
addressed for multiple tissue-engineered technologies. As the
technologies advance overtime, and the volume of the applica-
tion is considered, costs will naturally decrease.
5.539.4.2. Injectable Muscle Cells
The potential use of injectable cultured myoblasts for the treat-
ment of stress urinary incontinence has been investigated.82,83
Myoblasts were labeled with fluorescent latex microspheres
(FLM) in order to track them after injection. Labeled myoblasts
were directly injected into the proximal urethra and lateral
bladder walls of nude mice with a microsyringe in an open
surgical procedure. Tissue harvested up to 35 days postinjec-
tion contained the labeled myoblasts, as well as evidence
of differentiation of the labeled myoblasts into regenerative
myofibers. The authors reported that a significant portion of
the injected myoblast population persisted in vivo. Similar
techniques of sphincteric derived muscle cells have been used
for the treatment of urinary incontinence in a pig model.84 The
fact that myoblasts can be labeled and survive after injection
and begin the process of myogenic differentiation further sup-
ports the feasibility of using cultured cells of muscular origin as
an injectable bioimplant.
The use of injectable muscle precursor cells has also been
investigated for use in the treatment of urinary incontinence
due to irreversible urethral sphincter injury or maldevelop-
ment. Muscle precursor cells are the quiescent satellite cells
found in each myofiber that proliferate to form myoblasts
and eventually myotubes and new muscle tissue. Intrinsic
muscle precursor cells have previously been shown to play an
active role in the regeneration of injured striated urethral
sphincter.85 In a subsequent study, autologous muscle precur-
sor cells were injected into a rat model of urethral sphincter
injury, and both replacement of mature myotubes and restora-
tion of functional motor units were noted in the regenerating
sphincteric muscle tissue.86 This is the first demonstration of
the replacement of both sphincter muscle tissue and its inner-
vation by the injection of muscle precursor cells. As a result,
muscle precursor cells may be a minimally invasive solution
for urinary incontinence in patients with irreversible urinary
sphincter muscle insufficiency.
5.539.4.3. Endocrine Replacement
Patients with testicular dysfunction and hypogonadal disor-
ders are dependent on androgen replacement therapy to
restore and maintain physiological levels of serum testosterone
and its metabolites, dihydrotestosterone, and estradiol.87,88
Currently available androgen replacement modalities, such as
testosterone tablets and capsules, Depo-Provera injections, and
skin patches may be associated with fluctuating serum levels
and complications such as fluid and nitrogen retention, eryth-
ropoiesis, hypertension, and bone density changes.89 Since
Leydig cells of the testes are the major source of testosterone
in men, implantation of heterologous Leydig cells or gonadal
tissue fragments have previously been proposed as a method
for chronic testosterone replacement.90,91 These approaches,
however, were limited by the failure of the tissues and cells to
produce testosterone.
Encapsulation of cells in biocompatible and semiperme-
able polymeric membranes has been an effective method to
protect against a host immune response as well as to maintain
viability of the cells while allowing the secretion of desired
therapeutic agents.92,93 Alginate–poly-L-lysine-encapsulated
Leydig cell microspheres were used as a novel method for
testosterone delivery in vivo.88 Elevated stable serum testos-
terone levels were noted in castrated adult rats over the course
of the study, suggesting that microencapsulated Leydig cells

may be a potential therapeutic modality for testosterone
supplementation.
5.539.5. Tissue Engineering of Specific Structures
Investigators around the world, including those in our labora-
tory, have been working toward the development of several cell
types and tissues and organs for clinical application.
5.539.5.1. Urethra
Various strategies have been proposed over the years for the
regeneration of urethral tissue. Woven meshes of PGA without
cells94,95 or with cells96 were used to regenerate urethras in
various animal models. Naturally derived collagen-based mate-
rials such bladder-derived acellular submucosa,1 and an acellu-
lar urethral submucosa,97 have also been tried experimentally in
various animal models for urethral reconstruction.
The bladder submucosa matrix1 proved to be a suitable
graft for repair of urethral defects in rabbits. The neourethras
demonstrated a normal urothelial luminal lining and organized
muscle bundles. These results were confirmed clinically in a
series of patients with a history of failed hypospadias recon-
struction wherein the urethral defects were repaired with
human bladder acellular collagen matrices (Figure 2).98 The
neourethras were created by anastomosing the matrix in an
onlay fashion to the urethral plate. The size of the created
neourethra ranged from 5 to 15 cm. After a 3-year follow-up,
three of the four patients had a successful outcome with regard
to cosmetic appearance and function. One patient who had a
15-cm neourethra created developed a subglandular fistula.
The acellular collagen-based matrix eliminated the necessity
(a)
(b)
(c) (d)
Figure 2 Tissue engineering of the urethra using a collagen matrix.
(a) Representative case of a patient with a bulbar stricture. (b) During the
urethral repair surgery, strictured tissue is excised, preserving the
urethral plate on the left side, and matrix is anastamosed to the urethral
plate in an onlay fashion on the right. The boxes in both photos indicate
the area of interest, including the urethra, which appears white in the left
photograph. In the left photograph, the arrow indicates the area of
stricture in the urethra. On the right, the arrow indicates the repaired
stricture. Note that the engineered tissue now obscures the native
white urethral tissue in an onlay fashion in the right photograph.
