Bioengineered bladder patches constructed from multilayered adipose-derived

stem cell sheets for bladder regeneration

Ying Wang a,1, Shukui Zhou a,1, Ranxing Yang a, Qingsong Zou a, Kaile Zhang a,
Qinghua Tian b, Weixin Zhao c, Lijuan Zong d, Qiang Fu a,*

a Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s

Hospital, Shanghai Eastern Institute of Urologic Reconstruction, Shanghai Jiao Tong
University, 200233, Shanghai, China;
b Department of Radiology, Shanghai Jiao Tong University Affiliated Sixth People’s
Hospital, 200233, Shanghai, China
c Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA;
d Shanghai Key Laboratory of Tissue Engineering, 200011, Shanghai, China

1 These authors contributed equally.

* Corresponding author. Shanghai Jiao Tong University Affiliated Sixth People’s

Hospital, Shanghai Eastern Institute of Urologic Reconstruction, Shanghai Jiao Tong
University, No 600, Yishan Road, Xuhui District, Shanghai 200233, China.
E-mail address: jamesqfu@aliyun.com (Q, Fu)
Abstract
Cell-seeded scaffolds are a common route of cell transplantation for bladder repair
and reconstruction. However, when cell suspensions are harvested, proteolytic
enzymes often cause extracellular matrix damage and loss of intercellular junctions.
To overcome this problem, we developed a bioengineered three-dimensional bladder
patch comprising porous scaffolds and multilayered adipose-derived stem cell (ASC)
sheets, and evaluated its feasibility for bladder regeneration in a rat model. Adipose-
derived stem cells (ASCs) were labeled with ultrasmall super-paramagnetic iron oxide
(USPIO) nanoparticles. ASC patches were constructed using multilayered USPIO-
labeled ASC sheets and porous polyglycolic acid scaffolds. To monitor the
distribution and localization of bioengineered bladder patches in live animals,
magnetic resonance imaging (MRI) was performed 2 weeks, 4 weeks and 8 weeks
after transplantation. The bladder regenerative potential of ASC patches was further
evaluated by urodynamic and histological analysis. Scanning electron microscopy
indicated that cell sheets adhered tightly to the scaffold. MRI showed hypointense
signals that lasted up to 8 weeks at the site of USPIO-labeled ASC sheet transplants.
Immunofluorescence demonstrated that these tissue-engineered bladder patches
promoted regeneration of urothelium, smooth muscle, neural cells and blood vessels.
Urodynamic testing revealed that the ASC patch restored bladder function with
augmented capacity. The USPIO-labeled ASC patch provides a promising perspective
on image-guided tissue engineering and holds great promise as a safe and effective
therapeutic strategy for bladder regeneration.

Keywords: bladder regeneration; adipose-derived stem cell; cell sheet; ultrasmall

super-paramagnetic iron oxide; tissue engineering
1. Introduction
Reconstruction of the human bladder remains one of the greatest urological
challenges. Conventional bladder reconstruction using gastrointestinal segments is the
most frequently used therapeutic strategy [1]. However, because of the
incompatibility of gastrointestinal tissue with urine, a series of complications may
occur including mucus production, recurrent urinary tract infections, electrolyte
imbalances, and the chance of malignancy [2-4]. The application of transplantable
bladder neo-organs produced by tissue engineering may avoid many of these
complications and provide a potential alternative strategy for bladder repair and
reconstruction [5]. Various tissue-engineered scaffolds have been studied as bladder
patches for urinary bladder reconstruction [6-9]. However, constructing three-
dimensional scaffolds that contain multiple layers of seeded cells is still a crucial
issue for the creation of thick and viable bladder tissues.
Different types of cells have been used for bladder reconstruction in recent
studies [10-13]. Adipose-derived stem cells (ASCs) have been proposed as an ideal
cell source for bladder regeneration, and their use in the repair of bladder defects has
been successfully demonstrated [14-16]. However, when ASC suspensions are
harvested, proteolytic enzymes often cause extracellular matrix (ECM) damage and
loss of cell-to-cell junction proteins. ASC sheets have been developed using cell-sheet
technology, which avoids enzymatic dissociation and preserves cell-to-cell
interactions and ECM proteins [17]. However, because of their lack of mechanical
strength, transplantation of ASC sheets alone is insufficient to reconstruct bladder
tissues. To overcome this problem, we prepared a novel tissue-engineered construct
that incorporated porous scaffolds and multilayered ASC sheets. The three-
dimensional structure of these multilayered ASC sheets provides a nutritive and
structural microenvironment for cell adhesion and proliferation within the scaffold,
which might be useful for regulating tissue regeneration.
Non-invasive, in vivo characterization of the location of transplanted cell sheets
is important in identifying the mechanisms by which transplanted cells exert their
effects. Magnetic resonance imaging (MRI) of cell sheets labeled with ultrasmall
super-paramagnetic iron oxide (USPIO) can achieve this goal. USPIO was a highly
efficient label for various cell types in previous studies [18-20]. However, USPIO-
labeled cell sheets are rarely reported, and the fate of labeled ASC sheets after
transplantation is unclear.
In this study, the cytotoxicity of USPIO and the viability of USPIO-labeled
ASCs were evaluated, and the biocompatibility of porous scaffolds with ASC sheets
was analyzed in vitro. MRI was used as a noninvasive method to investigate the
location of transplanted USPIO-labeled cell sheets in vivo. Furthermore, we also
investigated the effects of this newly developed construct when implanted for bladder
regeneration in a rat model.

