Available online at www.sciencedirect.
com
Engineering organs
Anthony Atala1,2
Applications of regenerative medicine technology may offer
novel therapies for patients with injuries, end-stage organ
failure, or other clinical problems. Currently, patients suffering
from diseased and injured organs can be treated with
transplanted organs. However, there is a severe shortage of
donor organs that is worsening yearly as the population ages
and new cases of organ failure increase. Scientists in the field of
regenerative medicine and tissue engineering are now applying
the principles of cell transplantation, material science, and
bioengineering to construct biological substitutes that will
restore and maintain normal function in diseased and injured
tissues. The stem cell field is also advancing rapidly, opening
new avenues for this type of therapy. For example, therapeutic
cloning and cellular reprogramming may one day provide a
potentially limitless source of cells for tissue engineering
applications. Although stem cells are still in the research phase,
some therapies arising from tissue engineering endeavors have
already entered the clinical setting successfully, indicating the
promise regenerative medicine holds for the future.
Addresses
1
Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC
27157, United States
2
Department of Urology, Wake Forest University School of Medicine,
Medical Center Boulevard, Winston-Salem, NC 27157, United States
Corresponding author: Atala, Anthony (aatala@wfubmc.edu)
Current Opinion in Biotechnology 2009, 20:575–592
This review comes from a themed issue on
Tissue, cell and pathway engineering
Edited by Gail Naughton and Chandran Siddharthan
Available online 5th November 2009
0958-1669/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2009.10.003
Introduction
Patients suffering from diseased and injured organs may
be treated with transplanted organs. The use of one
body part for another or the exchange of parts from one
person to another was mentioned in the medical litera-
ture even in antiquity and captured the imagination of
many over time. The maintenance of organs in culture
was a major area of inquiry in the early 1900s. Charles
Lindbergh, the first pilot to successfully fly across the
Atlantic in the 1920s, joined forces with Alexis Carrel, a
Nobel Prize Winner in the field of Medicine, to inves-
tigate the potential of keeping organs alive ex vivo long
term [1].
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The kidney was the first entire organ to be replaced in
a human, in 1955 [2]. However, this transplant was
performed between identical twins, and thus it did not
address the immune response to transplanted organs. In
the early 1960s, Murray, who later received the Nobel
prize for his work, performed a nonrelated kidney trans-
plantation from a nongenetically identical patient into
another. This transplant, which overcame the immuno-
logic barrier, marked a new era in medical therapy and
opened the door for the use of transplantation as a means
of therapy for different organ systems. However, lack of
good immune-suppression, the inability to monitor and
control rejection, and the worsening donor shortage
spurred physicians and scientists to look for other
alternatives.
Synthetic materials were introduced to replace or rebuild
diseased tissues or parts in the human body. The advent
of new manmade materials, such as tetrafluoroethylene
(Teflon) and silicone, opened a new field which included
a wide array of devices that could be applied for human
use. However, although these devices could provide for
structural replacement, the functional component of the
original tissue was not achieved.
Meanwhile, new techniques for cell harvesting, culture,
and expansion were developed. Studies of the extracellu-
lar matrix and its interaction with cells, and with growth
factors and their ligands, led the way to a further un-
derstanding of cell and tissue growth and differentiation.
The concept of cell transplantation took hold in the
research arena, and culminated with the first human bone
marrow cell transplant in the 1970s. At this time, research-
ers began to combine the devices and materials with cell
biology concepts, creating a new field called tissue engin-
eering. As more scientists from different fields came
together with the common goal of tissue replacement,
the field of tissue engineering became more formally
established.
In the early 1980s, mouse embryonic stem cells were
discovered [3]. However, this new field remained rela-
tively dormant until the description of human embryonic
stem cells in 1998 [4]. The description of these cells led to
one of the most contested ethical debates in the field of
medicine. Then, in 1999, the world awoke to the startling
media announcement of the creation of the first cloned
mammal, a sheep named Dolly [5].
These fields — cell transplantation, tissue engineering,
stem cells, and nuclear transfer — had one unifying
concept: the regeneration of living tissues and organs.
Current Opinion in Biotechnology 2009, 20:575–592
576 Tissue, cell and pathway engineering
Thus in 1999, William Haseltine, then the Scientific
Founder and Chief Executive Officer of Human Genome
Sciences, coined the term ‘regenerative medicine’, in
effect bringing all these areas under one defining field
[6]. While organ transplantation remains a mainstay of
treatment for patients with severely compromised organ
function, the number of patients in need of treatment far
exceeds the organ supply, and this shortfall is expected to
worsen as the global population ages. However, recent
advances in regenerative medicine suggest that it can
provide new alternatives to donor organs. In the last two
decades, scientists have attempted to grow native and
stem cells, engineer tissues, and design treatment mod-
alities using regenerative medicine techniques for vir-
tually every tissue of the human body. This article
reviews some of the progress that has been achieved in
this field.
Cells for use in organ engineering
Stem cells
In 1981 pluripotent cells were found in the inner cell mass
of the human embryo, and the term ‘human embryonic
stem cell’ was coined [3]. Human embryonic stem cells
exhibit two remarkable properties: the ability to prolifer-
ate in an undifferentiated but pluripotent state (self-
renewal), and the ability to differentiate into many
specialized cell types [7]. This makes them an attractive
resource for regenerative medicine techniques. They can
be isolated by aspirating the inner cell mass from the
embryo during the blastocyst stage (five days postfertili-
zation), and are usually grown on feeder layers consisting
of mouse embryonic fibroblasts or human feeder cells [8].
More recent reports have shown that these cells can be
grown without the use of a feeder layer [9] and thus avoid
the exposure of these human cells to mouse viruses and
proteins. These cells have demonstrated longevity in
culture by maintaining their undifferentiated state for
at least 80 passages when grown using current published
protocols [4,10].
Human embryonic stem cells have been shown to differ-
entiate into cells from all three embryonic germ layers in
vitro. Skin and neurons have been formed, indicating
ectodermal differentiation [11–14]. Blood, cardiac cells,
cartilage, endothelial cells, and muscle have been formed,
indicating mesodermal differentiation [15–17]. Pancreatic
cells have been formed, indicating endodermal differen-
tiation [18]. In addition, as further evidence 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 [19].
The political controversy surrounding stem cells began in
1998 with the creation of human embryonic stem (hES)
cells derived from discarded embryos. In addition to the
ethical dilemma surrounding the use of embryonic stem
Current Opinion in Biotechnology 2009, 20:575–592
cells, their clinical application is also limited because they
represent an allogenic resource and thus have the poten-
tial to evoke an immune response. New stem cell tech-
nologies (such as cloning, or somatic cell nuclear transfer,
and reprogramming) promise to overcome this limitation.
Two types of nuclear cloning, reproductive cloning and
therapeutic cloning, have been described, and a better
understanding of the differences between the two types
may help to alleviate some of the controversy that sur-
rounds these technologies [20,21]. Banned in most
countries for human applications, reproductive cloning
is used to generate an embryo that has the identical
genetic material as its cell source. This embryo can then
be implanted into the uterus of a female to give rise to an
infant that is a clone of the donor. On the other hand,
therapeutic cloning is used to generate early stage
embryos that are explanted in culture to produce embryo-
nic stem cell lines whose genetic material is identical to
that of its source. These autologous stem cells have the
potential to become almost any type of cell in the adult
body, and thus would be useful in tissue and organ
replacement applications [22]. Therefore, therapeutic
cloning, which has also been called somatic cell nuclear
transfer, may provide an alternative source of transplan-
table cells. Figure 1 shows the strategy of combining
therapeutic cloning with tissue engineering to develop
tissues and organs.
While promising, somatic cell nuclear transfer technology
has certain limitations that require further improvements
before therapeutic cloning can be applied widely in
replacement therapy. Currently, the efficiency of the
overall cloning process is low. To improve cloning effi-
ciency, further improvements are required in the multiple
complex steps of nuclear transfer, such as enucleation and
reconstruction, activation of oocytes, and cell cycle syn-
chronization between donor cells and recipient oocytes
[23].
Recently, exciting reports of the successful transform-
ation of adult cells into pluripotent stem cells through a
type of genetic ‘reprogramming’ have been published.
Reprogramming is a technique that involves de-differen-
tiation of adult somatic cells to produce patient-specific
pluripotent stem cells, without the use of embryos. Cells
generated by reprogramming would be genetically iden-
tical to the somatic cells (and thus, the patient who
donated these cells) and would not be rejected. Yamanaka
was the first to discover that mouse embryonic fibroblasts
(MEFs) and adult mouse fibroblasts could be repro-
grammed into an ‘induced pluripotent state (iPS)’ [24].
The resultant iPS cells possessed the immortal growth
characteristics of self-renewing ES cells, expressed genes
specific for ES cells, and generated embryoid bodies in
vitro and teratomas in vivo. When iPS cells were injected
into mouse blastocysts, they contributed to a variety of
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 Engineering organs Atala 577

