EXPERIMENTAL

Auricular Tissue Engineering Using Osteogenic

Differentiation of Adipose Stem Cells with
Small Intestine Submucosa
Chih-Hsun Lin, M.D.
Background: Ear reconstruction remains a challenge for plastic surgeons.
I-Chen Yang, B.S.
A tissue-engineering approach could provide another route for obtaining
Chi-Han Tsai, M.S.
shape maintenance in neoauricular tissue.
Hsu-Wei Fang, Ph.D. Methods: The authors designed a novel tissue-engineering auricular construct
Hsu Ma, M.D., Ph.D. by culturing human adipose stem cells, which differentiated into osteocytes but
Taipei, Taiwan not chondrocytes, in small intestine submucosa scaffolds. The authors evalu-
ated cell growth potential and mechanical properties. An ear-shaped construct
was created in vitro and then implanted in the backs of nude mice. The his-
tology, cellularity, neovascularization, mechanical properties, and ear shape
maintenance were investigated.
Results: In vitro, human adipose stem cells could be successfully seeded in the
small intestine submucosa and differentiated toward osteogenesis. The ear-
shaped human adipose stem cell/small intestine submucosa construct could
maintain its shape in vivo up to 1 year. Alizarin Red S staining confirmed
osteogenic differentiation. CD31 stain showed prominent angiogenesis in the
human adipose stem cell/small intestine submucosa construct at 6 months and
persistence up to 1 year. h-MHC stain revealed the maintenance of cellularity at
6 months and persistence up to 1 year. The mechanical properties were similar
to those of native ear cartilage.
Conclusion: The authors study found that the combination of human adipose
stem cells and small intestine submucosa could provide a more durable ear-
shaped construct in vivo. The mechanical properties, shape, and cellularity
were maintained in the constructs for up to 12 months. (Plast. Reconstr. Surg.
140: 297, 2017.)
CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

C
ongenital auricular agenesis can lead to
physical and psychological impacts on
young patients.13 The use of autologous car-
tilage grafts has problems of donor-site availability
and morbidity, such as deformity of the thoracic
cage.4,5 Ear cartilage reconstruction remains a
challenge for plastic surgeons. The current tissue-
engineering techniques provide a new route for
From the Division of Plastic Surgery, Taipei Veterans Gener-
al Hospital; the Department of Surgery, School of Medicine,
National Yang-Ming University; and the Department of
Chemical Engineering and Biotechnology, National Taipei
University of Technology.
Received for publication November 2, 2016; accepted
February 2, 2017.
Presented at the 2016 Tissue Engineering and Regenerative
Medicine International SocietyAsia Pacific Meeting, in
Taipei, Taiwan, September 3 through 6, 2016.
Copyright 2017 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0000000000003522
ear reconstruction to obtain a precisely shaped
cartilage implant that avoids donor-site morbidity
and unsatisfactory cosmetic results.610
The greatest advantage of using reconstructed
ear is its compatibility with both mechanical
and biochemical properties. Tissue-engineering
Disclosure: The authors have no financial interest
to declare in relation to the content of this article.
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constructs have to withstand compression to pre-
vent deformation and should also have the prop-
erty of ease of cutting or sculpturing for shape
matching. An ideal cell-scaffold matrix should
maintain its shape for a longer period.11,12 How-
ever, a suitable combination of cell and scaffold
has yet to be developed. A variety of scaffolds and
cell resources have been investigated as alterna-
tives for autologous carved costal cartilage or
porous polyethylene implants.9,1215 Selection of
an appropriate scaffold and cell resource is impor-
tant because the physical cues of biomaterials will
direct cellular behavior and constantly interact
with the cells, resulting in dynamic transfer of
information between extracellular and intracellu-
lar environments.16,17
In the past, researchers used a combination
of chondrocyte and synthetic materials (e.g., poly-
glycolic acid, polylactic acid, polycaprolactone,
collagen, calcium alginate, chitosan) for in vitro
ear cartilage culture.1114,16,1820 Although there
were some promising results, some problems
remained, which included (1) less growth ability
in differentiated cells and (2) the loss of cartilage-
specific phenotype of the cells manipulated in
vitro.2123 The drawbacks associated with synthetic
scaffold include (1) lack of porosity for cell migra-
tion and penetration in the materials; (2) lack
of blood and nutrient supply of perichondrium,
resulting in cell death and unavoidable necrosis of
the engineered construct; and (3) inflammatory
reaction and altered in vivo tissue regeneration
because of the degradation products of synthetic
scaffolds.8,9,2430 Furthermore, no ideal tissue-engi-
neering constructs could withstand the contractile
forces exerted by the skin and surrounding tissue
during normal wound healing.6
To solve these problems, we introduce an alter-
native method of using a biological scaffold (small
intestine submucosa; Cook Biotech, Inc., Lafayette,
Ind.) with adipose progenitor cells (human adipose
stem cells) for ear reconstruction. The characteris-
tics of small intestine submucosa have been studied
for various types of tissue regeneration, such as ves-
sel, esophagus, tendon, and bladder.3136 Small intes-
tine submucosa has been approved by U.S. Food
and Drug Administration for clinical application.