2. Materials and methods

2.1. Materials
Vitamin C, Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine
serum (FBS) were purchased from Gibco (NY, USA) and Sigma (St. Louis, MO,
USA). Anti-α-Smooth muscle actin (α-SMA), and anti-CD31 monoclonal antibodies
were obtained from Abcam (Cambridge, UK). Female Sprague–Dawley (SD) rats at 6
weeks of age, body weight 160 ± 20 g, were obtained from the Animal Research
Center of Fudan University (Shanghai, China). The experimental procedures were
conducted according to local Guidelines on the Ethical Treatment of Animals.
2.2. Preparation of USPIO nanoparticles
The synthesis of USPIO followed protocols from previously reported studies
[21]. Briefly, a solution consisting of FeSO4·7H2O and ferric citrate in a molar ratio of
2:1 was mixed with vigorous stirring at room temperature, followed by gradual
addition of ascorbic acid. Next, the pH was brought to 10 with NaOH solution and the
resulting solution was dialyzed in a 14 kDa molecular weight cut-off dialysis bag. The
Fe3O4 nanoparticle solution was then filtered through a 0.22 μm nylon filter to remove
bacteria. Stable USPIO nanoparticles were obtained. The morphologies of the
synthesized USPIO nanoparticles were characterized using scanning electron
microscopy (SEM). The crystalline structure of the synthesized USPIO were
characterized by an X-ray diffractometer (XRD, D8 ADVANCE, BRUKER-AXS)
with Cu Kα radiation ( λ = 0.15418 nm) at a 2θ scan range from 20° to 70° and a scan
rate of 10 degrees per min. The Physical Property Measurement System (Quantum
Design, USA) was used to measure the magnetization loop.
2.3. Harvest and culture of ASCs
Adipose tissues were obtained from the inguinal regions of SD rats, which were
processed in accordance with a previously reported method [22]. Briefly, the fresh
adipose tissues were washed three times in phosphate-buffered saline (PBS) and then
cut into small pieces. They were digested with 0.1% collagenase I (Sigma) under
continuous oscillation at 37 °C for 1 h and then neutralized with basal DMEM
containing 10% FBS. The cell suspension was filtered through a 50 μm mesh filter
and then centrifuged. The primary ASCs were collected and maintained in DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco). The
characterization of ASCs was determined by cell surface markers and their ability to
differentiate into adipogenic and osteogenic lineages as previously reported [23].
2.4. Cell labeling and Prussian blue staining
ASCs at passage 2 were seeded into 6-well plates, and given a medium change
every two days until they reached 80%–90% confluence. Poly-L-lysine (PLL; Sigma)
was used as a transfection agent to boost intracellular uptake of the nanoparticles;
ASCs were incubated with USPIO and 0.75 μg/mL PLL. The USPIO concentrations
used for labeling were 12.5 μg/mL, 25 μg/mL or 50 μg/mL. Unlabeled ASCs were
used as a control. After incubation for 24 h, ASCs were washed with PBS and were
further cultured in basal DMEM. A small aliquot of labeled cells was fixed for 15 min
in 4% paraformaldehyde, and then incubated with 10% potassium ferrocyanide for 30
min. After washing with PBS, the cells were counterstained with nuclear fast red
(Solarbio science & Technology, China) for 10 min.
2.5. Viability and toxicity of labeled ASCs
Cell viability was assessed using a cell counting kit-8 (CCK-8; Dojindo, Tokyo,
Japan), following the manufacturer’s instructions. ASCs were added to 96-well plates
(2 × 103 cells/well) at 37 °C and 5% CO2. After 24 h, different concentrations of
USPIO (12.5 µg/mL, 25 µg/mL or 50 µg/mL) were added to each well for 24 h
incubation. Unlabeled ASCs served as a control group. CCK-8 (10 µL) was then
added to the wells for 2 h incubation. Optical density values were then measured at a
wavelength of 450 nm. The toxicity of USPIO labeling towards ASCs was further
assessed by flow cytometric detection of cell cycling and apoptosis (Beckman
Coutler, USA). USPIO-labeled and unlabeled ASCs were stained with propidium
iodide to analyze cell cycling and with annexin V (BD Biosciences, San Diego, CA,
USA) to assess apoptosis.
2.6. In vitro MRI of labeled ASCs
Cells labeled with different concentrations of USPIO (0 μg/mL, 12.5 μg/mL, 25
μg/mL or 50 μg/mL) were trypsinized and fixed with 4% paraformaldehyde. Next, 6 ×
105 cells were resuspended in 1.5-mL tubes and scanned with a 3 T MRI instrument
(PharmaScan, Bruker, Germany) to determine the concentration of USPIO required to
label cells and cause a sufficient decrease in MRI signal intensity.
2.7. Preparation and labeling of cell sheets
USPIO-labeled ASCs at the second passage were harvested for preparation of the
cell sheets. To create a cell sheet, a suspension of ASCs was plated into a 60-mm