Figure 1

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 six months after repair. (D)
Cystoscopic view of urethra before surgery on the left side, and four months after repair on the right side.

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 that they possessed a distinct gene
expression signature that was different from that of ES
cells. In addition, the epigenetic state of the iPS cells was
somewhere between that found in somatic cells and that
found in ES cells, suggesting that the reprogramming was
incomplete. These results were improved significantly by
Wernig and Jaenisch in July 2007 [25] and these iPS cells
were completely reprogrammed. It has recently been
shown that reprogramming of human cells is possible
[26,27].
An alternate source of stem cells is the amniotic fluid and
placenta. Amniotic fluid and the placenta are known to
contain 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 [28]. The undifferentiated stem
cells expand extensively without feeders and double
every 36 hours. 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 multi-
potent. Clonal human lines verified by retroviral marking
can be induced to differentiate into cell types represent-
ing each embryonic germ layer, including cells of adipo-
genic, osteogenic, myogenic, endothelial, neuronal, and
hepatic lineages. In this respect, they meet a commonly
accepted criterion for pluripotent stem cells, without
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 amniocent-
esis or chorionic villous sampling in the developing 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 approximately 4.5 million births per year in the
USA [28].
Finally, it may be possible to employ adult stem cells in
the regenerative process. Adult stem cells, especially
hematopoietic stem cells, are the best understood cell
type in stem cell biology [29]. However, adult stem cell
research remains an area of intense study, as their poten-
tial for therapy may be applicable to a myriad of degen-
erative disorders. Within the past decade, adult stem cell

