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3 months after grafting into CN injury rats, approximately twice as many cells were found on
seeded ADMT as on unseeded ADMT. The seeded ADMT also had various degrees of S100 and
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neuronal nitric oxide synthase expression, suggesting CN axonal ingrowth. Rats grafted with
seeded ADMT overall had the best erectile function recovery when compared with those grafted
with unseeded ADMT and those ungrafted. However, as a result of large variations, the
differences did not reach statistic significance (P � .07).
NCLUSIONS Grafting of ADSC-seeded matrix resulted in a substantial recovery of erectile function and
improvement of histology. However, further refinement of the matrix architecture is needed to
improve the success rate. UROLOGY 77: 1509.e1–1509.e8, 2011. © 2011 Elsevier Inc.
dipose tissue is unique in its ability to expand
and regress throughout life. In developed and
developing countries, the overall adipose tissue
pansion phase outpaces regression, resulting in an ever-
panding obese society. To shed the “extra pounds,” a
nificant number of patients undergo surgeries to re-
ve the unwanted fat. However, the resulting “medical
stes” are potential therapeutic treasures for 2 reasons.
e is that the adipose tissue is a rich source of multi-
tent mesenchymal cells called adipose-derived stem
cells (ADSCs),1,2 and the other is that the adipose
extracellular matrix (ECM) can be fabricated into
acellular scaffolds for tissue engineering.3-5 In regard to
ADSCs, these cells have been shown to differentiate
into Schwann cells that formed myelin sheath on
axons6 and have been used to seed nerve conduits for
peripheral nerve repair.7-9 In addition, we have shown
that ADSCs secrete neurotrophic factors that promote
cavernous nerve (CN) regeneration.10,11 In regard to
adipose ECM, the fabricated scaffolds have been shown to
support adipogenic differentiation of ADSCs.5 In the pres-
ent study, we combined ADSCs’ neuroregenerative poten-
tial and adipose ECMs’ scaffold potential to construct nerve
grafts for the repair of peripheral nerves; in this case, CN.
The CNs innervate penile erectile tissue and are es-
sential for erection. Because of their proximity to the
prostate, bladder, and rectum, these nerves are often
damaged during surgeries on these organs, resulting in
erectile dysfunction (ED).12-14 In particular, prostate
cancer is the most prevalent malignancy in men and is
often treated by radical prostatectomy, causing damage to
the CN and subsequent ED.15 To repair the damaged
s study was supported by a grant from the Department of Defense (PC030775 to
. MA has received research grants from the Belgische Vereniging voor Urologie,
opean Society of Surgical Oncology, Federico Foundation and Bayer Healthcare
ium. MA is a fellow of the Research Foundation (FWO) Flanders.
rom the Knuppe Molecular Urology Laboratory, Department of Urology, Univer-
of California, San Francisco, CA; Department of Urology, University Hospitals,
ven, Leuven, Belgium; Urology and Nephrology Center, Mansoura University,
pt; Division of Plastic Surgery, Department of Surgery, University of California,
Francisco, CA; and Department of Urologic Surgery, University of Minnesota,
neapolis, MN
eprint requests: Ching-Shwun Lin, M.D., Department of Urology, University of
ifornia, San Francisco, 533 Parnassus Avenue, Box 0738, CA 94143-0738.
ail: clin@urology.ucsf.edu
ubmitted: November 11, 2010, accepted (with revisions): December 22, 2010
2011 Elsevier Inc. 0090-4295/11/$36.00 1509.e1
avernous Nerve Repa
ith Allogenic Adipose
utologous Adipose-de
iting Lin, Maarten Albersen, Ahmed M. Ha
ary H. McGrath, Badrinath R. Konety, Tom
JECTIVES To investigate whether adipose-deriv
can facilitate the repair of injured ca
THODS Human and rat adipose tissues were
adipose tissue-derived acellular mat
transplanted into subcutaneous spac
ADMTs were then used to repair C
erectile function.
SULTS Adipose tissue can be fabricated int
threads and sheets. Seeding of ADMT
covered with ADSC and within 1 wee
into the subcutaneous space of an all
Rights Reserved
Matrix and
ved Stem Cells
, Thomas M. Fandel, Maurice Garcia,
Lue, and Ching-Shwun Lin
atrix seeded with adipose-derived stem cells (ADSC)
ous nerves (CNs).