(c) Urethrogram 6 months after repair. (d) Cystoscopic view of urethra
before surgery on the left side, and 4 months after repair on the
right side.
From Tissue to Organ Engineering 553
of performing additional surgical procedures for graft harvest-
ing, and both operative time and the potential morbidity from
the harvest procedure were decreased. Similar results were
obtained in pediatric and adult patients with primary urethral
stricture disease using the same collagen matrices.99 Another
study in 30 patients with recurrent stricture disease showed
that a healthy urethral bed (two or fewer prior urethral sur-
geries) was needed for successful urethral reconstruction using
the acellular collage-based grafts.100 More than 200 pediatric
and adult patients with urethral disease have been successfully
treated in an onlay manner with a bladder-derived collagen-
based matrix. One of its advantages over nongenital tissue grafts
used for urethroplasty is that the material is “off the shelf.” This
eliminates the necessity of additional surgical procedures for
graft harvesting, which may decrease operative time, as well as
the potential morbidity due to the harvest procedure.
The above techniques, using nonseeded acellular matrices,
were applied experimentally and clinically in a successful man-
ner for onlay urethral repairs. However, when tubularized
urethral repairs were attempted experimentally, adequate ure-
thral tissue regeneration was not achieved, and complications
ensued, such as graft contracture and stricture formation.101
Autologous rabbit bladder epithelial and smooth muscle cells
were grown and seeded onto preconfigured tubular matrices.
Entire urethra segments were resected and urethroplasties were
performed with tubularized collagen matrices either seeded
with cells, or without cells. The tubularized collagen matrices
seeded with autologous cells formed new tissue that was histo-
logically similar to native urethra. The tubularized collagen
matrices without cells lead to poor tissue development, fibro-
sis, and stricture formation. These findings were confirmed
clinically. A clinical trial using tubularized nonseeded SIS
for urethral stricture repair was performed in eight evaluable
patients. Two patients with short inflammatory strictures main-
tained urethral patency. Stricture recurrence developed in the
other six patients within 3 months of surgery.102 Other cell
types have also been tried experimentally in acellular bladder
collagen matrices, including foreskin epidermal cells and oral
keratinocytes.103,104 Vascular endothelial growth factor gene-
modified urothelial cells have also been used experimentally
for urethral reconstruction.105
The normal wound healing response to injury has been
studied extensively, and this knowledge has been helpful in
maximizing success for the engineering of tissues. At the time
of tissue injury, cell ingrowth is initiated from the wound edges
in order to cover the tissue defect. The cells from the edges of
the native tissue are able to traverse short distances without any
detrimental effects. If the wound is large, more than a few milli-
meters in distance or depth, increased collagen deposition, fibro-
sis, and scar formation ensue. Matrices implanted in wound beds
are able to lengthen the distances that cells can traverse without
initiating an adverse fibrotic response. However, these distances
are also limited. The maximum distance that adjacent cells from
the wound edge have to travel to create normal tissue over a
biologic matrix is approximately 1 cm.106 Tissue defects greater
than 1 cm, that are treated with a matrix alone, without cells,
usually have increased collagen deposition, increased fibrosis,
and scar formation. Cell-seeded matrices implanted in wound
beds are able to further lengthen the distance for normal tissue
formation, without initiating an adverse fibrotic response.
554 Organ Engineering
Studies in the field of regenerative medicine have shown that
very large defects, greater than 30 cm, can be successfully treated
using cell-seeded scaffolds. This explains the described experi-
mental and clinical results noted with urethral repair. Non-
seeded matrices are able to replace urethral segments when
used in an onlay fashion because of the short distances required
for tissue ingrowth. However, if a tubularized urethral repair is
needed, the matrices need to be seeded with autologous cells in
order to avoid the risk of stricture formation and poor tissue
development.
5.539.5.2. Bladder
Currently, gastrointestinal segments are commonly used as
tissues for bladder replacement or repair. However, gastroin-
testinal tissues are designed to absorb specific solutes, whereas
bladder tissue is designed for the excretion of solutes. Due to
the problems encountered with the use of gastrointestinal
segments, numerous investigators have attempted alternative
materials and tissues for bladder replacement or repair.
Over the last few decades, several bladder wall substitutes
have been attempted with both synthetic and organic materi-
als. Synthetic materials that have been tried in experimental
and clinical settings include polyvinyl sponge, Teflon, collagen
matrices, Vicryl (PGA) matrices, and silicone. Most of these
attempts have failed because of mechanical, structural, func-
tional, or biocompatibility problems. Usually, permanent syn-
thetic materials used for bladder reconstruction succumb to
mechanical failure and urinary stone formation, and use of
degradable materials lead to fibroblast deposition, scarring,
graft contracture, and a reduced reservoir volume overtime.7,107
Because of this, there has been an increase in the use of various
collagen-based matrices for tissue regeneration. Nonseeded
allogeneic acellular bladder matrices have served as scaffolds
for the ingrowth of host bladder wall components. The matri-
ces are prepared by mechanically and chemically removing all
cellular components from bladder tissue.3,4,108–110 The matri-
ces serve as vehicles for partial bladder regeneration, and rele-
vant antigenicity is not evident. However, in multiple studies
using various materials as nonseeded grafts for cystoplasty, the
urothelial layer was able to regenerate normally, but the muscle
layer, although present, was not fully developed.4,108,109,111–113
Studies involving acellular matrices that may provide the nec-
essary environment to promote cell migration, growth, and
differentiation are being conducted.114 With continued blad-
der research in this area, these matrices may have a clinical role
in bladder replacement in the future.