It is mainly composed of type I and type III colla-
gen and exerts both bioinductive and bioconduc-
tive properties by means of residual cytokines.37,38
It supports cell adhesion, migration, growth, and
differentiation.3841 In addition, the adipose stem
cells have great potential in tissue regeneration
because of their abundance and ability to differ-
entiate into different lineages. Adipose stem cells
298
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Plastic and Reconstructive Surgery August 2017
can repair damaged tissue, augment cellular dif-
ferentiation, and stimulate the release of multiple
growth factors.42,43 Their therapeutic effect has been
investigated in cell therapy for conditions such as
degenerative knee, renal failure, liver failure, and
myocardial infarction,4452 and applied in various
types of tissue engineering, such as bone, cartilage,
adipose tissue, tendon, and others.5358 Also, the bio-
compatibility of adipose stem cells in small intestine
submucosa had been reported for tunica albuginea
reconstruction.39
Here, we designed a tissue-engineered auric-
ular construct by culturing human adipose stem
cells for osteogenic differentiation in the small
intestine submucosa in vitro. We hypothesize that
the constructs have more potential in angiogen-
esis and longevity of shape maintenance in vivo.
We evaluated the growth potential of human adi-
pose stem cells in small intestine submucosa and
their mechanical properties after culturing. Then,
ear-shaped constructs were created in vitro and
implanted into the backs of nude mice. The histol-
ogy, differentiation, neovascularization, mechani-
cal properties, and ear shape maintenance were
investigated during follow-up studies.
MATERIALS AND METHODS
Adipose Stem Cell Culture
Theis study was approved by the institutional
review board and animal care/use committee at
Taipei Veterans General Hospital. Adipose stem
cells were obtained as described previously.52
Flow Cytometry
Cells were trypsinized, washed with phos-
phate-buffered saline, and incubated with the
following antibodies: phycoerythrinmouse anti-
human CD14, phycoerythrinmouse anti-human
CD29, phycoerythrinmouse anti-human CD34,
phycoerythrinmouse anti-human CD44, phyco-
erythrinmouse anti-human CD73, fluorescein iso-
thiocyanatemouse anti-human CD31, fluorescein
isothiocyanatemouse anti-human CD45, fluores-
cein isothiocyanatemouse anti-human CD90, fluo-
rescein isothiocyanatemouse anti-human CD105,
and fluorescein isothiocyanatemouse anti-human
human leukocyte antigenantigen D related (eBio-
science, Inc., San Diego, Calif.). Cell fluorescence
was evaluated using flow cytometry (FACS Canto II;
Becton Dickinson, Franklin Lakes, N.J.)
 Volume 140, Number 2 Tissue-Engineered Auricle
Differentiation of Adipose Stem Cells into
Adipocytes and Osteocytes
Adipogenesis
The adipose stem cells were subcultured
in a 6-cm dish with Dulbeccos Modified Eagle
Medium (10% fetal bovine serum) in an induc-
tion medium (0.5mM isobutyl methylxanthine, 1
M dexamethasone, 1 M indomethacin, 10 M
insulin) for 2 days and then in a medium with
insulin (10 g/ml) for 1 day. This induction pro-
tocol (indomethacin for 2 days and insulin for 1
day) was repeated three times for 3 weeks. Adi-
pogenesis was confirmed with Oil Red O stain-
ing (Sigma-Aldrich, St. Louis, Mo.) under light
microscopy (Olympus IX51; Olympus Corp.,
Tokyo, Japan).