llularized and fabricated into various forms, including
thread (ADMT). ADMT seeded with ADSC were
d examined for signs of inflammation. ADSC-seeded
jury in rats, followed by assessment of histology and
llular matrices of various shapes and sizes, including
urred rapidly: within 24 hours, 55% of the surface was
0% was covered. Transplantation of the seeded ADMT
ic host showed no signs of inflammatory reaction. At
doi:10.1016/j.urology.2010.12.076
CN, autologous nerve grafting with sural nerves has ini-
tially been shown to result in an erectile function recov-
ery
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Seeding of Acellular Matrix Thread with Adipose-
Derived Stem Cells
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rate of 43%.16 However, the harvest of the sural
rves causes donor site morbidity and requires the col-
orative support of plastic surgeons. By contrast, the
rvest of the genitofemoral nerves causes minimal mor-
ity and can be done by the urological surgeon. CN
air with genitofemoral nerve grafting has an erectile
ction recovery rate of approximately 50%.17,18 On an
perimental basis, grafting with decellularized CN from
nor rats has also been attempted.19 However, the clin-
l applicability of such a treatment procedure is rather
because of the need to harvest the CN from human
lunteers or cadavers.
In the present study, we processed lipectomized human
d rat adipose tissues into acellular matrices, seeded the
matrix with allogenic rat ADSCs, and grafted the
ded matrix into rats with transected CN. We observed
riable but substantial recovery of erectile function after
h grafting.
ATERIAL AND METHODS
ocurement of Adipose Tissues
ipose tissue samples were obtained from patients during
tine abdominoplasty after informed patient consent and
ording to the guidelines set by our institution’s Committee
Human Research. Rat adipose tissue was obtained from the
didymal fat pad of 2-month-old male Sprague-Dawley rats
harles River Laboratories, Wilmington, MA). All animal
e, treatments, and procedures were approved by our Institu-
nal Animal Care and Use Committee.
cellularization of Adipose Tissue
e adipose tissue in a 50-mL conical tube was dipped into
uid nitrogen for 5 minutes and then immediately placed in a
°C water bath for 10 minutes. After repeating this freeze-
d-thaw step 2 more times, the tissue was centrifuged at 1500
for 10 minutes at room temperature (RT). After removing
liquid fatty portion, the tissue was washed in 70% ethanol
d phosphate-buffered saline (PBS) 3 times each. It was then
ubated in 0.05% trypsin, 0.05% ethylenediaminetetraacetic
d, 20 ng/mL DNase I, and 20 ng/mL RNase for 4 hours with
w rotation at RT. After washing twice in PBS, the tissue was
ubated in 0.1% sodium dodecyl sulfate (SDS) for 12 hours at
. After washing 3 times in PBS, the tissue was incubated in
penicillin and streptomycin for 12 hours at 4°C.
eparation of Acellular Matrix
e decellularized tissue just described was further processed
o various forms of acellular matrices as follows. In one
mple, the decellularized tissue was cut to�0.5� 1� 8–mm
eads and dried for 12 hours for the preparation of adipose
ue–derived acellular matrix thread (ADMT). In another
mple, the decellularized tissue was homogenized in PBS,
ced into a 10-cm plastic dish, and dried for the preparation
adipose-derived acellular matrix sheet (ADMS). The result-
ADMT or ADMS was further ultraviolet-sterilized inside a
ue culture hood.
09.e2
MT was washed 3 times in PBS, dried at RT for 2 hours, and
ced in a 6-well cell culture dish. ADSCs were isolated as
cribed previously.20 Approximately 1 � 104 ADSCs in 200
of Dulbecco modified Eagle medium (DMEM) was then
ed evenly to the ADMT. The culture dish was then placed
a humidified 37°C incubator with 5% CO2. Four hours later,
L of DMEM supplemented with 10% fetal bovine serum was
ed to the ADMT, and the dish was returned to the incuba-
. At 24 hours and 1 week, the seeded ADMT was stained
h 1 �g/mL calcein AM (Invitrogen, Carlsbad, CA) for 10
nutes at 37°C and examined with a Nikon Eclipse E600
orescence microscope (Tokyo, Japan).
bcutaneous Transplantation of Seeded Matrix
proximately 5 � 104 ADSCs were grown to 60% confluence
then labeled with 10 �M of 5-ethynyl-2-deoxyuridine (EdU)
24 h as previously described.21 The cells were then washed
imes in PBS and then seeded onto an allogenic ADMT by
aforementioned procedure. Forty-eight hours later, the
ded ADMT was transplanted into an autologous host (from
ich the ADSC was isolated). The transplantation procedure
s as follows. With the rat under inhalant anesthesia, an
ision was made in the lower abdomen to expose the subcu-
eous space, into which the seeded ADMT was transplanted.
n days later, the rat was sacrificed and the transplanted tissue
mined by hematoxylin and eosin staining and microscopy.
e transplanted ADMT was also retrieved and stained with an
U detection cocktail that contained Alexa-594 (Invitrogen)
with 4=,6-diamidino-2-phenylindole (DAPI, for nuclear
ining, 1 �g/mL, Sigma-Aldrich). The stained matrix was
mined with a Nikon Eclipse E600 fluorescence microscope
d photographed with a Retiga 1300 Q-imaging camera (Sur-
, BC, Canada).