Cell-seeded allogeneic acellular bladder matrices were used
for bladder augmentation in dogs.4 The regenerated bladder
tissues contained a normal cellular organization consisting
of urothelium and smooth muscle and exhibited a normal
compliance. Biomaterials preloaded with cells before their
implantation showed better tissue regeneration compared
with biomaterials implanted with no cells, in which tissue
regeneration depended on ingrowth of the surrounding tissue.
The bladders showed a significant increase (100%) in capacity
when augmented with scaffolds seeded with cells, compared
to scaffolds without cells (30%). The acellular collagen matri-
ces can be enhanced with growth factors to improve bladder
regeneration.115
It has been well established for decades that the bladder is
able to regenerate generously over free grafts. Urothelium is
associated with a high reparative capacity.116 Bladder muscle
tissue is less likely to regenerate in a normal fashion. Both
urothelial and muscle ingrowth are believed to be initiated
from the edges of the normal bladder toward the region
of the free graft.117,118 Usually, however, contracture or resorp-
tion of the graft has been evident. The inflammatory response
toward the matrix may contribute to the resorption of the free
graft. It was hypothesized that building the three-dimensional
structure constructs in vitro, before implantation, would facili-
tate the eventual terminal differentiation of the cells after
implantation in vivo and would minimize the inflammatory
response toward the matrix, thus avoiding graft contracture
and shrinkage. The dog study demonstrated a major difference
between matrices used with autologous cells (tissue-engineered
matrices) and those used without cells.4 Matrices implanted
with cells for bladder augmentation retained most of their
implanted diameter, as opposed to matrices implanted without
cells for bladder augmentation, in which graft contraction
and shrinkage occurred. The histomorphology demonstrated a
marked paucity of muscle cells and a more aggressive inflamma-
tory reaction in the matrices implanted without cells. Epithelial-
mesenchymal signaling is important for the differentiation of
bladder smooth muscle.119
The results of initial studies showed that the creation of
artificial bladders may be achieved in vivo; however, it could
not be determined whether the functional parameters noted
were caused by the augmented segment or by the intact native
bladder tissue. To better address the functional parameters of
tissue-engineered bladders, an animal model was designed that
required a subtotal cystectomy with subsequent replacement
with a tissue-engineered organ.11 Cystectomy-only and non-
seeded controls maintained average capacities of 22% and 46%
of preoperative values, respectively. An average bladder capac-
ity of 95% of the original precystectomy volume was achieved
in the cell-seeded tissue-engineered bladder replacements.
These findings were confirmed radiographically (Figure 3).
The subtotal cystectomy reservoirs that were not reconstructed
and the polymer-only reconstructed bladders showed a marked
decrease in bladder compliance (10% and 42% total compli-
ance). The compliance of the cell-seeded tissue-engineered
bladders showed almost no difference from preoperative
values that were measured when the native bladder was present
(106%). Histologically, the nonseeded scaffold bladders pre-
sented a pattern of normal urothelial cells with a thickened
fibrotic submucosa and a thin layer of muscle fibers. The
retrieved tissue-engineered bladders showed a normal cellular
organization, consisting of a trilayer of urothelium, submu-
cosa, and muscle. Immunocytochemical analyses confirmed
the muscle and urothelial phenotype. S-100 staining indicated
the presence of neural structures.11 These studies, performed
with polyglocolic acid based-scaffolds, have been repeated by
other investigators, showing similar long-term results in a large
number of animals.111,120 The strategy of using biodegradable
scaffolds with cells can be pursued without concerns for local
or systemic toxicity.121 However, not all scaffolds perform well
if a large portion of the bladder needs replacement. In a study
using SIS for subtotal bladder replacement in dogs, both the
unseeded and cell-seeded experimental groups showed graft
 From Tissue to Organ Engineering 555

shrinkage and poor results.122 The type of scaffold used is

Subtotal Polymer only Tissue-engineered
cystectomy only implants neobladder
11M 11M 11M
Figure 3 Gross specimens and cystograms at 11 months of the
cystectomy-only, nonseeded controls, and cell-seeded tissue-engineered
bladder replacements in dogs. The cystectomy-only bladder had a
capacity of 22% of the preoperative value and a decrease in bladder
compliance to 10% of the preoperative value. The nonseeded controls
showed significant scarring with a capacity of 46% of the preoperative
value and a decrease in bladder compliance to 42% of the preoperative
value. An average bladder capacity of 95% of the original precystectomy
volume was achieved in the cell-seeded tissue-engineered bladder
replacements and the compliance showed almost no difference from
preoperative values that were measured when the native bladder was
present (106%).
(a) (b) (c)
Figure 4 Construction of engineered bladder. (a) Scaffold material
seeded with cells for use in bladder repair. (b) The seeded scaffold is
anastamosed to native bladder with running 4–0 polyglycolic sutures.
(c) Implant covered with fibrin glue and omentum.
critical for the success of these technologies. The use of bior-
eactors, in which mechanical stimulation is started at the time
of organ production, has also been proposed as an important
parameter for success.123
A clinical experience involving engineered bladder tissue for
cystoplasty reconstruction was conducted starting in 1998.