Osteogenesis
The cells were trypsinized, counted, and then
mixed with Dulbeccos Modified Eagle Medium to
a final concentration of 10 106/ml. The cell sus-
pension (10 l) was aspirated with a micropipette
to a 24-well dish and incubated for 2.5 hours.
Then, the medium was changed to the induction
medium (0.1 M dexamethasone, 50 M ascor-
bate-2-phosphate, 10mM -glycerophosphate,
and 1% antibiotic/antimycotics). The medium
was changed every 3 days for 3 weeks. Osteogen-
esis was checked with von Kossa stain (Sigma-
Aldrich) under light microscopy.
In Vitro Osteogenesis Induction of Adipose
Stem Cells on Small Intestine Submucosa
Sterilized small intestine submucosa measur-
ing 7 10cm was placed in a 15-cm dish. The
human adipose stem cell suspension (approxi-
mately 3 104 cells/cm2) was seeded onto the
small intestine submucosa sheet. The cells were
incubated overnight for cell attachment. Then,
the medium was changed to induction medium
(0.1 M dexamethasone, 50 M ascorbate-2-phos-
phate, 10mM -glycerophosphate, and 1% anti-
biotic/antimycotics). The medium was changed
every 3 days for 3 weeks.
In Vivo Biocompatibility Test
After osteogenesis induction, the adipose
stem cellseeded small intestine submucosa
(7 10cm) was rolled into a tube-like structure
(four layers). The tissue-engineered ear was cre-
ated by suturing the tubular structure into the ear-
shaped construct. This construct was transplanted
into the backs of nude mice (BALB/cAnN.Cg-
Foxn1nu/CrlNarl; National Laboratory Animal
Center, Nankang, Taiwan). The nude mice (n = 5)
were anesthetized with Zoletil 50 (Virbac, Carros,
Copyright 2017 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
France) (5mg/100g body weight). The animal was
held in prone position and a longitudinal incision
was performed to open the skin on the back. The
ear-shaped construct was implanted in the subcu-
taneous pocket and fixed with 6-0 nylon as an in
vivo bioreactor. After wound irrigation, the wound
was approximated with 6-0 nylon. For the control
groups, small intestine submucosa only (n = 3) and
small intestine submucosa with fibroblasts (n = 3,
the same cell number as human adipose stem cells)
were transplanted in the same way. The mice were
then sent back to the cage and allowed to feed ad
libitum. The mice were killed at 6 and 12 months
and the engineered ear was explanted for morphol-
ogy, compression testing, histology (hematoxylin
and eosin, Alizarin Red S), and immunohistology
(CD31, h-MHC). The cell number in CD31 or
h-MHC staining was presented as the average num-
ber of cells per 10 high-power fields.
Mechanical Testing
For the first test, human fibroblasts or adipose
stem cells (approximate 3 104 cells/cm2) were
seeded onto the 5 3-cm small intestine submucosa,
and the adipose stem cells were subjected to osteo-
genesis induction for 3 weeks. The medium was
changed every 2 days. Then, the small intestine sub-
mucosa, small intestine submucosa plus fibroblasts,
and small intestine submucosa plus adipose stem
cells were subjected to mechanical tests. Uniaxial
tensile testing was performed for the small intestine
submucosa and cell-seeded small intestine submu-
cosa constructs with a size of 30 4mm with an ini-
tial gauge length of 10mm. The tissue was extended
at a test speed of 0.08cm/second until broken.
For the second test, the unconfined uniaxial
compression test was performed for the ex vivo
explanted constructs. A block of construct with
an area of 4mm2 and a thickness of 4-mm was
prepared for the test. The compression test was
performed at a test speed of 2N/second until the
sample was broken.
For the two tests, the strength and displace-
ment were recorded. The data were loaded into
the software MATLAB (MathWorks, Natick, Mass.)
for stress-strain curve plotting. The Young modu-
lus was calculated.
Statistical Analysis
The group differences were calculated by a
two-tailed paired t test and a one-way analysis of
variance by using SPSS 12.0 (SPSS, Inc., Chicago,
Ill.). The quantitative results are presented as
mean standard deviation. Statistical significance
was indicated by a value of p < 0.05.