afting of Unseeded and Seeded Matrix in Nerve
ury Rats
irty 2-month-old male Sprague-Dawley rats were randomized
o 3 equal groups: control, ADMT, and ADMT�ADSC.
ith the rats under inhalant anesthesia, a midline incision was
de in the lower abdomen and the periprostatic space con-
ning the major pelvic ganglion (MPG) and the CNs were
osed. A 5-mm–long nerve segment, starting 5 mm distal
m the origin of the CN at the MPG was isolated and
ected. In the control group, the abdomen was then closed in
ers without further treatment. In the treatment groups, ei-
r acellular ADMT or ADMT�ADSC construct was micro-
gically interposed and fixed against the prostatic capsule
ng 10-0 nylon sutures to bridge the nerve gap.
termination of Erectile Function
ree months after CN injury with and without grafting, all
s were examined for erectile function by standard protocol.10
ith the rats under ketamine-midazolam anesthesia, the MPG
d CN were exposed bilaterally via a midline laparotomy. A
G butterfly needle was inserted into the proximal left corpus
ernosum, filled with 250 U/mL heparin solution, and con-
cted to a pressure transducer (Utah Medical Products, Mid-
e, UT) for intracavernous pressure (ICP) measurement. The
P was recorded at a rate of 10 samples/second using a com-
ter with LabVIEW 6.0 software (National Instruments, Aus-
UROLOGY 77 (6), 2011
tin, TX). A bipolar stainless-steel hook electrode was used to
stimulate the origin of the CN at the MPG proximally from the
ne
me
ato
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per
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cal
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ori
represented approximately 35% of the original tissue
mass. After drying under ambient conditions, the decel-
lul
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rve gap (control group) or the interposed construct (treat-
nt groups). The electrode was connected to a signal gener-
r (National Instruments) and a custom-built, constant-
rent amplifier generating monophasic rectangular pulses
h stimulus parameters of 1.5 mA, 20 Hz, pulse width 0.2 ms,
d duration 50 seconds. Three stimulations were conducted
side and the erection with the maximum increase in ICP
s included for statistical analysis in each animal. For the
culation of ICP increase/mean arterial pressure (MAP) ratio,
temic blood pressure was recorded using a 23G butterfly
edle inserted into the aorta at the level of the iliac bifurca-
n.
munohistochemical and Immunofluorescence
aining
sue samples were fixed in cold 2% formaldehyde and 0.002%
urated picric acid in 0.1-M phosphate buffer, pH 8.0, for 4
urs followed by overnight immersion in buffer containing
% sucrose. The specimens were then embedded in OCT
mpound (Sakura Finetec United States, Torrance, CA) and
red at –70°C until use. Fixed frozen tissue specimens were cut
7 �m, mounted onto SuperFrost-plus charged slides (Fisher
entific, Pittsburgh, PA), and air dried for 5 minutes. For
munostaining, the slides were placed in 0.3% H2O2/metha-
l for 10 minutes, washed twice in PBS for 5 minutes, and
ubated with 3% horse serum in PBS/0.3% Triton x-100 for
minutes at RT. After draining this solution, the slides were
ubated at RT with anti- neuronal nitric oxide synthase
ression (nNOS) antibody (Santa Cruz Biotechnology, Santa
uz, CA) or anti-S100 antibody (Leica Microsystems, Inc.,
nnockburn, IL) for 1.5 hours. After rinses in PBS, the sec-
ns were incubated with fluorescein isothiocyanate–
jugated secondary antibody (Jackson ImmunoResearch Lab-
tories, West Grove, PA). After rinses in PBS, the slides were
ubated with freshly made EdU detection cocktail (Invitro-
) for 30 minutes at RT followed by staining with DAPI. The
ined tissue was examined with a Nikon Eclipse E600 fluo-
cence microscope and photographed with a Retiga 1300
imaging camera.
atistical Analysis
ta were analyzed with Prism 4 (GraphPad Software, Inc., San
ego, CA) using one-way analysis of variance followed by
key-Kramer test for post hoc comparisons. All data are re-
rted as mean � standard deviation. Significance was set at P
05.