temperature-responsive cell culture dish (Thermo Fisher Scientific, San Jose, CA,
USA) at 1 × 105 cells/cm2. When the cells reached 90%–100% confluence, vitamin C
(50 μg/mL; Sigma, St. Louis, MO, USA) was added to the basal DMEM, to promote
the production of ECM. The culture medium was composed of low-glucose DMEM,
10% FBS, 50 μg/mL vitamin C, 1% penicillin/streptomycin, and 3.7 g/L sodium
bicarbonate (Sigma). The culture medium was replaced every two days. After the
ASCs had been cultured for 2 weeks at 37 °C in 5% CO2, an intact ASC sheet was
obtained by reducing the culture temperature to 20 °C for 30 min. The morphological
features of ASC sheets were evaluated by hematoxylin and eosin (HE) staining,
Masson’s trichrome staining and Prussian blue staining.
2.8. Preparation of ASC sheet–scaffold constructs
Polyglycolic acid (PGA) fibers were provided by Shanghai Ju Rui Biomaterials
Company Inc (Shanghai, China). PGA fibers (15 mg) were arranged into a cylindrical
shape of 9 mm diameter and 1.5 mm depth, disinfected with 75% ethanol overnight,
and then washed three times with sterile PBS. They were then pre-incubated in
DMEM supplemented with 10% FBS to test sterility and enhance cell attachment.
The medium was removed afterward and the scaffolds were air-dried for 45 min
under ultraviolet light before use.
To combine the ASC sheet and PGA, a prepared ASC sheet was allowed to
attach to the scaffold in an incubator at 37 °C for 1 h, to allow for complete adhesion.
Culture medium was then added to cover the constructs. After three days, part of cell–
scaffold construct was examined by SEM.
2.9. SEM examination
Both ASC sheets and ASC sheet–scaffold constructs were prepared for SEM.
Briefly, samples were washed three times with PBS and fixed with 2% glutaraldehyde
for 2 h followed by post-fixation with 1% osmic acid. After a thorough wash with
PBS, the samples were subjected to dehydration in an ethanol gradient and then dried
by lyophilization. The samples were sputter-coated with gold and examined by SEM.
2.10. Bladder reconstruction
ASC sheet–scaffold constructs were evaluated in a bladder augmentation model
using female SD rats. The animals were randomly divided into two groups: a
cystotomy group (the control group; n = 24) and an ASC patch group (the
experimental group; n = 24).
The dome of the bladder was incised longitudinally (approximately 1 cm) and the
ASC patch was grafted onto the dome in 24 rats (the experimental group). The grafted
area was covered with a piece of perivesical fat. Twenty-four rats were treated with
similar cystotomy and suture repair alone (the control group). Rats in the
experimental and control group were sacrificed at 2 weeks, 4 weeks and 8 weeks post-
implantation (n = 8 rats per time point).
2.11. MRI tracking and urodynamics
MRI and urodynamic studies were performed on the control and experimental
groups at 2 weeks, 4 weeks and 8 weeks after implantation. After general anesthesia
(pentobarbital, 30 mg/kg intraperitoneal injection; i.p.), rats were placed inside the
MRI receiver coil. T2-weighted MRI scans were obtained using the Rapid Acquisition
with Relaxation Enhancement sequence.
During urodynamic testing, the rats were anesthetized with pentobarbital (30
mg/kg i.p.). The bladder was exposed via a midline abdominal incision. A
polyethylene-90 catheter was inserted through the bladder dome. The other end of the
bladder catheter was connected to a pressure transducer and an infusion pump. The
urodynamic parameters were recorded spontaneously during continuous infusion of
sterile PBS. Bladder compliance was calculated by the ratio of the volume instilled
and the change in pressure.
2.12. Histological analysis
The rats were euthanized at 2 weeks, 4 weeks or 8 weeks postoperation, and their
bladders were removed for evaluation. Briefly, the bladders were rinsed with PBS to
remove blood. Portions of the regenerated tissue in the bladder dome were embedded
in optimum cutting temperature compound. Sections were cut at 5 μm and stained by
HE and Prussian blue staining. Immunofluorescence staining of cytokeratin (Abcam,
ab9377, 1:100 dilution), α-SMA (Abcam, ab32575, 1:150 dilution ), NeuN (Abcam,
ab177487, 1:100 dilution) and CD31 (Abcam, ab119339, 1:100 dilution) was
performed to examine the distribution of urothelium, smooth muscle cells, neurons
and blood vessels in the harvested specimens.
2.13. Statistical analysis
All quantitative data were evaluated and are presented as the mean ± standard
deviation. Statistical analysis was performed using Student’s-t test or one-way
analysis of variance, using SPSS 11.0 software (IBM, Armonk, NY, USA). A value of
P < 0.05 was considered statistically significant.