A small pilot study of seven patients was reported, using a
collagen scaffold seeded with cells either with or without omen-
tum coverage, or a combined PGA-collagen scaffold seeded
with cells and omental coverage (Figure 4). The patients
reconstructed with the engineered bladder tissue created with
the PGA-collagen cell-seeded scaffolds with omental coverage
showed increased compliance, decreased end-filling pres-
sures, increased capacities, and longer dry periods overtime
(Figure 5).124 It is clear from this experience that the engineered
bladders continued their improvement with time, mirroring
their continued development. Although the experience is
promising in terms of showing that engineered tissues can be
implanted safely, it is just a start in terms of accomplishing the
goal of engineering fully functional bladders. This was a limited
clinical experience, and the technology is not yet ready for wide
dissemination, as further experimental and clinical studies are
required. FDA Phase 2 studies have now been completed.
An area of concern in the field of tissue engineering in the
past was the source of cells for regeneration. The concept of
creating engineered constructs involves initially obtaining cells
for expansion from the diseased organ. However, it was not
known until recently whether the cell population obtained
for later autologous implantation was normal and would
lead to normal tissue formation. For example, would the
cells obtained from a neuropathic bladder lead to the engineer-
ing of another neuropathic bladder? Cultured neuropathic
bladder smooth muscle cells possess and maintain different
characteristics than normal smooth muscle cells in vitro, as
demonstrated by growth assays, contractility, adherence tests,
and microarray analysis.125–127 However, when neuropathic
smooth muscle cells were cultured in vitro, seeded onto matri-
ces and implanted in vivo, the tissue-engineered constructs

Pves
30 cnH20/Div

3 min/div
t1 t3 t4 t6 t8 t9 11 13
t2 t5 t7 10 12

Pves
30 cnH20/Div

5 min/div
t1 t2 t4 t5
t3

Figure 5 Cystograms and urodynamic studies of a patient before and after implantation of the tissue-engineered bladder. (a) Preoperative results
indicate an irregular-shaped bladder in the cystogram (left) and abnormal bladder pressures as the bladder is filled during urodynamic studies (right).
(b) Postoperatively, findings are significantly improved.
556 Organ Engineering
showed the same properties as the tissues engineered with
normal cells.128 The progenitor cells, which reside within
stem cell niches within each organ, are responsible for new
cell differentiation and tissue formation during the normal
process of tissue regeneration due to natural turnover, aging,
and tissue injury. It is known that genetically normal nonma-
lignant progenitor cells, which are the reservoirs for new cell
formation, are programmed to give rise to normal tissue
regardless of whether the niche resides in either normal or
diseased tissues.128–130 Therefore, although the mechanisms
for tissue self-assembly and regenerative medicine are not
fully understood, it is known that the progenitor cells are
able to “reset” their program for normal cell differentiation.
The stem cell niche and its role in normal tissue regeneration
remains a fertile area of ongoing investigation.
5.539.5.3. Kidney
The kidney is a complex organ with multiple cell types, derived
from combining anlagen and a complex functional anatomy
that renders it one of the most difficult to reconstruct.5,131
While the metanephros is responsible for the development of
the proximal section of the nephrons, the ureteric bud forms
the collecting ducts and distal structures. The large vessels
of the kidney are induced from extrarenal tissues. Divergent
embryologic origin converges to produce at least 26 distinct
functional cells in the kidney.132 Previous efforts in tissue
engineering of the kidney have been directed toward the devel-
opment of extracorporeal renal support systems made of
biological and synthetic components133–141 and these ex vivo
renal replacement devices are known to be life-sustaining.
However, there would be obvious benefits for patients with
end-stage kidney disease if these devices could be implanted
long term without the need for an extracorporeal perfusion
circuit or immunosuppressive drugs.
We have explored the possibility of seeding an implantable
biomaterial with a heterogeneous population of renal cells to
evaluate function and viability. Atala et al. plated donor rabbit
kidney cells, including distal tubules, glomeruli, and proximal
tubules in vitro and after expansion, these were seeded onto
biodegradeable PGA scaffolds and implanted subcutaneously
into athymic mice. The implants consisted of individual cell
types and a mixture of all three. When examined histologically,
progressive formation and organization of the nephron seg-
ments within the polymer fibers was noted. Additionally, BrdU
incorporation into the renal cell DNA was confirmed.142 It was
unclear if the tubular structures found on the scaffolds
occurred de novo from the implanted cells or if they merely
represented fragments of donor tubules that had survived
intact the original dissociation and culture process. To further
investigate this question, mouse renal cells were harvested and
expanded in culture. Subsequently, a single cell suspension was
created from the isolated cells, and these were seeded on bio-
degradable polymers and implanted into immunocompetent
synergic hosts. In this experiment, renal epithelial cells recon-
stituted tubular structures in vivo. The analyses of the retrieved
implants indicated that the renal epithelial cells first organized
into a structure with a solid center. Next, canalization into a
hollow tube could be seen at 2 weeks. Histologic examination
with nephron-specific lactins revealed successful reconstitution
of proximal tubules, distal tubules, loops of Henle, collecting
tubules, and collecting ducts. These results clearly showed that
single cell suspensions grown in vitro are capable of reconsti-
tuting tubule structures. The tubules contained homogenous
cell types within each tubule.143
Further investigations by our group have demonstrated that
renal tubular development from digested renal units is possible
in the murine model as well. Joraku et al. developed an in vitro
method for cultivation of renal cells that allowed for develop-
ment of tubular structures. This technique involves digestion of
the entire murine kidney followed by cultivation of the result-
ing cells in a specific renal media. Immunohistochemistry
revealed cells expressing proximal and distal tubule markers
as well as markers for glomerular and endothelial cells. When
these cells were allowed to grow on rat-tail type 1 collagen, cells
from the thick ascending loop of Henle stained positive for
Tamm–Horsfall protein in an architectural pattern reminiscent
of natural tubules.144
Yoo et al. evaluated whether murine renal cells grown
in vitro could produce functional results in vivo. They harvested
the cells, expanded them in culture and seeded them onto
a tubular device constructed from polycarbonate. At one
end of the tubular device was a silastic catheter, which termi-
nated into a reservoir. The device was subcutaneously
implanted into athymic mice. The implanted device demon-
strated extensive vascularization in addition to glomerular
formation and highly organized tubular architecture. Immu-
nohistochemistry for alkaline phosphatase showed positivity
in the proximal tubule-like structures. Osteopontin, which is
secreted by the proximal and distal tubular cells and the cells of
the thin loop of Henle, was found on immunocytochemical
staining of the tubular sections. In addition, the ECM of the
newly formed tubules stained uniformly positive for fibronec-
tin. Importantly, fluid collected in the reservoir of the device.