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578 Tissue, cell and pathway engineering
populations have been found in many adult tissues other
than the bone marrow and the gastrointestinal tract,
including the brain [30,31], skin [32], and muscle
[33]. Many other types of adult stem cells have been
identified in organs all over the body and are thought to
serve as the primary repair entities for their correspond-
ing organs [34]. The discovery of such tissue-specific
progenitors has opened up new avenues for research.
Research into adult stem cells has, however, progressed
slowly, mainly because investigators have had great
difficulty in maintaining adult non-mesenchymal stem
cells in culture.
Native targeted progenitor cells
One of the limitations of applying cell-based regenera-
tive medicine techniques to organ replacement has been
the inherent difficulty of growing specific cell types in
large quantities. Even when some organs, such as the
liver, have a high regenerative capacity in vivo, cell
growth and expansion in vitro may be difficult. By noting
the location of the progenitor cells, as well as by explor-
ing the conditions that promote differentiation and/or
self-renewal, it has been possible to overcome some of
the obstacles that limit cell expansion in vitro. One
example is the urothelial cell. Urothelial cells could
be grown in the laboratory setting in the past, but only
with limited success. It was believed that urothelial cells
had a natural senescence that was hard to overcome.
Several protocols have been developed over the last two
decades that have improved urothelial growth and
expansion [35–38]. A system of urothelial cell harvesting
was developed that does not use any enzymes or serum
and has a large expansion potential. Using these
methods of cell culture, it is possible to expand a
urothelial strain from a single specimen that initially
covers a surface area of 1 cm2 to one covering a surface
area of 4202 m2 (the equivalent area of one football field)
within eight weeks [35].
An additional advantage in using native cells is that they
can be obtained from the specific organ to be regenerated,
expanded, and used in the same patient without rejection,
in an autologous manner [35,39–55]. Bladder, ureter, and
renal pelvis cells can equally be harvested, cultured, and
expanded in a similar fashion. Normal human bladder
epithelial and muscle cells can be efficiently harvested
from surgical material, extensively expanded in culture,
and their differentiation characteristics, growth require-
ments, and other biologic properties can be studied
[35,37,38,48,49,56–63]. Major advances in cell culture
techniques have been made within the past decade,
and these techniques make the use of autologous cells
possible for clinical application. However, even now, not
all human cells can be grown or expanded in vitro. Liver,
nerve, and pancreas are examples of human tissues where
the technology is not yet advanced to the point where
these cells can be grown and expanded.
Current Opinion in Biotechnology 2009, 20:575–592
When native cells are used for tissue reconstitution, donor
tissue is dissociated into individual cells, which are either
implanted directly into the host or expanded in culture,
attached to a support matrix, and reimplanted after
expansion. The implanted tissue can be heterologous,
allogenic, or autologous. Ideally, this approach allows lost
tissue function to be restored or replaced in toto and with
limited complications [45]. Native cells and tissues are
usually preferable for reconstruction. In most cases, the
replacement of lost or deficient tissues with functionally
equivalent cells and tissues would improve the outcome
for these patients. This goal may be attainable with the
use of regenerative medicine techniques.
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 struc-
ture and function, and also can allow for the delivery of
cells and appropriate bioactive factors (e.g. cell-adhesion
peptides and growth factors), to desired sites in the body
[64]. As the majority of mammalian cell types are ancho-
rage-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 mech-
anical support against in vivo forces such that the pre-
defined 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.
Generally, three classes of biomaterials have been used
for engineering tissues and organs: naturally derived
materials, such as collagen and alginate; acellular tissue
matrices, such as bladder submucosa and small-intestinal
submucosa (SIS); and synthetic polymers, such as poly-
glycolic acid (PGA), poly-lactic acid (PLA), and poly(-
lactic-co-glycolic acid) (PLGA). Naturally derived
materials and acellular tissue matrices have the potential
advantage of biologic recognition, but synthetic polymers
can be produced reproducibly on a large scale with
controlled properties of strength, degradation rate, and
microstructure.
Collagen is the most abundant and ubiquitous structural
protein in the body, and it may be readily purified from
both animal and human tissues with an enzyme treatment
and salt/acid extraction [65]. Collagen has long been
known to exhibit minimal inflammatory and antigenic
responses [66], and it has been approved by the U.S. Food
and Drug Administration (FDA) for many types of
medical applications, including wound dressings and
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artificial skin [67]. This material can be processed into a
wide variety of structures such as sponges, fibers, and
films [68–70].
Alginate, a polysaccharide isolated from seaweed, has
been used as an injectable cell delivery vehicle [71]
and a cell immobilization matrix (Lim and Sun, 1980)
owing to its gentle gelling properties in the presence of
divalent ions such as calcium. Efforts have been made to
synthesize biodegradable alginate hydrogels with mech-
anical properties that are controllable in a wide range by
intermolecular covalent cross-linking and with cell-
adhesion peptides coupled to their backbones [72].
Recently, natural materials such as alginate and collagen
have been used as ‘bio-inks’ in a newly developed bio-
printing technique based on inkjet technology [73,74].
Using this technology, these scaffold materials can be
‘printed’ into a desired scaffold shape using a modified
inkjet printer. In addition, several groups have shown that
living cells can also be printed using this technology
[75,76]. This exciting technique can be modified so that
a three-dimensional construct containing a precise
arrangement of cells, growth factors, and extracellular
matrix material can be printed [77–79]. Such constructs
may eventually be implanted into a host to serve as the
backbone for a new tissue or organ.
Acellular tissue matrices are collagen-rich matrices pre-
pared by removing cellular components from tissues. The
matrices are often prepared by mechanical and chemical
manipulation of a segment of bladder tissue [51,80–82].
The matrices slowly degrade after implantation and are
replaced and remodeled by ECM proteins synthesized
and secreted by transplanted or ingrowing cells. Acellular
tissue matrices have been proved to support cell ingrowth
and regeneration of genitourinary tissues, including ure-
thra and bladder, with no evidence of immunogenic
rejection [82,83]. Because the structures of the proteins
(e.g. collagen and elastin) in acellular matrices are well
conserved and normally arranged, the mechanical proper-
ties of the acellular matrices are not significantly different
from those of native bladder submucosa [80].
Polyesters of naturally occurring a-hydroxy acids, in-
cluding PGA, PLA, and PLGA, are widely used in regen-
erative medicine. These polymers have gained FDA
approval for human use in a variety of applications,
including sutures [84]. The degradation products of
PGA, PLA, and PLGA are nontoxic, natural metabolites
that are eventually eliminated from the body in the form
of carbon dioxide and water [84]. Because these polymers
are thermoplastics, they can easily be formed into a three-
dimensional scaffold with a desired microstructure, gross
shape, and dimension by various techniques, including
molding, extrusion [85], solvent casting [86], phase sep-
aration techniques, and gas foaming techniques [87].
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Engineering organs Atala 579
More recently, techniques such as electrospinning have
been used to quickly create highly porous scaffolds in
various conformations [88–91]. However, one drawback
of the synthetic polymers is lack of biologic recognition.
As an approach toward incorporating cell recognition
domains into these materials, copolymers with amino
acids have been synthesized [92–94]. Other biodegrad-
able synthetic polymers, including poly(anhydrides) and
poly(ortho-esters), can also be used to fabricate scaffolds
with controlled properties [95].
Nanotechnology, which exploits the ability to use small
molecules that have distinct properties in a small scale,
has been used to create smart biomaterials for regenera-
tive medicine [96,97]. Nanoscaffolds have been manu-
factured specifically for bladder applications [98]. The
manufacturing of biomaterials can also lead to enhanced
cell alignment and tissue formation [89].
Tissue engineering of specific structures
Investigators around the world, including our laboratory,