3. Results
3.1. Synthesis and characterization of USPIO
The size and morphology of the synthesized USPIO nanoparticles was
characterized by transmission electron microscopy (TEM; Fig. 1A). TEM images
showed that the USPIO nanoparticles were approximately spherical and fell within a
relatively narrow diameter range of 2–11 nm (Fig. 1B). The USPIO nanoparticles
were further characterized through X-ray power diffraction (XRD). Fig. 1C shows that
the XRD pattern of the USPIO nanoparticles was consistent with that of the standard
cubic Fe3O4 structure. The calculated diameter of the nanoparticles was 8.02 nm,
based on the strongest peak (311) observed in XRD, which is in good agreement with
the TEM data. The saturation magnetization of the USPIO nanoparticles was
approximately 59.2 emu g−1 (Fig. 1D).
3.2. Prussian blue staining and MRI examination
Prussian blue staining showed increasing iron accumulation in the ASCs as the
USPIO concentration was increased (Fig. 2A). The biocompatibility of the
synthesized USPIO is an important consideration for in vivo applications. The
potential cytotoxicity of the USPIO was examined using a standard CCK-8 assay. Fig.
2B shows that cell viability rarely decreased with different USPIO concentrations
(12.5 µg/mL and 25 µg/mL), but cytotoxicity was observed at doses up to 50 µg/mL
(P < 0.05). There were notable differences in MRI signal intensity with varying
concentrations of USPIO used to label cells. With increasing USPIO concentration,
the MRI signal was progressively decreased. The reduced MRI signal could be easily
observed by the naked eye at USPIO concentrations greater than or equal to 25 µg/mL
(Fig. 2C). Hence, we chose 25 µg/mL as an appropriate concentration to efficiently
label ASCs.
3.3. Cell viability of labeled ASCs
Cell cycling is an important aspect of cell behavior. Cell cycle analysis showed
no significant differences in the proliferation indices of labeled versus unlabeled
ASCs (P > 0.05; Fig. 3A–C, Supplementary Table S1). To investigate the influence of
USPIO on cell apoptosis, labeled ASCs were stained with annexin V and analyzed by
flow cytometry. The percentages of apoptotic labeled and unlabeled ASCs were less
than 5%, with no significant differences (Fig. 3D–F, Supplementary Table S1). These
results further confirmed that 25 µg/mL USPIO did not affect cell viability.
3.4. TEM and size characterization of labeled ASCs
 TEM imaging of cells was performed to characterize the USPIO distribution in
subcellular compartments. After incubation with 25 µg/mL USPIO, the cell
membranes of labeled ASCs were clear and intact, and the ultramicrostructure did not
appear to be damaged (Fig. 4A). Higher-magnification images further demonstrated
that the USPIO particles were incorporated intracellularly and predominantly
accumulated in the endosomes/lysosomes (Fig. 4A). The size of labeled ASCs in
suspension ranged from 13 μm to 23 μm diameter (mean = 20.2 μm; Fig. 4B), which
is comparable with the unlabeled ASCs (Fig. 4C).
3.5. Characterization of ASC sheets
Labeled ASCs proliferated and formed a continuous cell sheet at 14 days (Fig.
5A). Inverted phase contrast microscopy showed that ASCs exhibited stratified
growth (Fig. 5B). SEM images confirmed that the cell sheets contained multilayered
cells and abundant ECM (Fig. 5C). HE staining and Masson’s trichrome staining
showed that the cultured cell sheets were composed of five or six layers of cells, and a
large amount of ground substance was observed outside the ASCs (Fig. 5D–E).
Prussian blue staining revealed blue-stained USPIO distributed in the cell sheets (Fig.
5F). Intact, labeled ASC sheets were transferred onto the highly porous PGA scaffolds
to construct an ASC patch (Fig. 5G); the cell sheets gradually adhered to the PGA
scaffolds after transfer (Fig. 5H). SEM observations showed that multilayered cells