This fluid was yellow and the uric acid concentration was
66 mg dl 1 (as compared to 2 mg dl 1 in plasma). The creati-
nine concentration of the fluid (27.91  7.56 mg dl 1) was
8.2 times higher than that found in the serum (4.49  0.08 mg
dl 1).145 These studies demonstrate that single cells can form
multicellular structures, become organized into functional
renal units, and are capable of unidirectional excretion of
solutes through production of a urine-like fluid.
Next, we applied the principles of both tissue engineering
and therapeutic cloning in an effort to produce genetically
identical renal tissue in a large animal model, the cow (Bos
taurus).146 Bovine skin fibroblasts from adult Holstein steers
were obtained by ear notch, and single donor cells were isolated
and microinjected into the perivitelline space of donor enu-
cleated oocytes (nuclear transfer). The resulting blastocysts were
implanted into progestin-synchronized recipients to allow for
further in vivo growth. After 12 weeks, cloned renal cells were
harvested, expanded in vitro, then seeded onto biodegradable
scaffolds consisting of polycarbonate membranes and three
collagen-coated cylindrical silastic catheters (Figure 6(a)). The
ends of the three membranes of each scaffold were connected to
catheters that terminated into a collecting reservoir. This created
a renal neoorgan with a mechanism for collecting the excreted
urinary fluid (Figure 6(b)). These devices were transplanted
subcutaneously into the same steer from which the genetic mate-
rial originated, and then retrieved 12 weeks after implantation.

Porous membrane
Silastic Coated
catheter collagen Glomerulus
Renal
units
Tubule
Reservoir
(a) (b)
Membrane
Allogeneic Cloned Autologous (c)
(d)
Figure 6 Combining therapeutic cloning and tissue engineering to
produce kidney tissue. (a) Illustration of the tissue-engineered renal unit.
(b) Renal unit seeded with cloned cells, 3 months after implantation,
showing the accumulation of urine-like fluid. (c) Clear unidirectional
continuity between the mature glomeruli, their tubules, and silastic
catheter. (d) Elispot analyses of the frequencies of T cells that secrete IFN
FX after stimulation with allogeneic renal cells, cloned renal cells, or
nuclear donor fibroblasts. Cloned renal cells produce fewer IFNg spots
than the allogeneic cells, indicating that the rejection response to cloned
cells is diminished. The presented wells are single representatives of
duplicate wells.
Chemical analysis of the collected urine-like fluid, includ-
ing urea nitrogen and creatinine levels, electrolyte levels,
specific gravity, and glucose concentration, revealed that
the implanted renal cells possessed filtration, reabsorption,
and secretory capabilities. Histological examination of the
retrieved implants revealed extensive vascularization and
self-organization of the cells into glomeruli and tubule-like
structures. A clear continuity between the glomeruli, the
tubules, and the silastic catheter was noted (Figure 6(c)).
Immunohistochemical analysis with renal-specific antibo-
dies revealed the presence of renal proteins, RT-PCR analysis
confirmed the transcription of renal-specific RNA in the
cloned specimens, and Western blot analysis confirmed the
presence of elevated renal-specific protein levels.
Since previous studies have shown that bovine clones harbor
oocyte mitochondrial DNA,147–149 the donor egg’s mitochon-
drial DNA (mtDNA) was thought to be a potential source of
immunologic incompatibility. Differences in mtDNA-encoded
proteins expressed by cloned cells could stimulate a T cell
response specific for mtDNA-encoded minor histocompatibility
antigens when the cloned cells are implanted back into the
original nuclear donor.150 Maternally transmitted minor histo-
compatibility antigens in mice have been shown to stimulate
both skin allograft rejection in vivo and cytotoxic T lympho-
cytes expansion in vitro150 that could prevent the use of these
cloned constructs in patients with chronic rejection of major
histocompatibility matched human renal transplants.151,152
We tested for a possible T cell response to the cloned renal
devices using delayed-type hypersensitivity testing in vivo and
EliSPOT analysis of interferon-g secreting T cells in vitro.