have been working toward the development of several
cell types and tissues and organs for clinical application.
Urethra
Various strategies have been proposed over the years for
the regeneration of urethral tissue. Woven meshes of
PGA without cells [99,100] or with cells (Atala et al.
[39] #208) were used to regenerate urethras in various
animal models. Naturally derived collagen-based
materials such bladder-derived acellular submucosa
[82], and an acellular urethral submucosa [101], have also
been tried experimentally in various animal models for
urethral reconstruction.
The bladder submucosa matrix [82] was proved to be a
suitable graft for the repair of urethral defects in rabbits.
The neourethras demonstrated a normal urothelial lumi-
nal lining and organized muscle bundles. These results
were confirmed clinically in a series of patients with a
history of failed hypospadias reconstruction wherein the
urethral defects were repaired with human bladder acel-
lular collagen matrices (Figure 1) [44]. 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 three-year
follow-up, three of the four patients had a successful
outcome in regard to cosmetic appearance and function.
One patient who had a 15-cm neourethra developed a
subglanular fistula. The acellular collagen-based matrix
eliminated the necessity of performing additional surgical
procedures for graft harvesting, 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 [102]. Another study in
30 patients with recurrent stricture disease showed that a
Current Opinion in Biotechnology 2009, 20:575–592
580 Tissue, cell and pathway engineering
healthy urethral bed (two or fewer prior urethral
surgeries) was needed for successful urethral reconstruc-
tion using the acellular collage-based grafts [103]. 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 advan-
tages 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 har-
vesting, 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 manner for onlay urethral repairs. However,
when tubularized urethral repairs were attempted exper-
imentally, adequate urethral tissue regeneration was not
achieved, and complications ensued, such as graft con-
tracture and stricture formation [104]. 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 seeded
either with cells, or without cells. The tubularized col-
lagen matrices seeded with autologous cells formed new
tissue which was histologically similar to native urethra.
The tubularized collagen matrices without cells lead to
poor tissue development, fibrosis, 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 maintained
urethral patency. Stricture recurrence developed in the
other six patients within three months of surgery [105].
Other cell types have also been tried experimentally in
acellular bladder collagen matrices, including foreskin
epidermal cells and oral keratinocytes [106,107]. Vascular
endothelial growth factor gene-modified urothelial cells
have also been used experimentally for urethral recon-
struction [108].
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 millimeters in
distance or depth, increased collagen deposition, fibrosis,
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 [109]. Tissue defects greater than
Current Opinion in Biotechnology 2009, 20:575–592
1 cm, which 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 initiat-
ing an adverse fibrotic response. 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
experimental and clinical results noted with urethral
repair. Nonseeded 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.
Bladder
Over the last few decades, several bladder wall substitutes
have been attempted with both synthetic and organic
materials. 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, functional, or biocompatibility pro-
blems. Usually, permanent synthetic materials used for
bladder reconstruction succumb to mechanical failure and
urinary stone formation, and the use of degradable
materials leads to fibroblast deposition, scarring, graft con-
tracture, and a reduced reservoir volume over time
[46,110]. Because of this, there has been an increase in
the use of various collagen-based matrices for tissue regen-
eration. Nonseeded allogenic acellular bladder matrices
have served as scaffolds for the ingrowth of host bladder
wall components. The matrices are prepared by mechani-
cally and chemically removing all cellular components
from bladder tissue [51,81,83,111,112]. The matrices serve
as vehicles for partial bladder regeneration, and relevant
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
[51,83,111,113–115]. Studies involving acellular matrices
that may provide the necessary environment to promote
cell migration, growth, and differentiation are being con-
ducted [116]. With continued bladder research in this area,
these matrices may have a clinical role in bladder replace-
ment in the future.
Human urothelial and muscle cells can be expanded in
vitro, seeded onto polymer scaffolds, and allowed to
attach and form sheets of cells. The cell–polymer scaffold
can then be implanted in vivo. Histologic analysis indi-
cated that viable cells were able to self-assemble back
into their respective tissue types, and would retain their
native phenotype [41]. These experiments demonstrated,
for the first time, that composite layered tissue-engineered
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structures could be created de novo. Before this study, only
nonlayered structures had been created in the field of
regenerative medicine.
Cell-seeded allogenic acellular bladder matrices were
used for bladder augmentation in dogs [51]. The regen-
erated bladder tissues contained a normal cellular organ-
ization 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 matrices can
be enhanced with growth factors to improve bladder
regeneration [117].
It has been well established for decades that the bladder
is able to regenerate generously over free grafts. Urothe-
lium is associated with a high reparative capacity [118].
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 [119,120].
Usually, however, contracture or resorption 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
facilitate the eventual terminal differentiation of the cells
after implantation in vivo and would minimize the inflam-
matory 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 [51]. Matrices implanted with cells for bladder
augmentation retained most of their implanted diameter,
as opposed to matrices implanted without cells for blad-
der augmentation, in which graft contraction and shrink-
age occurred. The histomorphology demonstrated a
marked paucity of muscle cells and a more aggressive
inflammatory reaction in the matrices implanted without
cells. Epithelial–mesenchymal signaling is important for
the differentiation of bladder smooth muscle [121].
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 seg-
ment 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 [54]. Cystectomy-only and non-
seeded controls maintained average capacities of 22% and
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Engineering organs Atala 581
46% of preoperative values, respectively. An average
bladder capacity of 95% of the original precystectomy
volume was achieved in the cell-seeded tissue-engin-
eered bladder replacements. These findings were con-
firmed radiographically. 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 compliance). 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
presented 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, submucosa, and muscle. Immunocytochemi-
cal analyses confirmed the muscle and urothelial pheno-
type. S-100 staining indicated the presence of neural
structures [54]. These studies, performed with poly-gly-
colic acid based scaffolds, have been repeated by other
investigators, showing similar results in large numbers of
animals long term [114,122]. The strategy of using bio-
degradable scaffolds with cells can be pursued without
concerns for local or systemic toxicity [123]. 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 shrinkage
and poor results [124]. The type of scaffold used is critical
for the success of these technologies. The use of bio-
reactors, in which mechanical stimulation is started at the
time of organ production, has also been proposed as an
important parameter for success [125].
A clinical experience involving engineered bladder tis-
sue 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 omentum coverage, or a combined
PGA-collagen scaffold seeded with cells and omental
coverage (Figure 2). The patients reconstructed with the
engineered bladder tissue created with the PGA–col-
lagen cell-seeded scaffolds with omental coverage
showed increased compliance, decreased end-filling
pressures, increased capacities and longer dry periods
over time (Figure 3) [126]. It is clear from this experience
that the engineered bladders continued their improve-
ment with time, mirroring their continued development.
Although the experience is promising in terms of show-
ing that engineered tissues can be implanted safely, it is
just a start in terms of accomplishing the goal of engin-
eering 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.
Current Opinion in Biotechnology 2009, 20:575–592
582 Tissue, cell and pathway engineering