with secreted matrices covered the PGA fibers (Fig. 5I).
3.6. Rat bladder reconstruction and MRI tracking in vivo
The bladder was mobilized from the peritoneal cavity, which was incised
longitudinally to perform augmentation cystoplasty. A bioengineered bladder patch
(the ASC patch) was developed for bladder reconstruction (Fig. 6A). To determine the
efficacy of USPIO for detecting and tracking ASCs, MRI was performed on weeks 2,
4 and 8 after transplantation of a labeled ASC patch (Fig. 6B). The USPIO-labeled
bioengineered bladder patch could be effectively visualized by MRI. Relative
hypointensity representing labeled cell sheets was observed at the transplantation site
of bladder, whereas no hypointense area was detected in the cystotomy group. In the
ASC patch group, MRI revealed that the majority of hypointense areas could persist
for a long time, which was especially obvious at 2 and 4 weeks after transplantation.
The relative signal intensity of the transplantation site of bladder at 8 weeks after
transplantation was higher than that at 2 and 4 weeks after transplantation, but was
still lower than that in the cystotomy group. No urinary stones were observed by
macroscopic examination after euthanasia. HE staining showed that the ASC patch
was covered by a thin layer of urothelium and trivial smooth muscle at 2 weeks. A
multilayered urothelium with organized smooth muscle bundles gradually
accumulated over time. At 8 weeks after ASC patch implantation, PGA fibers
degraded completely and the tissue-engineered de novo bladder wall showed an inner
stratified urothelium and an outer layer of well-organized muscle fascicles, which
resembled the tissue structure of native bladder tissue (Fig. 6B). Prussian blue staining
revealed blue-stained iron particles in tissue sections, which matched the MRI images
(Fig. 6B).
3.7. Histology of engineered tissues
Immunofluorescence analysis revealed regeneration of the urothelium
(cytokeratin), smooth muscle (α-SMA), neural cells (NeuN), and neo-angiogenesis
(CD31) at different time points. Discontinuous urothelium could be observed at
implanted sites at 2 weeks (Fig. 7A). After 8 weeks in vivo, multilayered urothelium
was detected, which presented more extensive hyperplasia compared with the
cystotomy group (P < 0.05; Fig. 7B). Furthermore, immunofluorescence staining
revealed α-SMA expression at the graft site, indicating smooth muscle differentiation
(Fig. 7A). Histomorphometric analysis revealed that the extent of smooth muscle
bundles was similar to that of the cystotomy group at 8 weeks (Fig. 7C). Positive
staining for NeuN demonstrated the presence of mature neural cells (Fig. 7A). The
number of NeuN-positive cells in the experimental group was lower than that in the
cystotomy group (Fig. 7D). The number and diameter of CD31-positive vessels
steadily increased over time (Fig. 7A). The diameter of vessels finally achieved a
greater level at 8 weeks after bladder reconstruction, which was still less favorable
than the cystotomy group (P < 0.05; Fig. 7E).
3.8. Evaluation of bladder capacity and compliance
 Bladder capacity and compliance were evaluated in an urodynamic study.
Bladder capacity and compliance increased continuously in both the cystotomy group
and the bladder patch group. In the bladder patch group, a significant increase in
bladder capacity was observed after implantation. At 8 weeks postoperation, the
capacity in the bladder patch group reached 152.87% of that in the control group (0.44
± 0.04 ml versus 0.29 ± 0.01 ml, P < 0.05; Fig. 8A). In addition, although bladder
compliance in the bladder patch group was slightly lower than that in the control
group at 2 weeks after implantation, it recovered to nearly the same level as control
group at 8 weeks postoperation (9.82 ± 1.20 mL/cm H2O versus 7.38 ± 0.44 mL/cm
H2O, P > 0.05; Fig. 8B).