Both analyses revealed no evidence of a T cell response, indi-
cating that rejection of cloned renal cells will not necessarily
occur in the presence of oocyte-derived mtDNA (Figure 6(d)).
This finding may represent a step forward in overcoming the
histocompatibility problem of stem cell therapy.152
From Tissue to Organ Engineering 557
These studies demonstrated that cells derived from nuclear
transfer can be successfully harvested, expanded in culture, and
transplanted in vivo with the use of biodegradable scaffolds on
which the single suspended cells can organize into tissue struc-
tures that are genetically identical to that of the host. These
studies were the first demonstration of the use of therapeutic
cloning for regeneration of tissues in vivo.
Investigations into the possibility of using other cell types to
produce renal tissue have also been promising. Perin et al. have
been able to induce AFPSC to differentiate into renal tissues.
They labeled human AFPSC with a green fluorescent protein
and microinjected the cells into developing murine embryonic
kidneys. They found that the labeled cells assisted in forming
both C- and S- shaped bodies, and the cells expressed RNA for
early markers of renal development.153 This study demon-
strates that AFPSC can differentiate into renal lineages when
cultured in vitro with renal precursors. Although it looks
promising, further in vivo studies and functional assays are
required prior to clinical applications with AFPSC. For more
information on renal tissue engineering strategies, please see
Chapter 5.540, Kidney Tissue Engineering in this book.
5.539.5.4. Male Reproductive Organs
Reconstructive surgery is required for a wide variety of patho-
logic penile conditions, including penile carcinoma, trauma,
severe erectile dysfunction, and congenital conditions such as
ambiguous genitalia, hypospadias, and epispadias. One of the
major limitations of phallic reconstructive surgery is the avail-
ability of sufficient autologous tissue. Nongenital autologous
tissue sources have been used for decades. Phallic reconstruc-
tion was initially attempted in the late 1930s, with rib cartilage
used as a stiffener for patients with traumatic penile loss,154,155
but this process produced unsatisfactory functional and cos-
metic results. Silicone rigid prostheses were popularized in the
1970s and have been used widely.156,157 However, biocompat-
ibility issues have been a problem in selected patients.158,159
Tissue transfer techniques with flaps from various nongenital
sources such as the groin and forearm have been used for genital
reconstruction.160 However, operative complications such as
infection, graft failure, and donor site morbidity are not negligi-
ble. Phallic reconstruction with autologous tissue, derived from
the patient’s own cells, may be preferable in selected cases.
One of the major components of the phallus is corporal
smooth muscle. Initial experiments have shown that cultured
human corporal smooth muscle cells may be used in conjunc-
tion with biodegradable polymers to create corpus cavernosum
tissue de novo.161 In a subsequent study, human corporal
smooth muscle cells and endothelial cells seeded on biode-
gradable polymer scaffolds were able to form vascularized
cavernosal muscle when implanted in vivo.162 Later, in order
to minimize any immune reactions that a synthetic biomaterial
might cause, naturally derived acellular corporal tissue matrix
with the same architecture as native corpora was developed.
Acellular collagen matrices were derived from processed donor
rabbit corpora using cell lysis techniques. Human corpus caver-
nosal muscle and endothelial cells were derived from donor
penile tissue, and the cells were expanded in vitro and seeded
on the acellular matrices. The matrices were covered with the
appropriate cell architecture 4 weeks after implantation.163
558 Organ Engineering

In order to look at the functional parameters of the 5.539.5.5. Female Reproductive Organs
engineered corpora, acellular corporal collagen matrices were
Congenital malformations of the uterus may have profound
obtained from donor rabbit penis and autologous corpus
implications clinically. Patients with cloacal exstrophy and
cavernosal smooth muscle and endothelial cells were har-
intersex disorders may not have sufficient uterine tissue pres-
vested, expanded, and seeded on the matrices. An entire
ent for future reproduction. We investigated the possibility
cross-sectional segment of protruding rabbit phallus was
of engineering functional uterine tissue using autologous
excised, leaving the urethra intact. Cell-seeded matrices were
cells.167 Autologous rabbit uterine smooth muscle and epithe-
interposed into the excised corporal space. Functional and
lial cells were harvested, then grown, and expanded in culture.
structural parameters (cavernosography, cavernosometry, mat-
These cells were seeded onto preconfigured uterine-shaped
ing behavior, and sperm ejaculation) were followed, and
biodegradable polymer scaffolds, which were then used for
histological, immunocytochemical, and Western blot analyses
subtotal uterine tissue replacement in the corresponding autol-
were performed up to 6 months after implantation. The engi-
ogous animals. Upon retrieval 6 months after implantation,
neered corpora cavernosa achieved adequate structural and
histological, immunocytochemical, and Western blot analyses
functional parameters.164 This technology was further con-
confirmed the presence of normal uterine tissue components.
firmed when the entire rabbit corpora was removed and
Biomechanical analyses and organ bath studies showed that
replaced with the engineered scaffolds. Most interestingly, mat-
the functional characteristics of these tissues were similar to
ing activity in the animals with the engineered corpora
those of normal uterine tissue. Breeding studies using these
appeared normal by 1 month after implantation. The presence
engineered uteri are currently being performed.