299

RESULTS
Characteristics of Adipose Stem Cells
Flow cytometric analysis showed that adipose
stem cells were positive for the cell surface markers
CD29, CD44, CD73, and CD90. The morphology
of adipose stem cells and the successful differen-
tiation of adipose stem cells into adipogenic and
osteogenic cells were confirmed by Oil-Red O
and von Kossa staining, respectively. [See Figure,
Supplemental Digital Content 1, which shows the
profile of adipose stem cells. (Left) Human adi-
pose stem cell morphology (original magnifica-
tion, 100; scale bar = 50 m). (Center) Induction
of human adipose stem cells to adipogenesis (Oil-
Red O stain; original magnification, 100; scale
bar = 50 m). (Right) Induction of human adi-
pose stem cells to osteogenesis (von Kossa stain;
original magnification, 100; scale bar = 50 m),
http://links.lww.com/PRS/C258.]
In Vivo Biocompatibility Test
Morphology
The ear-shaped construct using the human
adipose stem cells and small intestine submu-
cosa could maintain an upright position on the
backs of nude mice for up to 1 year. There was no
shrinkage or collapse of the shape during follow-
up. The skin was adherent to the construct closely
and was soft and pliable. However, the construct
using small intestine submucosa only or small
intestine submucosa with fibroblasts could not
maintain an upright position and shape during
follow-up. [See Figure, Supplemental Digital Con-
tent 2, which shows the morphology of a tissue-
engineered construct. (Left) The morphology of
the implanted human adipose stem cell/small
intestine submucosa construct on the back of a
nude mouse at 1 year. (Above, right) Small intes-
tine submucosaonly implantation at 4 months.
(Below, right) Fibroblast plus small intestine sub-
mucosa implantation at 4 months. Both showed
collapse, shrinkage, and deformation of the con-
structs, http://links.lww.com/PRS/C259.]
Histology
Hematoxylin and Eosin Staining
Histologic evaluation of small intestine sub-
mucosa only and small intestine submucosa with
fibroblasts showed only fibrous structures. In
particular, the structures were much more dis-
organized in the small intestine submucosa with
fibroblast group. In the group with small intestine
submucosa and adipose stem cells, an intact oste-
oid-like structure was noted, which persisted for 1
300
Copyright 2017 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Plastic and Reconstructive Surgery August 2017
year. [See Figure, Supplemental Digital Content 3,
which shows in vivo osteogenic induction of adi-
pose stem cells in the small intestine submucosa.
(Left) Fibroblasts (at 6 months, no induction),
(center) adipose stem cells (at 6 months, osteo-
genic induction), and (right) adipose stem cells
(at 1 year, osteogenic induction) (above, hematox-
ylin and eosin, original magnification, 200, scale
bar = 100 m; below, Alizarin Red S stain, original
magnification, 200, scale bar = 100 m), http://
links.lww.com/PRS/C260.]
Alizarin Red S Staining
The staining confirmed the osteogenic dif-
ferentiation in the small intestine submucosa and
adipose stem cell groups. The process persisted in
vivo and became more prominent at 1 year. There
was no osteogenic differentiation in the small
intestine submucosa with fibroblast group. (See
Figure, Supplemental Digital Content 3, http://
links.lww.com/PRS/C260.)
Immunohistology
h-MHC Staining
The results revealed the maintenance of the
adipose stem cells or fibroblasts in the construct
at 6 months. The viability of adipose stem cells
persisted for 1 year. Quantification of cellular-
ity showed significant cellularity in the adipose
stem cell/small intestine submucosa construct
at 6 months. [See Figure, Supplemental Digital
Content 4, which shows in vivo cell viability of
fibroblasts/adipose stem cells (ASC) in the small
intestine submucosa. (Above, first row) Control
(small intestine submucosa only, at 6 months);
(above, second row) fibroblasts plus small intestine
submucosa (at 6 months, no induction); (above,
third row) adipose stem cells plus small intestine
submucosa (at 6 months, osteogenic induction);
and (above, fourth row) adipose stem cells plus
small intestine submucosa (at 1 year, osteogenic
induction). (Above, left) h-MHC stain, immunohis-
tochemistry (original magnification 400; scale
bar = 50, m); (above, second from left) h-MHC
stain, IF; (above, second from right) 4,6-diamidino-
2-phenylindole stain; and (above, right) merge
(original magnification, 400; scale bar = 50, m;
white arrow, human cells). (Below) Quantification
of in vivo cell viability of fibroblasts/adipose stem
cells in the small intestine submucosa, http://links.
lww.com/PRS/C261.]