Figure 2

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 poly-glycolic sutures. (C) Implant covered with fibrin glue and omentum.

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 engineering 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 micro-
array analysis [127–129]. However, when neuropathic
smooth muscle cells were cultured in vitro, seeded onto
matrices and implanted in vivo, the tissue-engineered
constructs showed the same properties as the tissues
engineered with normal cells [130]. 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, ageing, and tissue injury. It is
known that genetically normal nonmalignant 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 [130–132]. 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 differen-
tiation. The stem cell niche and its role in normal tissue
regeneration remains a fertile area of ongoing investigation.
Genital tissues and organs
Reconstructive surgery is required for a wide variety of
pathologic penile conditions, including penile carcinoma,
trauma, severe erectile dysfunction, and congenital con-
ditions such as ambiguous genitalia, hypospadias, and
epispadias. One of the major limitations of phallic recon-
structive surgery is the availability of sufficient autologous

Figure 3

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.

Current Opinion in Biotechnology 2009, 20:575–592 www.sciencedirect.com


tissue. Nongenital autologous tissue sources have been
used for decades. Phallic reconstruction was initially
attempted in the late 1930s, with rib cartilage used as a
stiffener for patients with traumatic penile loss [133,134],
but this process produced unsatisfactory functional and
cosmetic results. Silicone rigid prostheses were popular-
ized in the 1970s and have been used widely [135,136].
However, biocompatibility issues have been a problem in
selected patients [137,138]. Tissue transfer techniques
with flaps from various nongenital sources such as the
groin and forearm have been used for genital reconstruc-
tion [139]. However, operative complications such as
infection, graft failure, and donor site morbidity are not
negligible. 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 conjunction with biodegradable polymers to
create corpus cavernosum tissue de novo [140]. In a
subsequent study, human corporal smooth muscle cells
and endothelial cells seeded on biodegradable polymer
scaffolds were able to form vascularized cavernosal
muscle when implanted in vivo [55]. Later, in order to
minimize any immune reactions that a synthetic bioma-
terial 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 tech-
niques. Human corpus cavernosal 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 four weeks after implantation [141].
In order to look at the functional parameters of the
engineered corpora, acellular corporal collagen matrices
were obtained from donor rabbit penis and autologous
corpus cavernosal smooth muscle and endothelial cells
were harvested, expanded and seeded on the matrices. An
entire cross-sectional segment of protruding rabbit phal-
lus was excised, leaving the urethra intact. Cell-seeded
matrices were interposed into the excised corporal space.
Functional and structural parameters (cavernosography,
cavernosometry, mating behavior, and sperm ejaculation)
were followed, and histological, immunocytochemical,
and Western blot analyses were performed up to six
months after implantation. The engineered corpora
cavernosa achieved adequate structural and functional
parameters [142]. This technology was further confirmed
when the entire rabbit corpora was removed and replaced
with the engineered scaffolds. Most interestingly, mating
activity in the animals with the engineered corpora
appeared normal by one month after implantation. The
presence of sperm was confirmed during mating, and was
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Engineering organs Atala 583
present in all the rabbits with the engineered corpora.
The female rabbits mated with the animals implanted
with engineered corpora and they conceived and deliv-
ered healthy pups. These studies demonstrate that penile
corpora cavernosa tissue can be engineered. The engin-
eered tissue is able to achieve adequate structural and
functional parameters sufficient for erection, copulation,
ejaculation, and conception in rabbits. Further studies
will be needed to confirm the long-term functionality of
these organs. In addition, further studies are needed to
show that comparable human structures can also be
engineered.
Testis
Patients with testicular dysfunction require androgen
replacement for somatic development. Conventional
treatment for testicular dysfunction consists of periodic
intramuscular injections of chemically modified testos-
terone or, more recently, skin patch applications. How-
ever, long-term nonpulsatile testosterone therapy is not
optimal and can cause multiple problems, including
excessive erythropoiesis and bone density changes. To
address the problem, a system was designed in which
Leydig cells, which produce most of the testosterone in
the male, were microencapsulated in an alginate-poly-L-
lysine solution and injected into castrated animals. Serum
testosterone was measured serially; the animals were able
to maintain testosterone levels in the long term [50].
These studies suggest that microencapsulated Leydig
cells may be able to replace or supplement testosterone
in situations where anorchia or testicular failure is present.
Microencapsulated Leydig cells offer several advantages.
For example, the encapsulation process creates a semi-
permeable barrier between the transplanted cells and the
host’s immune system, and it allows for the long-term
physiologic release of testosterone.
Other studies have shown that testicular prostheses cre-
ated with chondrocytes in bioreactors could be loaded
with testosterone, and that these prostheses could provide
controlled testosterone release into the bloodstream
when implanted. The prostheses were implanted in
athymic mice with bilateral anorchia, and testosterone
was released long term, maintaining the androgen level at
a physiologic range [143]. One could envision combining
the Leydig cell technology described above with engin-
eered prostheses for the long-term functional replace-
ment of androgen levels.
Female genital and reproductive tissues
Congenital malformations of the uterus may have pro-
found implications clinically. Patients with cloacal exstro-