of sperm was confirmed during mating, and was present
Similarly, several pathologic conditions, including congeni-
in all the rabbits with the engineered corpora. The female
tal malformations and malignancy, can adversely affect normal
rabbits mated with the animals implanted with engineered
vaginal development or anatomy. Vaginal reconstruction has
corpora and they conceived and delivered healthy pups.
traditionally been challenging due to the paucity of available
These studies demonstrate that penile corpora cavernosa tissue
native tissue. The feasibility of engineering vaginal tissue in vivo
can be engineered. The engineered tissue is able to achieve
was investigated.168 Vaginal epithelial and smooth muscle cells
adequate structural and functional parameters sufficient for
of female rabbits were harvested, grown, and expanded in cul-
erection, copulation, ejaculation, and conception in rabbits.
ture. These cells were seeded onto biodegradable polymer scaf-
Further studies will be needed to confirm the long-term func-
folds, and the cell-seeded constructs were then implanted into
tionality of these organs. In addition, further studies are
nude mice for up to 6 weeks. Immunocytochemical, histologi-
needed to show that comparable human structures can also
cal, and Western blot analyses confirmed the presence of vaginal
be engineered.
tissue phenotypes. Electrical field stimulation studies in the
Patients with testicular dysfunction require androgen
tissue-engineered constructs showed similar functional proper-
replacement for somatic development. Conventional treat-
ties to those of normal vaginal tissue. When these constructs
ment for testicular dysfunction consists of periodic intramus-
were used for autologous total vaginal replacement, patent vagi-
cular injections of chemically modified testosterone or, more
nal structures were noted in the tissue-engineered specimens,
recently, skin patch applications. However, long-term non-
while the noncell-seeded structures were noted to be stenotic.168
pulsatile testosterone therapy is not optimal and can cause
multiple problems, including excessive erythropoiesis and
bone density changes. To address the problem, a system was 5.539.5.6. Blood Vessels
designed in which Leydig cells, which produce most of Xenogenic or synthetic materials have been used as replace-
the testosterone in the male, were microencapsulated in an ment blood vessels for complex cardiovascular lesions. How-
alginate–poly-L-lysine solution and injected into castrated ani- ever, these materials typically lack growth potential, and may
mals. Serum testosterone was measured serially; the animals place the recipient at risk for complications such as stenosis,
were able to maintain testosterone levels in the long term.165 thromboembolization, or infection.169 Tissue-engineered vas-
These studies suggest that microencapsulated Leydig cells may cular grafts have been constructed using autologous cells and
be able to replace or supplement testosterone in situations biodegradable scaffolds and have been applied in dog and
where anorchia or testicular failure is present. Microencapsu- lamb models.170–173 The key advantage of using these auto-
lated Leydig cells offer several advantages. For example, the grafts is that they degrade in vivo, and thus allow the new tissue
encapsulation process creates a semipermeable barrier between to form without the long-term presence of foreign material.169
the transplanted cells and the host’s immune system, and it Application of these techniques from the laboratory to
allows for the long-term physiologic release of testosterone. the clinical setting has begun, with autologous vascular cells
Other studies have shown that testicular prostheses created harvested, expanded, and seeded onto a biodegradable scaf-
with chondrocytes in bioreactors could be loaded with testos- fold.174 The resultant autologous construct was used to replace
terone, and that these prostheses could provide controlled a stenosed pulmonary artery that had been previously repaired.
testosterone release into the bloodstream when implanted. Seven months after implantation, no evidence of graft occlu-
The prostheses were implanted in athymic mice with bilateral sion or aneurysmal changes was noted in the recipient.
anorchia, and testosterone was released long term, maintain-
ing the androgen level at a physiologic range.166 One could
5.539.5.7. Heart
envision combining the Leydig cell technology described
above with engineered prostheses for the long-term functional In the United States, over 5 million people currently live with
replacement of androgen levels. some form of heart disease, and many more are diagnosed each

year. While many medications have been developed to assist the
ailing heart, the treatment for end-stage heart failure still remains
transplantation. Unfortunately, as with other organs, donor
hearts are in short supply, and even when a transplant can be
performed, the patient must endure the side effects created by
lifelong immunosuppression. Thus, alternatives are desperately
needed, and the development of novel methods to regenerate or
replace damaged heart muscle using tissue engineering and
regenerative medicine techniques is an attractive option.
Cell therapy for infarcted areas of the heart is attractive, as
these methods involve a rather simple injection into the dam-
aged area of a patient’s heart, rather than a rigorous surgical
procedure, to complete. Various types of stem cells have been
investigated for their potential to regenerate damaged or dead
heart tissue in this manner. Skeletal muscle cells, bone marrow
stem cells (both mesenchymal and hematopoietic), amniotic
fluid stem cells, and embryonic stem cells have been used for
this purpose. In this technique, cells are suspended in a bio-
compatible matrix that can range from simple normal saline to
complex yet biocompatible hydrogels depending on the type
of injection to be performed. The cells are either injected into
the damaged area of the heart itself, or they are injected
into the coronary circulation with the hope that they will
home to the damaged area, take up residence there, and
begin to repair the tissue. However, injectable therapies have
been shown to be relatively inefficient, and cell loss is quite
substantial. Newer methods of tissue engineering include the
development of engineered “patches,” which comprise cells
adhered to a biomaterial that can theoretically be used to
replace the damaged area of the heart. These techniques hold
promise, but require further research into the optimal cell types
and biomaterials for this purpose before they can be used
extensively in the clinic (see Jawad et al.175 for an excellent
review of these methods).