4. Discussion
Tissue-engineered bladder grafts previously have been used as patches for
functional bladder reconstruction [24]. In previous reports, investigators reconstructed
functional bladders with acellular matrices or synthetic scaffolds seeded with
autologous cells [25-27]. A porous structure of synthetic scaffolds has been proven to
be beneficial for the incorporation of cells [28]. Before cells could be seeded onto
scaffolds, a large number of cells had to be produced and subsequently harvested by
the use of proteolytic enzymes [29]. However, this conventional tissue-engineering
procedure disrupts cell-to-cell interactions and ECM proteins [30-32]. In contrast, the
cell sheet technique allowed us to harvest an intact sheet of cells by a non-enzymatic
process, without destroying cell-to-cell networks and the deposition of ECM. This
technique has been applied in various areas of regenerative medicine [17, 29, 33-36].
The dense cell sheets play an important role in preventing urine leakage into the
abdominal cavity. Thus, we have established a tissue-engineered bladder patch
combining the advantages of cell sheets and porous scaffolds, and we further proved
the feasibility of creating thick and viable bladder tissues. We also showed that
transplanted USPIO-labeled ASC sheets can be tracked with MRI for long-term
monitoring in vivo.
The localization and long-term tracking of transplanted cells in living organisms
is needed in pre-clinical trials. Although MRI tracking of cells labeled with optimized
superparamagnetic iron oxide nanoparticles has been reported in many studies [19,
37], there have been few reports on USPIO-labeled cell sheets. In this study, we
describe the characteristics of USPIO as an MRI contrast agent. Prussian blue staining
confirmed that ASC labeling efficiency increased with increasing concentrations of
USPIO. We further investigated the cytotoxicity of USPIO in labeled ASCs. The
results of the CCK-8 assay suggest that the USPIO was not significantly cytotoxic
until a concentration of 50 µg/mL was reached. Therefore, we chose to use USPIO at
the safer concentration of 25 µg/mL for labeling the ASCs. The TEM results confirm
that the USPIO particles were internalized by the cells and aggregated in endosomes
or lysosomes, and that the internalization of USPIO particles did not affect cell size.
Apoptosis and cell cycle analyses showed that 25 µg/mL USPIO did not impair cell
proliferation capacity, and so should be suitable for further experiments.
A large number of growth factors and adhesion molecules are found in cell
sheets [38, 39]. Thus, ASC sheets have a good ability to adhere to porous structures,
providing a specific cellular microenvironment suitable for tissue regeneration. In
previous reports, the rough surface of scaffolds used for bladder reconstruction often
resulted in bladder stone formation [40]. ASC sheets were observed to reduce the
incidence of stone formation in this study, which might be explained through two
mechanisms: ASC sheets transferred onto the porous scaffold might have acted as a
waterproof barrier that prevented penetration of urine and indirectly decreased the
inflammatory response; the intact ASC sheets might have provided the bladder patch
with a flat, smooth surface that hampered urine crystal precipitation.
In this study, we observed that USPIO-labeled ASCs can be clearly detected by
MRI in vitro. To assess the fate of transplanted USPIO-labeled bladder patches, MRI
was used to monitor the localization of USPIO labeling in the transplanted area of the
bladder, which presented a hypointense signal on T2 images. However, the relative
hypointense areas gradually became less contrasted over time. We speculated that
prolonged implantation time led to remodeling of the bladder patch into neobladder,