phy and intersex disorders may not have sufficient uterine
tissue present for future reproduction. We investigated
the possibility of engineering functional uterine tissue
using autologous cells [144]. Autologous rabbit uterine
smooth muscle and epithelial cells were harvested, then
Current Opinion in Biotechnology 2009, 20:575–592
584 Tissue, cell and pathway engineering
grown, and expanded in culture. These cells were seeded
onto preconfigured uterine-shaped biodegradable poly-
mer scaffolds, and the constructs were used for subtotal
autologous uterine tissue replacement. Six months after
implantation, analyses confirmed the presence of uterine
tissue components. Biomechanical analyses and organ
bath studies showed that the functional characteristics
of the engineered tissues were similar to those of normal
uterine tissue. Breeding studies using these engineered
uteri are currently being performed.
Similarly, several pathologic conditions, including con-
genital malformations and malignancy, can adversely
affect normal vaginal development or anatomy. Vaginal
reconstruction has traditionally been challenging due to
the paucity of available native tissue. Acellular materials
have been used experimentally for vaginal reconstruction
in rats [145]. The feasibility of using cells to engineer
vaginal replacements was also investigated [146]. Vaginal
epithelial and smooth muscle cells of female rabbits were
harvested, grown, and expanded in culture. These cells
were seeded onto biodegradable polymer scaffolds, and
the cell-seeded constructs were then implanted into
mice. Functional studies in the tissue-engineered con-
structs showed similar properties to normal vaginal tissue.
When these constructs were used for autologous total
vaginal replacement in a rabbit model, patent functional
vaginal structures were noted in the tissue-engineered
specimens, while the non-cell-seeded structures were
noted to be stenotic [147]. These studies indicated that
a regenerative medicine approach to clinical vaginal re-
construction would be a realistic possibility. Clinical trials
are currently being conducted.
Kidney
Although the kidney was the first organ to be substituted
by an artificial device and the first successfully trans-
planted organ [2], current modalities of treatment are far
from satisfactory. Renal tissue is arguably one of the most
difficult tissues to replicate in the laboratory. The kidney
is very complex and the unique structural and cellular
heterogeneity present within it creates many challenges.
The system of nephrons and collecting ducts within the
kidney is composed of multiple functionally and morpho-
logically distinct segments. For this reason, appropriate
conditions must be provided to ensure the long-term
survival, differentiation, and growth of many types of
cells at once. Efforts in the area of kidney tissue regen-
eration have focused on the development of a reliable cell
source [148–153]. Moreover, optimal growth conditions
have been extensively investigated to provide adequate
enrichment to achieve stable renal cell expansion systems
[154,155–157].
Isolation of particular cell types that produce specific
factors may be a good approach for selective cell therapies
for renal failure. For example, cells that produce erythro-
Current Opinion in Biotechnology 2009, 20:575–592
poietin have been isolated in culture, and these cells
could eventually be used to treat anemia that results from
end-stage renal failure [158]. Other more ambitious
approaches tackle the goal of total renal function replace-
ment. To create kidney tissue that would deliver full
renal function, a culture containing all of the cell types
comprising the functional nephron units should be used.
Optimal culture conditions to nurture renal cells have
been extensively studied and cells grown under these
conditions have been reported to maintain their cellular
characteristics [159]. Cells obtained through the initial
process of nuclear transfer were retrieved and expanded
from cloned tissue. Moreover, renal cells placed in a
three-dimensional culture environment are able to recon-
stitute into renal structures.
Although isolated renal cells are able to retain their
phenotypic and functional characteristics in culture,
transplantation of these cells in vivo may not result in
structural remodeling. In addition, cell or tissue com-
ponents cannot be implanted in large volumes due to
limited diffusion of oxygen and nutrients [160]. Thus, a
cell-support matrix, preferably one that encourages angio-
genesis, is necessary to allow diffusion across the entire
implant. A variety of synthetic and naturally derived
materials have been examined in order to determine
the ideal support structures for regeneration
[43,54,102,126,161]. Biodegradable synthetic materials,
such as poly-lactic and poly-glycolic acid polymers, have
been used to provide structural support for cells. Syn-
thetic materials can be easily fabricated and configured in
a controlled manner, which make them attractive options
for regenerative medicine. However, naturally derived
materials, such as collagen, laminin, and fibronectin, are
much more biocompatible and provide a similar extra-
cellular matrix environment to normal tissue. For this
reason, collagen-based scaffolds have been used increas-
ingly in many applications [102,104,126,141,162].
The feasibility of achieving renal cell growth, expansion,
and in vivo reconstitution using regenerative medicine
techniques was investigated [43]. Donor rabbit kidney
cells, including distal tubules, glomeruli, and proximal
tubules were plated separately in vitro and after expan-
sion, were seeded onto biodegradable poly-glycolic acid
scaffolds and implanted subcutaneously into host athymic
mice. This included implants of proximal tubular cells,
glomeruli, distal tubular cells, and a mixture of all three
cell types. Histologic examination demonstrated pro-
gressive formation and organization of the nephron seg-
ments within the polymer fibers with time. BrdU
incorporation into renal cell DNA was confirmed. These
results demonstrated that renal specific cells can be
successfully harvested and cultured, and can sub-
sequently attach to artificial biodegradable polymers.
However, it was unclear whether the tubular structures
reconstituted de novo from dispersed renal elements, or if
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 Engineering organs Atala 585