SULTS
cellularization and Molding
cellularization was performed with adipose tissue iso-
ed from both human and rat. Data shown in Figure 1
for a tissue sample obtained from a patient who
derwent elective abdominoplasty. After a 5-day ex-
ction procedure, the original tissue (Fig. 1A) was
nsformed into a loose matrix that was devoid of cells
d cell debris (Fig. 1B). Qualitatively, the decellularized
ipose tissue had similar dimensions and shape as the
ginal tissue (Fig. 1A, B). Quantitatively, the matrix
OLOGY 77 (6), 2011
arized adipose tissue could be cut into various sizes and
pes, e.g., threads (Fig. 1C). It could also be homoge-
ed and then molded into various three-dimensional
hitectures; in this case, a sheet (Fig. 1D).
eding of Matrix with ADSCs
has been shown that nerve conduits seeded with AD-
s produced better results than unseeded ones for pe-
heral nerve repair.7-9,22 We thus examined how well
SCs could attach and grow on acellular adipose ma-
x; in this case, threads to be used as nerve conduits. We
o considered that, for future clinical application, the
st ideal matrix-based nerve conduit should combine
ogenic matrix with autologous ADSCs. Thus, data
wn in Figure 2 are for a nerve conduit that will be
fted into a rat host, and it was constructed by seeding
allogenic matrix with autologous ADSCs. Twenty-
r hours after seeding, the autologous ADSCs adhered
the allogenic matrix and covered about 55% of the
face. One week later, the cells covered about 90% of
matrix surface (Fig. 2A–C). As a pregrafting test, the
ded matrix was transplanted into the subcutaneous
ce of an allogenic host, and during a 10-day course, no
n of inflammatory reaction was observed. Furthermore,
tologic examination of the transplanted tissue showed
t the matrix remained intact and was covered with a
er of cells (Fig. 2D). Thus, autologous ADSC-seeded
ogenic adipose matrix was tolerated by the host and
ained intact for an extended period in vivo.
afting of Seeded and Unseeded Matrix
simulate postprostatectomy nerve injury, male rats
derwent bilateral CN transection. These rats then
eived no graft, unseeded adipose matrix, or ADSC-
ded adipose matrix (Fig. 3A). Three months later,
se rats were examined for erectile function and then
rificed. Histologic examination of the grafts showed
t both unseeded and seeded matrices were covered
th numerous cells (Fig. 3B–F). On average, the cell
nsity was nearly twice as high on seeded matrix as on
seeded matrix (Fig. 3B). S100, a general nerve marker,
d nNOS, an erectile nerve–specific nerve marker, were
th absent and present on the unseeded and seeded
trices, respectively (Fig. 3C–F). On the seeded matri-
, S100 and nNOS were expressed at various degrees
t mostly colocalized near the MPG, indicating various
grees of CN axonal extension. One of the seeded
trices contained an S100/nNOS-positive nerve across
entire length, and several EdU-positive cells (ADSCs)
re found along this nerve fiber (Fig. 3E, F).
sessment of Erectile Function
ctile function of the aforementioned rats was assessed
measuring the ICP after electrostimulation of CN,
ich was normalized against mean MAP. The results
1509.e3
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wed that the ADMT group had better erectile func-
n than the control, and the ADMT�ADSC group was
tter than the ADMT group (Fig. 4). However, despite
lear trend toward functional recovery, especially in the
MT�ADSC group, the difference (P � .07 between
MT�ADSC and control) did not reach statistical
nificance because of large variations.
MMENT
tologous nerve grafting inevitably requires sacrificing a
ctional nerve; in addition, the procurement of such a
ft often requires a secondary surgery and causes donor site
rbidity. By contrast, xenografting materials do not have
se problems but carry the risks of host rejection and
nsmitting zoonotic diseases.23 Thus, allogenic grafting
y have the best potential but still requires careful con-
eration. For example, allogenic graft procurement should
t be harmful to the living donor and should be agreeable
the family of the deceased donor. However, these criteria
difficult to fulfill because few tissues are graft-quality
terial that can be removed from a living person without
sing harm. In addition, cultural confines may prevent
wide adoption of cadaveric tissues.
ure 1. Decellularization and fabrication of adipose tissue.
protocol shown in Materials and Methods and transform
tologic images of the adipose tissue before and after dec
mogenized and then molded into a sheet (D).
09.e4
Adipose tissue is one of the rare tissues that can be
rtially removed from a living person without causing
rm. Its superficial location makes it more accessible than
st other tissues, and its removal is an intervention desired
many patients. As the volume of surgeries for obesity and
st–weight loss contouring continues to increase, adipose
ue is removed in an increasing rate. Thus, there is no
ubt that there is an abundance of adipose tissue, and a
ent study by Flynn5 has demonstrated its potential as an
llular matrix for adipose tissue reconstruction. Specifi-
ly, scanning electron microscopic examination of the
llular adipose matrix identified regions of network-type
llagen, consistent with the rich basement membrane
mponent in adipose tissue. Thus, the decellularized adi-
se tissue appears to be an ideal material for tissue recon-
uction.
The present study was initiated more than 1 year
fore the publication of the aforementioned study by
nn. Therefore, both the idea of developing an acellu-
adipose matrix and the formulation of the decellular-
tion protocol were conceived independently. In fact,
h