as evidenced by the change of urothelium and smooth muscle density in the grafts;
Meanwhile, USPIO-labeled cells dispersed gradually in the remodeled grafts wall
over time. The MRI results were further confirmed by Prussian blue staining of
corresponding tissue. Histological and immunofluorescence staining indicated that
bladder reconstruction with ASC patches achieved histological regeneration of
urothelium, smooth muscle and vessels at each time point. However, nerve
regeneration in the de novo bladder wall was unsatisfactory, which was in agreement
with the previous literature [40, 41]. It is reasonable to speculate that the 8-week
evaluation period may have been too short for nerve regeneration to occur in the
transplanted bladder patches, and thus we would need a longer evaluation period to
observe any innervation of the reconstructed bladder wall.
Vascularization is one of the key elements for the survival of the implanted cells.
The porosity and pore size of scaffolds affect angiogenesis of implanted tissue. One of
the major advantages of PGA is its high porosity, which accelerates angiogenesis and
improves implanted cell survival. In the present study, neovascularization was
detected in the transplanted bladder patch in the tissue regeneration process and no
remnant of undegraded PGA could be observed histologically after 8 weeks of
implantation, which indicated PGA with high porosity could render a positive effect
on the vascularization of the implanted bladder patch and suitable degradation rate of
PGA matched the formation of an engineered neobladder.
Urodynamic parameters illustrated that bladder volume was augmented with the
ASC patches. Bladder compliance in the experimental group was lower than that in
the cystotomy group at week 2, but gradually increased over time. We hypothesize
that the transplanted ASC sheet contributed to the regeneration of a well-organized
muscular layer, which would improve compliance of the bladder.
Our results indicate that ASC sheets together with porous scaffolds are a
promising approach for bladder tissue regeneration. However, a limitation of our
study is the lack of comparable results with simple porous scaffolds, as simple porous
scaffolds have the drawbacks of urine leakage directly into the abdominal cavity and
increased mortality of rats. Future studies of bladder reconstruction in larger animals
are necessary to confirm and validate the efficacy of bioengineered bladder patches
for clinical application.

5. Conclusions
A promising strategy, employing the techniques of USPIO synthesis,
construction of ASC sheets, and fabrication of porous scaffolds, was successfully
established for the construction of a bioengineered bladder patch. The USPIO
nanoparticles can be used to label ASCs efficiently without affecting cell viability,
proliferation or apoptosis. Furthermore, USPIO-labeled bladder patches can be
dynamically monitored in vivo by noninvasive MRI for long periods of time.
Transplantation of the ASC sheets promoted rapid regeneration of the urothelium,
smooth muscle and vessels. Taken together, our results suggest that this
bioengineered bladder patch could effectively restore the structure and function of an
engineered neobladder.

6. Conflict of interest
The authors declare no conflict of interest.

Acknowledgements
This work was supported by the National Natural Science Foundation of China
(Grant No. 81270780, 81800593 and 81470917), the Science and Technology
Commission of Shanghai (14JC1492100), Shanghai Jiao Tong University Biomedical
Engineering Cross Research Foundation (YG2014ZD07) and Leaders Training
Program of Shanghai. The authors are grateful to Institute of Nano Biomedicine and
Engineering, Shanghai Jiao Tong University for their technical support.

Supplementary data
Supplementary Fig. S1 and Table S1

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