Figure 4

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, three 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 IFNg after stimulation with allogenic
renal cells, cloned renal cells, or nuclear donor fibroblasts. Cloned renal cells produce fewer IFNg spots than the allogenic cells, indicating that the
rejection response to cloned cells is diminished. The presented wells are single representatives of duplicate wells.

they merely represented fragments of donor tubules
which survived the original dissociation and culture pro-
cesses intact. Further investigation was conducted in
order to examine the process [163]. Mouse renal cells
were harvested and expanded in culture. Subsequently,
single isolated cells were seeded on biodegradable poly-
mers and implanted into immune competent syngeneic
hosts. Renal epithelial cells were observed to reconstitute
into tubular structures in vivo. Sequential analyses of the
retrieved implants over time demonstrated that renal
epithelial cells first organized into a cord-like structure
with a solid center. Subsequent canalization into a hollow
tube could be seen by two weeks. Histologic examination
with nephron segment specific lactins showed successful
reconstitution of proximal tubules, distal tubules, loop of
Henle, collecting tubules, and collecting ducts. These
results showed that single suspended cells are capable of
reconstituting into tubular structures, with homogeneous
cell types within each tubule.
In a subsequent study mouse renal cells were harvested,
expanded in culture, and seeded onto a tubular device
constructed from polycarbonate [164]. The tubular device
was connected at one end to a silastic catheter which
terminated into a reservoir. The device was implanted
subcutaneously in athymic mice. Histological examin-
ation of the implanted device demonstrated extensive
vascularization as well as formation of glomeruli and
highly organized tubule-like structures. Immunocyto-
chemical staining with anti-osteopontin antibody, which
is secreted by proximal and distal tubular cells and the
cells of the thin ascending loop of Henle, stained the
tubular sections. Immunohistochemical staining for
alkaline phosphatase stained proximal tubule-like struc-
tures. Uniform staining for fibronectin in the extracellular
matrix of newly formed tubes was observed. The fluid
collected from the reservoir was yellow and contained
66 mg/dl uric acid (as compared to 2 mg/dl in plasma)
suggesting that these tubules are capable of uni-
directional secretion and concentration of uric acid.
The creatinine assay performed on the collected fluid
showed an 8.2-fold increase in concentration, as compared
to serum. These results demonstrated that single cells
forming multicellular structures can become organized
into functional renal units that are able to excrete high
levels of solutes through a urine-like fluid [164].
To determine whether renal tissue could be formed using
an alternative cell source, nuclear transplantation (thera-
peutic cloning) was performed to generate histocompa-
tible tissues, and the feasibility of engineering syngeneic
renal tissues in vivo using these cloned cells was inves-
tigated (Figure 4) [159]. Nuclear material from bovine
dermal fibroblasts was transferred into unfertilized enu-
cleated donor bovine eggs. Renal cells from the cloned
embryos were harvested, expanded in vitro, and seeded
onto three-dimensional renal devices. The devices were
implanted into the back of the same steer from which the
cells were cloned, and were retrieved 12 weeks later. This
process produced functioning renal units. Urine pro-
duction and viability were demonstrated after transplan-
tation back into the nuclear donor animal. Chemical