CD31 Staining
The results showed angiogenesis in the con-
struct in the three groups. However, dominant
angiogenesis was noted in the small intestine
 Volume 140, Number 2 Tissue-Engineered Auricle
submucosa and adipose stem cell group at 6
months and persisted at 1 year. [See Figure, Sup-
plemental Digital Content 5, which shows in vivo
angiogenesis of fibroblasts/adipose stem cells in
the small intestine submucosa. (Above, left) Small
intestine submucosa; (above, center) fibroblasts
plus small intestine submucosa (no induction);
and (above, right) adipose stem cells plus small
intestine submucosa (osteogenic induction), all at
6 months (CD31 stain; original magnification,
400; scale bar = 50 m). (Below) Quantification of
the CD31 staining. ASC, adipose stem cells; HPF,
high-power field, http://links.lww.com/PRS/C262.]
Mechanical Test
The uniaxial tensile test showed more extensi-
bility in the adipose stem cell/small intestine sub-
mucosa construct, whereas more stiffness was noted
in the fibroblast/small intestine submucosa con-
struct. [See Figure, Supplemental Digital Content
6, which shows uniaxial tensile stress-strain curves
of small intestine submucosa (SIM), fibroblasts
plus small intestine submucosa, and adipose stem
cells (ASC) plus small intestine submucosa (in vitro
cultured samples), http://links.lww.com/PRS/C263.]
The unconfined compression test showed that the
Young modulus for the construct with small intes-
tine submucosa and adipose stem cells at 1 year was
approximately 2.59 0.49MPa, which was within
the normal range of native ear cartilage.59,60
DISCUSSION
The past decade has witnessed an explosion of
scientific advances in the field of tissue engineering.
The goal is to bridge the gap between basic research
and clinical practice, with a special focus on specific
tissue or organ regeneration. The emergence of
several key concepts, such as novel stem cell devel-
opment, biomaterial properties, cell-biomaterial
interaction, angiogenesis, and immune reactions to
graft or material, has resulted in an increase in the
number of clinical studies.6163 Our method of auric-
ular reconstruction using human adipose stem cells
with small intestine submucosa adheres to these
key concepts of tissue engineering. In addition, this
method is safe and ethically conducive, and uses a
biomaterial and cell source that is easily accessible
by surgeons. The in vivo results of the present work
might pave the way for translation of this proposed
method of ear reconstruction for human use in the
future through further studies.
At present, using autologous costal cartilage
is the gold standard for microtia reconstruction.
Despite donor-site morbidity, it involves an inher-
ent difficulty in sculpting an anatomically correct,
Copyright 2017 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
patient-specific auricular replica,9,64,65 and the
inability of costal cartilage to adequately approxi-
mate the complex biomechanical properties of
native auricular elastic cartilage9,11 Using only
alloplastic implants usually leads to failure unless
cellular components are used to coat the sur-
face.66 The tissue-engineering approach provides
a potential alternative that may advantageously
reduce donor-site morbidity resulting from surgi-
cal reconstruction.67
The major concept in past studies focused on
applying chondrocytes as the cell source. However,
chondrocytes usually dedifferentiated during in
vitro culture. Although there are some methods
of increasing cell expansion and maintaining the
differentiation status of chondrocytes,7,6971 the
process is more complex and time-consuming,
and inevitably, the donor-site impairment hinders
their translation into clinical application, unless
using the discarded microtia ear.
The scaffolds should be hydrophilic, porous,
biodegradable, and mechanically matched. There
are two main techniques of tissue engineering
for auricular reconstruction. The first method is
the incorporation of chondrocytes with synthetic
degradable polymer, which is fabricated as an ear-
shaped structure. The key issues in this method
are (1) porosity for cell infiltration and vascular
ingrowth and (2) compatibility of the degrada-
tion rate of polymer with the in vivo cartilage
regeneration rate to prevent the structure from
collapsing. The most investigated biodegradable
polymers are polylactic acid,6,11,65 polyglycolic
acid,18,72 polyhydroxybutyrate,6 polycaprolactone,6
chitosan,19 alginate,73 and hydrogel.15,16 The diffi-
culty is related to various parameters that need to
be tuned, such as pore size, porosity, hydrophilic-
hydrophobicity, and surface topography60 Differ-
ent polymer combinations are known to allow
tuning of the mechanical properties, the degrada-
tion rate of the scaffold, and shape maintenance.74
The second method is a scaffold-free approach
where chondrocytes are cultured in a layer struc-
ture or membrane structure and then molded
into the ear shape.8,75,76 Although this approach
may avoid the problems of scaffold degradation, it
requires an internal stent or external mode to sup-
port the shape of the construct when implanted
in vivo. Shape maintenance without such a mode
is not possible because of poor vascularity.8,70 Fur-
thermore, it involves risks of extrusion or infec-
tion during use of an alloplastic implant or stent.