However, the methods described above could only be used
in cases where a relatively small section of heart muscle was
damaged. In cases where a large area or even the whole heart
has become nonfunctional, a more radical approach may be
required. In these situations, the use of a bioartificial heart
would be ideal, as rejection would be avoided and the pro-
blems associated with a mechanical heart (such as throm-
boembolus formation) would be abolished. To this end, Ott
et al. recently developed a novel heart construct in vitro using
decellularized cadaveric hearts. By reseeding the tissue scaffold
that remained after a specialized decellularization process
with various types of cells that make up a heart (cardiomyo-
cytes, smooth muscle cells, endothelial cells, and fibrocytes)
and culturing the resulting construct in a bioreactor system
designed to mimic physiologic conditions, this group was
able to produce a construct that could generate pump function
on its own.176 This study suggests that production of bioartifi-
cial hearts may one day be possible.
5.539.5.8. Liver
The liver can sustain a variety of insults, including viral infection,
alcohol abuse, surgical resection of tumors, and acute drug-
induced hepatic failure. The current therapy for liver failure is
liver transplantation. However, this therapy is limited by the
shortage of donors and the need for lifelong immunosuppressive
From Tissue to Organ Engineering 559
therapy. Cell transplantation has been proposed as a potential
solution for liver failure. The fact that the liver has enormous
regenerative potential in vivo suggests that in the right envi-
ronment, it may be possible to expand liver cells in vitro in
sufficient quantities for tissue engineering.177 Many approaches
have been tried, including development of specialized media,
coculture with other cell types, identification of growth factors
that have proliferative effects on these cells, and culture on
three-dimensional scaffolds within bioreactors.177
Extracorporeal bioartificial liver devices that use porcine
hepatocytes have been designed and applied. These devices
are designed to filter and purify the patient’s blood as would
the patient’s own liver, and the blood is returned to the patient
in a manner similar to kidney dialysis. Another cell-based
approach is the injection of liver cell suspensions. This has
been performed in animal models. Intraportal hepatocyte
injection has also been used in patients with Crigler–Najjar
Syndrome Type 1178; however, complications such as portal
vein thrombosis and pulmonary embolism are major con-
cerns, especially when large cell numbers are used.179 Finally,
cells including stem cells, oval progenitor cells, and mature
hepatocytes have been seeded onto liver-shaped biocompati-
ble matrices to engineer artificial, implantable livers. These
have been tested in various animal models;180,181 however,
the transplantation efficiency as well as the functionality of
these constructs must be improved substantially before the
technology can be moved into the clinic.
5.539.5.9. Articular Cartilage and Trachea
Full-thickness articular cartilage lesions have limited healing
capacity and thus represent a difficult management issue for
the clinicians who treat adult patients with damaged articular
cartilage.182,183 Large defects can be associated with mechani-
cal instability and may lead to degenerative joint disease if left
untreated.184,185 Chondrocytes were expanded and cultured
onto biodegradable scaffolds to create engineered cartilage
for use in large osteochondral defects in rabbits.186 When
sutured to a subchondral support, the engineered cartilage
was able to withstand physiologic loading and underwent
orderly remodeling of the large osteochondral defects in
adult rabbits, providing a biomechanically functional tem-
plate that was able to undergo orderly remodeling when sub-
jected to quantitative structural and functional analyses.
Few treatment options are currently available for patients
who suffer from severe congenital tracheal pathology, such as
stenosis, atresia, and agenesis, due to the limited availability of
autologous transplantable tissue in the neonatal period. Tissue
engineering in the fetal period may be a viable alternative for
the surgical treatment of these prenatally diagnosed congential
anomalies, as cells could be harvested and grown into trans-
plantable tissue in parallel with the remainder of gestation.
Chondrocytes from both elastic and hyaline cartilage speci-
mens have been harvested from fetal lambs, expanded
in vitro, then dynamically seeded onto biodegradable scaf-
folds.187 The constructs were then implanted as replacement
tracheal tissue in fetal lambs. The resultant tissue-engineered
cartilage was noted to undergo engraftment and epithelializa-
tion, while maintaining its structural support and patency.
Furthermore, if native tracheal tissue is unavailable, engineered
560 Organ Engineering
cartilage may be derived from bone marrow-derived mesen-
chymal progenitor cells as well.188
5.539.6. Summary and Conclusion
Regenerative medicine efforts are currently underway experi-
mentally for virtually every type of tissue and organ within the
human body. As regenerative medicine incorporates the fields of
tissue engineering, cell biology, nuclear transfer, and materials
science, personnel who have mastered the techniques of cell
harvest, culture, expansion, transplantation, as well as polymer
design are essential for the successful application of these tech-
nologies to extend human life. Various tissues are at different
stages of development, with some already being used clinically,
a few in preclinical trials, and some in the discovery stage.
Recent progress suggests that engineered tissues may have an
expanded clinical applicability in the future and may represent a
viable therapeutic option for those who would benefit from the
life-extending benefits of tissue replacement or repair.
Acknowledgment
The author wishes to thank Jennifer L. Olson, Ph.D., for edito-
rial assistance with this manuscript.
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