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586 Tissue, cell and pathway engineering
analysis suggested unidirectional secretion and concen-
tration of urea nitrogen and creatinine. Microscopic
analysis revealed the formation of organized glomeruli
and tubular structures. Immunohistochemical and RT-
PCR analysis confirmed the expression of renal mRNA
and proteins. These studies demonstrated that cells
derived from nuclear transfer can be successfully har-
vested, expanded in culture, and transplanted in vivo with
the use of biodegradable scaffolds on which the single
suspended cells can organize into tissue structures that
are genetically identical to that of the host. These studies
were the first demonstration of the use of therapeutic
cloning for the regeneration of tissues in vivo.
A naturally derived tissue matrix with existing three-
dimensional kidney architecture would be preferable to
the artificial matrix used in the above experiments,
because it would allow for transplantation of a larger
number of cells, resulting in greater renal tissue volumes.
Thus, an acellular collagen-based kidney matrix, which is
similar to the native renal architecture, was developed. In
a subsequent study it was investigated whether these
collagen-based matrices could accommodate large
volumes of renal cells and form kidney structures in vivo
[165].
Acellular collagen matrices, derived from porcine kid-
neys, were obtained through a multi-step decellulariza-
tion process. During this process, serial evaluation of the
matrix for cellular remnants was performed using histo-
chemistry, scanning electron microscopy (SEM), and RT-
PCR. Mouse renal cells were harvested, grown, and
seeded on decellularized collagen matrices. Cell-matrix
constructs grown in vitro and implanted in the subcu-
taneous space of 20 athymic mice were analyzed over
time. Renal cells seeded on the matrix adhered to the
inner surface and proliferated to confluency by seven days
after seeding. Renal tubular and glomerulus-like struc-
tures were observed eight weeks after implantation.
Blood vessels
Xenogenic or synthetic materials have been used as
replacement blood vessels for complex cardiovascular
lesions. However, these materials typically lack growth
potential, and may place the recipient at risk for compli-
cations such as stenosis, thromboembolization, or infec-
tion [166]. Tissue-engineered vascular grafts have been
constructed using autologous cells and biodegradable
scaffolds and have been applied in dog and lamb models
(Figure 5) [167–170]. The key advantage of using these
autografts is that they degrade in vivo, and thus allow the
new tissue to form without the long-term presence of
foreign material [166]. Application of these techniques
from the laboratory to the clinical setting has begun, with
autologous vascular cells harvested, expanded, and
seeded onto a biodegradable scaffold [171]. The resultant
autologous construct was used to replace a stenosed
Current Opinion in Biotechnology 2009, 20:575–592
pulmonary artery that had been previously repaired.
Seven months after implantation, no evidence of graft
occlusion or aneurysmal changes was noted in the reci-
pient.
Heart
In the United States, over five million people currently
live with 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. Unfortu-
nately, 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 regen-
erate or replace damaged heart muscle using tissue engin-
eering and regenerative medicine techniques is an
attractive option.
Cell therapy for infracted areas of the heart is attractive, as
these methods involve a rather simple injection into the
damaged 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 regen-
erate 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
biocompatible 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
injected either into the damaged area of the heart itself, or
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
are comprised of cells adhered to a biomaterial, that can
theoretically be used to replace the damaged area of the
heart. These techniques have promise, but require
further research into the optimal cell types and biomater-
ials for this purpose before they can be used extensively
in the clinic (see [172] for an excellent review of these
methods).
However, the methods described above could be used
only 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 problems associated with a mechanical
heart (such as thromboembolus formation) would be
abolished. To this end, Ott et al. recently developed a
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 Engineering organs Atala 587

Figure 5

Studies of an engineered blood vessel implanted in a sheep. (A) An arteriogram showing the engineered vessel graft 130 days after implantation (arrows
indicate graft position). (B) A macroscopic view of the engineered vessel, which consists of a decellularized porcine iliac artery reseeded with endothelial
progenitor cells (EPC) derived from the peripheral blood of the sheep to receive the graft. (C) Microscopic view of graft explanted at day 15
postimplantation indicates a poorly organized thrombotic deposit over the acellular graft media. (D) Microscopic view of the graft at 130 days
postimplantation indicates cellular intimal thickening as pannus from adjacent arterial segments, infiltration of cells into the graft, and a confluent, flattened
cell monolayer lining the luminal surface of the graft (arrow). (E) Section of the 130-day graft stained for extracellular matrix shows preservation of anatomic
features, especially elastin (black, indicated with arrow). (F) Cell staining within the intimal layer (indicated by the letter ‘I’ and in the inset) for smooth muscle
a-actin at 130 days. Note that the medial layer, indicated by the letter ‘m’ does not stain for a-actin. (G) Staining for von Willebrand factor (vWF), an
endothelial cell marker. (H) Preimplant graft seeded with endothelial cells labeled with a fluorescent tracer. (I and J) Fifteen-day and 130-day explanted
grafts still contain a layer of seeded endothelial cells, as indicated by the presence of the fluorescent cells along the lumen of the graft. In H, I, and J, the
lumen is oriented to the left of the photomicrograph. Magnifications in B, C, and the F inset, 200, in D and F, 100, in G, 400, and in H, I, and J, 400.

novel heart construct in vitro using decellularized cada-
veric hearts. By reseeding the tissue scaffold that
remained after a specialized decellularization process
with various types of cells that make up a heart (cardi-
omyocytes, smooth muscle cells, endothelial cells, and
fibrocytes) and culturing the resulting construct in a
bioreactor system designed to mimic physiologic con-
ditions, this group was able to produce a construct that
could generate pump function on its own [173]. This
study suggests that production of bioartificial hearts may
one day be possible.
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 therapy. Cell transplan-
tation has been proposed as a potential solution for liver
failure. This is based on the fact that the liver has
enormous regenerative potential in vivo, which suggests
that in the right environment, it may be possible to
expand liver cells in vitro in sufficient quantities for tissue
engineering [174]. 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 [174].
Extracorporeal bioartificial liver devices that use porcine
hepatocytes have been designed and applied. These

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588 Tissue, cell and pathway engineering
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 1
[175]; however, complications such as portal vein throm-
bosis and pulmonary embolism are major concerns,
especially when large cell numbers are used [176].
Finally, cells including stem cells, oval progenitor cells,
and mature hepatocytes have been seeded onto liver
shaped biocompatible matrices to engineer artificial,
implantable livers. These have been tested in various
animal models [177,178]; 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.
Summary and conclusions
Regenerative medicine efforts are currently underway
experimentally for virtually every type of tissue and organ
within the human body. As regenerative medicine incorp-
orates 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 technol-
ogies 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, PhD, for editorial assistance
with this manuscript.
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