Vascularized auricular cartilage engineering with
pedicle implantation has also been proposed to
create a capsule or cross over a synthetic structure
301

seeded with chondrocytes to maintain oxygen and
nutrition supply.10,77 However, an external mode is
still required for shape maintenance.
In our study, angiogenesis is more prominent
in the differentiation of adipose stem cells toward
osteogenesis, because of the paucity of vasculature
of the cartilage tissue that is devoid of perichon-
drium.78,79 In our experience, the osteocytes dif-
ferentiated from adipose stem cells have a more
stable status in vitro and maintained growth on
small intestine submucosa for at least 3 weeks.
Histologic evaluation of explanted constructs con-
firmed angiogenesis; furthermore, organized col-
lagen fibers and MHC-stained positive cellularity
were observed inside the neotissue in vivo for sev-
eral months to 1 year. The gross morphology and
the ear-shape remained similar until the end of
implantation. There was no extrusion, skin break-
down, or shrinkage. Collectively, these findings
show that combining osteogenic differentiation
from human adipose stem cells and small intes-
tine submucosa could provide a biocompatible,
angiogenic, viable, ear-shaped structure in vivo.
The scenario of the tissue-engineered auricle
is different from the cell-seeded scaffold for bone
repair such as the cranial bone defect model.58,80,81
In the bone repair model, because the defect is
adjacent to healthy bone, the adipose stem cells
showed a paracrine effect to recruit the native
osteoblasts into the defect for osteogenesis. In
contrast, for microtia, there is no environment for
the normal cartilage tissue to induce adipose stem
cells to secrete cytokines, to initiate the migra-
tion of chondroblasts or chondrocytes into the
selected site of the ear. Our results of implanta-
tion in the backs of nude mice demonstrated that
in vitro adipose stem cell induction of osteogen-
esis on the small intestine submucosa is an impor-
tant and effective step for creating an ear-shaped
construct, as there is no cartilage tissue, bone tis-
sue, or internal rigid support in the backs of the
mice initially. This construct had the ability to pro-
vide angiogenesis and maintain ear shape. The
expression of h-MHC marker also confirmed that
the human adipose stem cellinduced osteocytes
could survive in the construct in vivo for several
months to 1 year. This also hinted that the neo-
auricle construct had not only a paracrine effect
but also an autocrine effect for maintaining cell
viability in vivo for longer periods.
In contrast to engineering of new tissue from
synthetic materials, some of the recent, most
high-profile studies on tissue engineering have
focused on translating simple approaches, such
as use of decellularized extracellular matrix, into
302
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Plastic and Reconstructive Surgery August 2017
preclinical and clinical studies.82 Small intestine
submucosa is a decellularized extracellular matrix
of porcine small intestine submucosa. The devel-
opment of decellularization technique has pro-
vided a method of using a biological scaffold,
instead of synthetic polymers, for tissue regen-
eration. Theoretically, removal of the cell compo-
nents could decrease the immunologic reaction
from xenogenic tissue. The decellularized extra-
cellular matrix preserved most of the collagen,
elastin, proteoglycans, and other macromolecules
such as growth factors and cytokines.83 The envi-
ronment is suitable for cell adhesion, migration,
proliferation, and differentiation.84 Ahn et al.
showed that small intestine submucosa provided
a natural environment for human bone marrow
stem cell adhesion and proliferation.37 Our results
also demonstrated that human adipose stem
cells could be easily attached, infiltrated, prolif-
erated, and differentiated into osteocytes in the
small intestine submucosa. Moreover, small intes-
tine submucosa is bioactive and thus stimulates
angiogenesis through an assortment of matrix-
embedded angiogenic factors, including connec-
tive tissue growth factor, fibroblast growth factor,
transforming growth factor-, and fibronectin.85
Based on our results, we found that small intestine
submucosa had a similar effect on adipose-stem
cells that enhanced angiogenesis.
The ideal biomaterial scaffold for auricular
reconstruction should have structural stability,
match the mechanical properties of the native tis-
sue, and have porosity to enable vascularization
and host tissue integration. Substrate stiffness
also affects cellular behavior, including migration,
growth, and differentiation, through various mech-
anotransduction processes.86 For the mechanical
property, the Young modulus of our neoear con-
struct was similar to that of the native ear cartilage
(2.59MPa versus 1 to 5MPa).59,60 This means that
our tissue-engineered ear could avoid deformation
easily and has ability to resist compression force.
Although some studies have shown positive results
with regard to mechanical properties while using
chondrocytes as the cell source in biodegradable
synthetic scaffolds, several earlier studies dem-
onstrated construct shrinkage or deformation at
approximately 3 to 6 months.6,11,14,27,64,79 Further-
more, material elastic modulus mismatch with the
native tissue, inadequate cellular adhesion, and
delayed fibrovascular ingrowth into the porous
implant may all contribute to the failure rate.24 The
potential limitations from using bone rather than
cartilage in the construct could be related to the
composition of the extracellular matrix. Constructs
 Volume 140, Number 2 Tissue-Engineered Auricle
formed of cartilage have more type II collagen,
elastin, and glycosaminoglycans, which are highly
polar and hydrophilic. Thus, these constructs may
demonstrate not only elasticity but also viscos-
ity, represented by their stress-relaxation or creep
properties. Shock absorbance could be an advan-
tage of these constructs.6,79,87 In contrast, in the
case of bone, the hydroxyapatite content is higher.
Because of crystal formation, hydroxyapatite can
result in stiffness in the bone tissue. Although we
induced the adipose stem cells toward osteogen-
esis, the Young modulus of our construct was com-
parable to that of native cartilage. We presume that
the percentage of hydroxyapatite in our construct
is much lower than that present in native bone tis-
sue. It is still possible for this method to maintain
that of stiffness close to native tissue and to main-
tain shape for longer periods.
Although the results of our pilot study were
promising, it has a few limitations and poses ques-
tions for future research. First, the sample size
per group used in the study was small. Second,
the animal model used is small for implanting a
human-sized ear and mimicking soft-tissue con-
traction. Third, we used nude mice for the in vivo
test, which could result in exclusion of T-cell adap-
tive immune responses. In addition, long-term
follow-up is required to detect inflammatory reac-
tions against the construct and understand their
mechanisms. Lastly, we did not quantify the bio-
chemical contents, such as collagen, elastin, gly-
cosaminoglycans, or calcium composites, of the
tissue-engineered construct.
CONCLUSIONS
Most research has differentiated chondro-
cytes for auricular cartilage engineering, but lim-
ited angiogenesis was the obstacle in moderate
to large size tissue regeneration. Although the
addition of an external mold or pedicle cross-
over provided an alternative route for ear tis-
sue engineering, shape maintenance remained
difficult. Our results showed that, although not
using chondrocytes, the combination of human
adipose stem cells with small intestine submu-
cosa could provide a relatively vascularized ear-
shaped construct in vivo where angiogenesis is de
novo in the process of osteogenic differentiation
of human adipose stem cells. In addition, the
mechanical properties were similar to those of
native ear cartilage and the shape was maintained
for several months. This method could be a novel
alternative tissue-engineering method for auricu-
lar reconstruction.
Copyright 2017 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Hsu Ma, M.D., Ph.D.
Division of Plastic Surgery
Taipei Veterans General Hospital
19F, No. 201, Sec. 2, Shi-Pei Street
Beitou District
Taipei 112, Taiwan
sma@vghtpe.gov.tw
ACKNOWLEDGMENTS
The authors acknowledge a grant from the Taipei
Veterans General Hospital (no. V105C-139) and also
thank the Taipei Zhisong Social Welfare Foundation
(Taiwan), the Sanyo Electric Social Welfare Foundation
(Taiwan), and the Medical Scholarship Foundation in
memory of Professor Albert Ly-Young Shen (Taiwan).
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